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Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2015. Tomato spotted wilt virus (TSWV) bem como outras espécies de tospovírus (Família Bunyaviridae) é responsável por perdas substanciais na produção de vegetais ao redor do mundo. Tospovírus são transmitidos de maneira circulativa e propagativa por tripes (Ordem Thysanoptera). Como para outros patógenos, ações contra tospovírus exigem um olhar holístico, envolvendo uma combinação de táticas culturais, fitosanitárias, químicas e biológicas, quando apropriadas, além do uso de cultivares resistentes. Entretanto, o controle de doenças causadas por tospovírus tem se mostrado difícil, dado o grande espectro de hospedeiros desses vírus e a grande eficiência dos tripes vetores em transmiti-los. Além disso, a dependência e o aumento no uso de inseticidas de baixo custo tem exacerbado a presença de tospovírus pela forte pressão de seleção sobre os tripes vetores que se tornam resistentes a diferentes inseticidas. Para solucionar de forma simples todas as questões financeiras e ambientais associadas ao uso abusivo de inseticidas para o controle de tripes, esforços têm aumentado para a obtenção de cultivares geneticamente resistentes como um componente integral das estratégias de controle de doenças. Até o momento existem duas fontes de resistência disponíveis para o melhoramento genético de hortaliças à TSWV. Uma dessas fontes é o cluster genético Sw-5, o qual foi encontrado em Solanum peruvianum L., uma espécie de tomate selvagem do Peru, e tem sido introduzido em cultivares de S. lycopersicum L. (tomate comercial). Após mapeamento gênico, pelo menos cinco parálogos compõem o cluster genético Sw-5 em S. peruvianum, os quais foram nomeados de Sw-5a a Sw-5e. Esses parálogos codificam receptores do tipo NB-LRR, uma classe de proteínas citoplasmática que ativam resistência após reconhecimento direto ou indireto de patógenos que tenham ultrapassado as primeiras barreiras de defesa do sistema imune vegetal. Transformação de plantas de tabaco com os parálogos Sw-a e Sw-5b revelou que somente o último ativa resistência contra isolados de TSWV. Com a disponibilidade do genoma do tomate, genes ortólogos também foram mapeados em S. lycopersicum. Esta tese inicia-se com uma descrição detalhada do sistema imune vegetal e descobertas anteriores sobre o cluster genético Sw-5 (Capítulo 1). Como parte da introdução, os problemas causados por tospovírus e suas características são descritas. Tendo essas informações como ponto de partida, esta tese focou no entendimento de pontos-chaves da resistência mediada pela proteína Sw-5b contra tospovírus com uma atenção especial nos eventos moleculares e celulares envolvidos atrás desse mecanismo de resistência. Análises funcionais foram realizadas para esclarecer as delimitações genéticas entre os ortólogos funcionais e não funcionais do cluster Sw-5 no reconhecimento de tospovírus e ativação de resistência. Já que transformantes de N. tabacum com o gene Sw-5b feitos anteriormente não apresentaram resposta hipersensitiva (HR) após inoculação com TSWV, plantas de N. benthamiana foram transformadas com o gene Sw-5b buscando linhas transgênicas que poderiam responder com HR e assim facilitar a identificação do determinante de avirulência (Avr) de TSWV (Capítulo 2). Enquanto N. tabacum foi transformada com o gene Sw-5b sob controle de seus próprios elementos regulatórios, N. benthamiana foi transformada com o gene Sw-5b sob controle do promotor 35S do Cauliflower mosaic virus (CaMV). De forma interessante, as plantas transformadas de N. benthamiana apresentaram forte HR após inoculação de suas folhas com TSWV e outras quatro espécies de tospovírus: Alstroemeria necrotic streak virus (ANSV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Impatiens necrotic spot virus (INSV) (Capítulos 2 e 7). Para identificação do Avr, os genes de TSWV foram clonados e expressos individualmente em folhas de N. benthamiana expressando Sw-5b e em isolinhas de tomate resistentes (contendo Sw-5) para monitoramento de HR. Como resultado, HR foi induzida após expressão da proteína não-estrutural NSM do isolado BR-01 de TSWV que induz resistência, mas não do isolado GRAU de TSWV que quebra resistência (Capítulo 2). Foco sobre a proteína NSM seguiu no capítulo 3. Versões truncadas dessa proteína foram transientemente co-expressas com Sw-5b em folhas de N. benthamiana (wild type). Tais versões truncadas excluíam domínios anteriormente associados com formação de túbulos, movimento viral célula-célula e sistêmico. Proteínas NSM truncadas faltando até 50 aminoácidos (aa) de ambos N e C terminais (NSM inteira contém 301 aa) ainda induziram HR, sugerindo que funções associadas ao movimento viral e comportamento como Avr são características independentes para NSM. No capítulo 4 experimentos foram realizados para caracterizar uma nova espécie de tospovírus observado em plantas de feijão que apresentavam sintomas de mosaico necrótico em São Paulo, Brasil (2006). Microscopia eletrônica de folhas sintomáticas revelou partículas pleiomórficas agrupadas em vesículas. Devido ao fato de feijão ser um hospedeiro não usual e pelo resultado negativo em testes de ELISA para outras espécies já conhecidas por circular no Brasil, testes biológicos, sorológicos e moleculares foram realizados para caracterização de uma provável nova espécie de tospovírus. Este vírus apresentou um estreito espectro de hospedeiros, infectando sistemicamente três de vintes espécies de plantas indicadoras e apresentou propriedades sorológicas únicas quando comparado com outras espécies de tospovírus encontradas no Brasil. O sequenciamento do genoma deste tospovírus revelou uma nova espécie que, junto com Soybean vein necrosis-associated virus (SVNaV), representa uma nova linhagem evolutiva de tospovírus circulando no continente americano. Este tospovírus foi tentativamente chamado Bean necrotic mosaic virus (BeNMV). Já que a proteína Sw-5b reconhece pelo menos cinco espécies de tospovírus de origem “americana”, a proteína NSM do BeNMV também foi testada para monitoramento de HR através de sua co-expressão com Sw- 5b em folhas de N. benthamiana. O resultado, entretanto, mostrou que a proteína NSM do BeNMV não é um Avr cognato da proteína Sw-5b. No capítulo 5 é mostrado que a proteína Sw-5b induz HR dependentemente e independentemente da presença de NSM. A forma independente foi observada através da coexpressão da proteína Sw-5b com supressores de silenciamento gênico (p19 e NSS), os quais aumentaram a acumulação celular da proteína Sw-5b, induzindo auto-HR. Esta observação permitiu o screening de indução de HR e reconhecimento de NSM como eventos independentes para as outras proteínas Sw-5. Enquanto Sw-5a induziu auto-HR, ela não foi capaz de reconhecer NSM como Avr. De forma diferente, o ortólogo mais conservado das proteínas Sw- 5a e Sw-5b de S. lycopersicum susceptível a TSWV, nomeado Sw-5aS, não induziu qualquer tipo de HR. Através da co-expressão dos domínios individuais de Sw-5b, CC, NB-ARC, LRR ou de versões combinadas (CC-NB-ARC e NB-ARC-LRR) com NSM e com o supressor de silenciamento gênico p19, o papel desses domínios na indução de HR e no reconhecimento de NSM foram determinados. Enquanto NB-ARC foi suficiente para induzir auto-HR, NB-ARC-LRR induziu tanto auto quanto HR dependente de NSM, indicando que o domínio LRR especifica o reconhecimento do Avr. Já o domínio CC suprimiu HR induzida por NB-ARC em cis e trans, apontando para uma função regulatória de CC. A superexpressão do domínio NB-ARC de Sw- 5aS não resultou em HR, similar ao resultado da proteína Sw-5aS completa. Após alinhamento dos domínios NB-ARC, três variações de aminoácidos (aa) foram encontradas entre as proteínas Sw-5a, Sw-5b e Sw-5aS, os quais foram revertidos no último. Quando a glutamina da posição 599 foi convertida em uma arginina (Q599R), igual a proteína Sw-5b nesta posição, o domínio NB-ARC da proteína Sw-5aS tornou-se funcional para indução de HR. Modelagem deste domínio revelou que esta mutação encontra-se fora do domínio de ligação de ADP/ATP, o qual é importante para o switching entre os estados on e off dos domínios NB-ARC. Finalmente, a fusão do domínio LRR da proteína Sw-5b na variante Q599R do domínio NBARC de Sw-5aS resultou em auto-HR e HR dependente de NSM. Os construtos codificando os domínios da proteína Sw-5b também foram usados para estudos subcelulares no capítulo 6. Para este propósito, todas as proteínas estavam fusionadas a Green Fluorescence Protein (GFP) na porção N-terminal para tornar possível a visualização por microscopia confocal em tecido folhear. Enquanto a proteína Sw-5b inteira, os domínios CC, NB-ARC e LRR e o combinado CC-NB-ARC apresentaram distribuição nucleocitoplasmática, NB-ARC-LRR localizou-se somente no citoplasma, sugerindo que CC sinaliza o importe nuclear. Os domínios NB-ARC e LRR também foram investigados para uma possível interação direta com NSM através da técnica Bimolecular Fluorescence Complementation (BiFC). Enquanto interação direta não foi observada entre LRR e NSM, o domínio NB-ARC aparentou interagir com NSM. As especificidades para o reconhecimento de NSM como Avr são discutidas no capítulo 7, levando em consideração aspectos evolutivos de tomates e tospovírus. Um modelo da ativação da proteína Sw-5b é postulado no texto tendo como base a dissecção desta proteína e os ensaios de localização subcelular, além de sua putativa interação direta com NSM. Tomato spotted wilt virus (TSWV) along with other tospovirus species (family Bunyaviridae) is responsible for substantial losses in crop production around the world. Tospoviruses are transmitted in a propagative and circulative manner by thrips vectors (Order Thysanoptera). As for other pathogens, disease management against tospoviruses pursues a holistic approach involving a combination of cultural, phytosanitary, chemical, and biological tactics, when suitable in addition to the use of resistant crops. Nevertheless, successful control has proven difficult given the broad host range of these viruses and their effective spread by thrips vectors. More importantly, increased reliance on the use of low-cost insecticides has exacerbated tospovirus spread by causing thrips resistance. To ease the financial and environmental constraints associated with insecticide abuse to control thrips, efforts have been increased to obtain genetically resistant cultivars as an integral component of disease management strategies. So far there are two resistance sources available for commercial breeding of vegetables against TSWV. One of these sources is the Sw-5 gene cluster, which has been found in Solanum peruvianum L., a wild species of tomato from Peru, and has been introgressed in S. lycopersicum L. cultivars (commercial tomatoes). After gene mapping, it has been reported that at least five paralogs compose the Sw-5 gene cluster in S. peruvianum, which were named Sw- 5a to Sw-5e. These paralogs encode NB-LRR receptors, a class of cytoplasmic proteins that activate disease resistance upon direct or indirect recognition of pathogens that have overcome the first lines of defense of the plant immune system. Transformation of tobacco plants with the paralogs Sw-5a and Sw-5b, revealed that only the latter triggers resistance against TSWV isolates. With the availability of the tomato genome, highly conserved orthologs have been mapped in S. lycopersicum as well. This thesis started off with a detailed description of the plant immune system and previous findings about the Sw-5 gene cluster (Chapter 1). As part of this introduction, the problems caused by tospoviruses, to which Sw-5b locus confers resistance and the characteristics of these viruses have been described. With this knowledge as a starting point, this thesis focused on unraveling the key features of Sw-5b-mediated resistance against TSWV with special attention to the molecular and cellular events underlying the resistance mechanism. Functional analyses have been performed towards clarification of the genetic delimitations between functional and non-functional Sw-5 orthologs considering tospovirus recognition and resistance triggering. Since earlier made N. tabacum transformants containing Sw-5b did not show a hypersensitive response (HR) upon challenging with TSWV, N. benthamiana have been transformed with Sw-5b aiming to obtain transgenic lines that would respond with a visual HR and thereby facilitate the identification of the avirulence determinant (Avr) from TSWV (Chapter 2). While N. tabacum plants were transformed with Sw-5b gene under control of its own regulatory elements, the N. benthamiana plants were transformed with the Sw-5b gene under control of the Cauliflower mosaic virus (CaMV) 35S promoter. Interestingly, the Sw-5btransformed N. benthamiana plants presented a strong HR upon challenging with TSWV and four other tospoviruses: Alstroemeria necrotic streak virus (ANSV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Impatiens necrotic spot virus (INSV) (Chapter 2 and 7). To identify the Avr, TSWV genes were cloned and expressed individually in leaves of Sw-5b-transformed N. benthamiana plants and Sw-5-resistant tomato isolines for HR monitoring. As a result, HR was triggered upon expression of the non-structural protein NSM from the resistance-inducing TSWV isolate BR-01, but not from the resistance-breaking TSWV isolate GRAU (Chapter 2). The research on NSM was continued in Chapter 3, in which truncated versions of this protein were transiently co-expressed with Sw-5b in wild type N. benthamiana leaves. These truncations lacked domains previously associated with tubule formation, cell-tocell and systemic viral movement. Truncated NSM proteins lacking up to 50 amino acids (aa) from either the N- or C-terminus (full NSM is 301 aa in size) still triggered Sw-5b-mediated HR, suggesting that viral movement functions of NSM and its fate as Avr are independent from each other. Chapter 4 described experiments to characterize a new tospovirus collected from bean plants showing necrotic mosaic symptoms during a field survey in São Paulo, Brazil (2006). Electron microscopy of symptomatic leaves revealed pleomorphic particles packed in vesicles. Due to its unusual natural host for a “Brazilian” tospovirus and being negative in ELISA for tospovirus species known to be circulating in Brazil, biological, serological, and molecular tests were performed to further characterize this putatively new tospovirus species. The virus appeared to have a narrow host-range, systemically infecting only three out of twenty test-plants of various species and presented unique serological properties when compared to other known tospovirus species. The genome sequencing of this tospovirus revealed a completely new species, which, together with Soybean vein necrosis-associated virus (SVNaV), represents a second evolutionary lineage of tospoviruses circulating in the American continent. This new tospovirus was tentatively named Bean necrotic mosaic virus (BeNMV). Since the Sw-5b protein recognizes at least five tospovirus species of “American” origin, the NSM protein of BeNMV was also tested for HR-triggering through co-expression with Sw-5b in N. benthamiana leaves. The outcome, however, indicated that the NSM protein of BeNMV is not a cognate Avr of the Sw-5b protein. In Chapter 5 it is shown that the Sw-5b protein triggers both NSM-dependent and - independent HR. The latter was achieved by co-expression of Sw-5b with RNA silencing suppressors (p19 and NSS), which increased the Sw-5b cellular accumulation and lead to auto- HR. This observation allowed the screening of HR-triggering and NSM-recognition as uncoupled events for other Sw-5 proteins as well. While Sw-5a could trigger auto-HR, it lacked the ability to recognize NSM as Avr. On the other hand, the highest conserved ortholog of Sw-5a and Sw-5b from susceptible S. lycopersicum, named here as Sw-5aS, lacked both auto-HR and NSMdependent HR. By co-expression of the individual Sw-5b domains CC, NB-ARC, LRR or combined versions (CC-NB-ARC and NB-ARC-LRR) with NSM and the silencing suppressor p19, the role of these domains in HR-triggering and NSM-recognition was determined. While NBARC was sufficient for auto-HR, NB-ARC-LRR triggered both auto- and NSM-dependent HR, indicating that LRR specifies the Avr recognition. The CC domain suppressed HR triggered by NB-ARC in cis and trans, pointing towards a regulatory function for CC. The overexpression of the NB-ARC domain from Sw-5aS did not result in HR, similar to the outcome with the full-length Sw-5aS protein. After alignment of the NB-ARC domain, three mismatches were found between Sw-5a, Sw-5b, and Sw-5aS which were reverted in the latter. When the glutamine at position 599 was converted into an arginine (Q599R), to mimic the situation in the Sw-5b protein at this position, the Sw-5aS NB-ARC domain became functional for auto-HR triggering. Modeling of this domain revealed that this mutation was outside of the ADP/ATP binding site, which is important for the switching between “on” and “off” states of NB-ARC domains. Finally, the fusion of the LRR domain of Sw-5b to the Q599R variant of the Sw-5aS NB-ARC domain resulted in both auto- and NSM-dependent HR. The constructs encoding the various Sw-5b domains were used for subcellular studies in Chapter 6. To this end, all constructs encoded proteins were fused with Green Fluorescence Protein (GFP) at their N-terminus, to enable easy visualization by confocal microscopy in leaf tissue. Whereas the full-length Sw-5b protein, the individual domains CC, NB-ARC, and LRR and the combined CC-NB-ARC version showed a nucleocytoplasmic distribution, NB-ARC-LRR localized only in the cytoplasm, suggesting that CC signalizes nuclear import. The subdomains NB-ARC and LRR were also investigated for a possible direct interaction with NSM using Bimolecular Fluorescence Complementation (BiFC). While for LRR interaction with NSM was not observed, the NB-ARC domain seemed to directly bind to NSM. The specificities for NSM recognition have been discussed in Chapter 7 taking into consideration evolutionary aspects of tomatoes and tospoviruses. A model for Sw-5b activation is postulated throughout the text based on the dissection of this protein in HR-triggering and subcellular localization assays and on its putative direct interaction with NSM.

Rice is the most important food crop in the world, feeding over half the world’s population. However, rice water use efficiency, defined by units of yield produced per unit of water used, is the lowest of all crops. The aim of this thesis was to study the effect of plant hormones and the root microbiome on drought tolerance in rice. The new plant hormone, strigolactone, was shown to be upregulated under drought and to regulate drought tolerance in interaction with the drought-hormone abscisic acid. Using a large collection of rice genotypes grown in the field, we showed that the composition of the root associated fungal microbiome is determined by the rice genotype and can contribute to drought tolerance.&nbsp

Vegetative propagation is very important for the survival of species with long juvenile and adult vegetative phases, as it is the case for bulbous plants. Bulbous plants are ornamental geophytes with a bulb as an underground storage organ. Among flower bulbs, tulip and lily are the two commercially leading plants in The Netherlands. Tulip propagates vegetatively via axillary bud outgrowth, while lily propagates via adventitious bulblet formation. The vegetative propagation rate in tulip is very low due to the limited amount of axillary buds that will grow successfully. Moreover, tulip is very recalcitrant to in vitro regeneration. On the other hand, lily propagates efficiently via adventitious bulblet formation, either naturally from the underground portion of the stem of the apical bud, or artificially from detached bulb scales. This thesis study aimed to understand how axillary bud outgrowth is controlled in tulip bulbs and how regeneration capacity is established in lily bulb scales. As a first step towards these goals, the state of the art of the molecular control of sexual and vegetative reproduction was reviewed for model species. Moreover, two approaches, “bottom-up” and “top-down”, to transfer the knowledge from model to non-model species were described (Chapter 2). In short, the “bottom-up” approach usually goes from individual genes to systems, assuming conservation of molecular pathways and using sequence homology searches to identify candidate genes. ”Top-down” methodologies go from systems to genes, and are based on large scale transcriptome profiling via e.g. microarrays or RNA sequencing, followed by the identification of associations between phenotypes, genes, and gene expression patterns and levels. Next (Chapter 3), two sets of high quality transcriptomes, one for tulip and one for lily were generated from a collection of several tissues using the Illumina HiSeq 2000 platform. Several assembly filtering parameters were applied, to highlight the limitations of stringent but routinely used filtering in de novo transcriptome assembly. The final created transcriptomes were made publicly available via a user friendly Transcriptome browser (http://www.bioinformatics.nl/bulbs/db/species/index) and their usefulness was exemplified by a search for all potential transcription factors in lily and tulip, with special focus on the TCP transcription factor family. One TCP member was of special interest because it has proven to integrate several pathways that control axillary bud outgrowth in a wide range of species. It is called TEOSINTE BRANCHED 1 (TB1) in monocots and BRNACHED 1 (BRC1) in dicots. A Tulipa gesneriana TB1 transcript was identified from the generated transcriptome and subsequently, tulip axillary bud outgrowth was studied through a “bottom-up” approach (Chapter 4). The degree of axillary bud outgrowth in tulip determines the success of their vegetative propagation. However the number of axillary meristems in one bulb is low –six on average– and not all of them seem to have the same growth capacity. The combination of physiological and targeted molecular experiments indicated that the first two inner located buds do not seem to experience dormancy (assessed by weight increase and TgTB1expression) at any point of the growth cycle, while mid-located buds enter dormancy by the end of the growing season. Moreover it was shown that TgTB1 expression in tulip bulbs can be modulated by sucrose, cytokinin and strigolactone, just as it has been reported for other species. However, the limited growth of mid-located buds even when their TgTB1 expression was naturally or artificially downregulated, pointed at other factors, probably physical, inhibiting their growth. Next, the remarkable regeneration capacity of lily by initiating de novo shoot meristems from excised bulb scales without the addition of exogenous hormones or growth regulators was studied using a “top-down” approach (Chapter 5). An extensive and comprehensive transcriptome set was generated from lily bulb scales in a time-series using two cultivars and two explant types, all differing in regeneration capacity. This set up provided first insight in the key molecular process underlying pro-meristem induction and meristem initiation in lily. We found that wounding activates a very fast regeneration response, probably mediated by APETALA2/ETHYLENE RESPONSIVE FACTORS (AP2/ERF,) such as LoERF115 and WOUND INDUCED DEDIFFERENTIATION 2 (LoWIND2), which in turn might mediate polar auxin re-distribution, cell proliferation and de-differentiation. Moreover, the timing and level of induction of shoot meristem regulators, such as ENHANCER OF SHOOT REGENERATION 2 (LoESR2) and SHOOT MERISTEMLESS (LoSTM) correlated with the regeneration capacity of the scale. Regardless the regeneration capacity of the different explants e.g. cultivar or position within the scale, regeneration occurs at the proximal-adaxial side of the bulb scale, right on top of the excision line. Thus the possible cellular and physiological factors granting lily bulb scales their competence to regenerate was investigated (Chapter 6). We found that the adaxial parenchyma tissue seems to be more competent than the abaxial tissue, partially because of higher number of secondary veins and larger cell population than the abaxial parenchyma region. It was proposed that upon explant excision, the polar auxin transport is disrupted, creating an auxin maximum at the excision line, which might create a gradient of cell divisions favouring the adaxial parenchyma tissue. The direction of this cell division gradient proved to be negatively affected by the absence of the adaxial epidermis. Moreover, explants without epidermis reduced dramatically their regeneration capacity, and lost the typical proximal-adaxial orientation of regeneration. Thus, a better understanding of the composition and physiology of the epidermis in lily bulb scales is essential to identify the regeneration stimulating signals originating from this tissue layer in Lilium sp. Finally in Chapter 7, integration of all the results was done and I addressed how this may contributes to the fundamental and applied understanding of vegetative propagation in bulbous plants. Also, some challenges are discussed, for example, the complexity in the architecture of tulip bulbs and how this influences ways for improving its rate of axillary bud outgrowth. The challenge to prove the findings of this thesis through functional analysis is also discussed and the possibility of using transient virus-induced gene silencing is highlighted. Moreover, the potential of lily bulb scales as a model system to study some aspects of de novo regeneration, as well as to study the recalcitrance of in vitro propagation is highlighted, supporting the idea that more “omics” data and biotechnological tools for bulbous plant research are necessary.

As a consequence of a sessile lifestyle, plants are constantly facing a fluctuating environment. In order to both profit maximally and protect themselves from these environmental cues, plants evolved ways to sense and respond to signals. Ambient temperature is one of the cues for which plants have acquired a strategy to enhance their chance of survival and reproduction. Small changes in ambient temperature can have major effects on plant architecture and development, such as the transition from the vegetative to the reproductive flowering phase. The moment of flowering is an important event in the life cycle of a plant, since reproductive success depends on it. In Chapter 1, I introduced the concept of alternative splicing, a molecular mechanism with a pivotal role in ambient temperature regulation of flowering time. In the model plant Arabidopsis thaliana, approximately 60% of the intron-containing genes show alternative splicing. Gene splicing varies depending on developmental stage and tissue type, but also environmental changes trigger differential splicing. Splicing is conducted by a large cellular machinery called the spliceosome, which recognizes intron-defining sequences and other cis-regulatory elements acting as splicing enhancers or silencers. Moreover, factors like chromatin structure, histone marks, RNA polymerase II (polII) elongation speed and the secondary structure of the pre-mRNA all play a role in the splicing outcome. Due to alternative splicing, a single gene can yield various transcripts. However, this does not cause an equal expansion of the proteome. Part of the transcripts are targeted for nonsense-mediated decay, or will be translated into unstable proteins. This is a way of regulating gene expression at the post-transcriptional or –translational level. Other transcripts will be translated into functional proteins that may be structurally and functionally different. Hence, alternative splicing creates additional complexity in the transcriptome, providing plants with molecular tools to respond to their environment, including the translation of ambient temperature alterations into a flowering time response. In Chapter 2, we reviewed the current knowledge on molecular mechanisms that control the ambient-temperature directed flowering time pathway in the plant model species Arabidopsis thaliana. Several different mechanisms have been proposed, like alternative splicing of FLOWERING LOCUS M (FLM) (described in Chapter 4) and protein degradation of SHORT VEGETATIVE PHASE (SVP), two mechanisms that probably work in a cooperative manner to release floral repression at higher ambient temperatures. Another mechanism that is involved at high ambient temperature is the replacement of the canonical histone H2A by the variant H2A.Z. As a consequence of this replacement, chromatin becomes less tightly wrapped around the nucleosomes, which allows transcription of flowering time activators, such as PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Lastly, we discuss microRNAs (miRNA) that can either repress or activate flowering (miR156 and miR172, respectively). These miRNAs have been proposed to be regulated by low and high ambient temperature. However, due to the lack of mutant analyses, more research is necessary to show the true involvement of these factors. Altogether, there are several mechanisms acting partly in cooperation to regulate thermosensitive floral timing. In Chapter 3, we analysed ambient temperature-directed alternative splicing events that occur after a temperature shift by RNAseq. We performed the experiment in two different accessions of A. thaliana, and in one variant of B. oleracea (cauliflower). We showed that flowering time genes are overrepresented amongst the ambient temperature induced alternatively spliced genes, but also genes encoding components of the splicing machinery itself, indicating that alternative splicing is one of the potential mechanisms by which plants are able to sense temperature and adapt floral timing. Analysis of a mutant for one of these alternatively-spliced splicing related factors, ATU2AF65A, showed a temperature-dependent flowering time phenotype, confirming its proposed role in the flowering time response upon temperature fluctuations. Based on these findings, we proposed a two-step model in which splicing related genes are targeted for differential splicing upon ambient temperature fluctuations, which results in changes in the composition of the spliceosome, causing differential splicing of downstream genes that affect the development and architecture of the plant, including flowering time. In Chapter 4, we investigated the molecular mode-of-action of FLM, one of the differentially spliced flowering time regulating genes that we identified in Chapter 3. We showed that in A. thaliana Col-0, the main splice forms of FLM are FLMβ and FLMδ. FLMβ forms an obligate heterodimer with SVP, and this complex represses floral integrators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) by binding to the regulatory regions of these genes. FLMδ also dimerizes with SVP, but this complex is not able to bind to DNA. When temperature rises, more FLMδ is produced at the cost of FLMβ. Hence, less repressive complexes can be formed. However, the fact that FLMδ is still able to binds SVP makes it function as a dominant negative form, titrating out SVP and preventing repressive SVP/FLMβ-complex formation. Chapter 5 is a short comment written to clarify the concept of thermoplasticity in flowering time control. Occasionally, this concept is confused with adaptation to different ambient temperature environments on the long term. Thermoplasticity is the ability to adapt flowering time to fluctuations in ambient temperature within one life cycle. Furthermore, some genes have been marked as players in the ambient temperature response, whereas these appear to be general flowering repressors or activators, affecting flowering time in a similar manner at low and high ambient temperature. In order to interpret novel findings on thermosensitive flowering time control, it is essential to distinguish between these various concepts. In chapter 6, we unveiled the first indications that differential splicing of FLM can be caused by differences in polymerase II elongation rate. We mimicked a situation in which FLM is transcribed at a higher rate, by expressing the genomic FLM gene under a strong artificial promoter. Preliminary results showed that plants harbouring this construct have altered flowering time and temperature-responsiveness, which can be explained by the altered FLMβ/FLMδ ratio that we observed. In chapter 7, we assessed the functional conservation between FLM and closely related genes at the intraspecific level in A. thaliana. FLM (also called MAF1) is a member of the FLC-clade, that consists of FLC, FLM and MAF2-5. FLC is widely known for its function in the vernalization pathway, whereas MAF2 has been shown to regulate flowering time through alternative splicing in a way very similar to FLM. For the other MAF genes, not much is known. We showed that all of these genes produce splicing isoforms that function in a more or less similar way to FLM and MAF2. Despite the high functional conservation at the intraspecific level, FLM and MAF orthologues are not widely present. Through synteny analysis, we showed that FLM and MAF2 are very recent genes, which are only present in a small group of Brassicaceae species. MAF3-5 originated less recently, but are not present outside the Brassicaceae. For FLC, it was previously shown that it originated from an ancestor of the seed plants, and in many plant species belonging to other families, presence of more than one FLC-like gene has been reported. This raises the question what the function of these genes is. In tomato, we showed that the FLC-like gene MBP8 becomes differentially spliced upon temperature changes, suggesting a function in the ambient temperature pathway. A binding assay showed high similarities of the different MBP8 isoforms to FLM and MAF isoforms, but suggests a slightly different functionality, since all three isoforms showed binding to the DNA. Further research is necessary to confirm the role of MBP8 in thermosensitive flowering time control, and elucidate the functionality of the different splice forms. In Chapter 8, I discussed the finding of this thesis in a broader perspective, and make suggestions for future research. Over the last few years, several mechanisms that act in the temperature-directed floral pathway have been revealed. In this thesis, we showed that alternative splicing plays an important role, and we demonstrated how temperature may affect the splicing outcome directly through the effect of temperature on transcription elongation rate. It is becoming clear that most likely a single thermosensor does not exist in plants, and a model in which temperature is sensed through thermodynamic properties of DNA, RNA and proteins, is gaining support. Future research is assigned to the exiting task to elucidate the exact mechanisms by which temperature-sensing is achieved in different plant species and to determine how conserved the currently identified molecular mechanisms are.

Potato is affected by several diseases. Although, resistance can be obtained by introgression of major resistance genes from wild species, this has rarely been durable. Hence, other sources of resistance are highly needed. New research with a focus on loss of function mutations has led to the identification of disease susceptibility (S) genes in plants. The research in this thesis was aimed at the identification and characterization of potato S genes involved in the interaction with Phytophthora infestans and Botrytis cinerea. We selected 11 Arabidopsis thaliana S genes and silenced their potato orthologs by RNAi in the potato cultivar Desiree. The silencing of six genes resulted in resistance to P. infestans. Moreover, silencing of StDND1 reduced the infection of B. cinerea. Microscopic analysis showed that spore attachment and/or germination of P. infestans and B. cinerea was hampered on the leaf surface of StDND1-silenced potato plants. On StDMR1- and StDMR6-silenced potato plants, hyphal growth of P. infestans was arrested by the hypersensitive response-like cell death. Our results demonstrate that impairment of plant S genes may open a new way for breeding potatoes with resistance to pathogens like P. infestans and B. cinerea.

Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor. A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells. Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis. In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis. Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis. Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis. Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context. In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.

Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

Powdery mildew (PM) is a major fungal disease that threatens thousands of plant species. PM is caused by Podosphaera leucotricha in apple and Erysiphe necator in grapevine. Powdery mildew is controlled by frequent applications of fungicides, having negative effects on the environment, and leading to additional costs for growers. To reduce the amount of chemicals required to control this pathogen, the development of resistant apple and grapevine varieties should become a priority. PM pathogenesis is associated with up-regulation of specific MLO genes during early stages of infection, causing down-regulation of plant defense pathways. These up-regulated genes are responsible for PM susceptibility (S-genes) and their knock-out causes durable and broad-spectrum resistance. All MLO S-genes of dicots belong to the phylogenetic clade V. In grapevine, four genes belong to clade V. VvMLO7, 11 and 13 are up-regulated during PM infection, while VvMLO6 is not. Chapter 2 reports the genome-wide characterization and sequence analysis of the MLO gene family in apple, peach and woodland strawberry, and the isolation of apricot MLO homologs. Twenty-one homologues were found in apple, 19 in peach and 17 in woodland strawberry. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Candidate genes for causing PM susceptibility were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen P. leucotricha. In apple, clade V genes MdMLO11 and 19 were up-regulated, whereas the two other members of clade V, MdMLO5 and 7, were not up-regulated. The clade VII gene MdMLO18 was also up-regulated upon P. leucotricha infection. Chapter 3 reports the knock-down, through RNA interference, of MdMLO11 and 19, as well as complementation of the mutant phenotype by expression of the MdMLO18 gene in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 resulted in a reduction of PM disease severity up to 75%, whereas the knock-down of MdMLO11, alone or combined with MdMLO19, did not cause any reduction or additional reduction of susceptibility compared to MdMLO19 alone. Complementation by MdMLO18 did not restore susceptibility. Cell wall appositions (papillae), a response to PM infection, were found in both susceptible plants and PM resistant plants where MdMLO19 was knocked-down, but were larger in resistant lines. The expression analysis of 17 genes related to plant defense, and quantification of phenolic metabolites in resistant lines revealed line-specific changes compared to the control. Chapter 4 evaluates the presence of non-functional alleles of the MdMLO19 S-gene in apple germplasm. The screening of the re-sequencing data of 63 apple genotypes led to the identification of 627 SNP in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18 and MdMLO19). Insertion T-1201 in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino-acids and the calmodulin-binding domain. The presence of the insertion was evaluated in a collection of 159 apple genotypes: it was homozygous in 53 genotypes, 45 of which were resistant or very resistant to PM, four partially susceptible and four not assessed. These results strongly suggest that this insertion is causative for the observed PM resistance. The absence of a clear fitness cost associated to the loss-of-function of MdMLO19, might have contributed to the high frequency of the mutation in breeding germplasm and cultivars. Among the genotypes containing the homozygous insertion, ‘McIntosh’ and ‘Fuji’ are commonly used in apple breeding. After barley and tomato, apple is the third species with a reported natural non-functional mlo allele in its germplasm, with the important difference that the allele is present in a relatively large number of apple genotypes, most of which not related to each other. Chapter 5 reports the knock-down through RNA interference of four grapevine MLO genes, all members of clade V. VvMLO7, 11 and 13 are up-regulated in early stages of infection, whereas VvMLO6 is not responsive to the pathogen. Knock-down of VvMLO6, 11 and 13, alone or combined, did not decrease PM severity, whereas the knock-down of VvMLO7, alone or in combination with VvMLO6 and VvMLO11, caused a reduction of severity of 77%. Cell wall appositions (papillae), a response to PM attack, were present in both resistant and susceptible lines, but were larger in resistant lines. Thirteen genes involved in defense were less up-regulated in resistant plants, highlighting the reduction of PM disease severity. In Chapter 6 we discuss the results presented in this thesis. The pivotal role of MLO genes in the interaction of PM pathogens with apple and grapevine is described and further experiments aimed at addressing open questions are proposed. The results described in this thesis open interesting avenues in MLO genes research, particularly the finding that a natural mlo mutation in apple appeared to be more common than expected. This mutation is directly applicable in marker assisted breeding for durable PM resistance in apple.

Noccaea caerulescens is the only known Zn/Cd/Ni hyperaccumulator. The Ganges accession (2n = 14) has an, yet unpublished, genome size of ~319 Mb, with 29,712 predicted genes representing 15,874 gene families. This species is distributed mainly in Europe. Three ecotypes can be distinguished: two metallicolous ecotypes, resident to serpentine soil (Ni enriched) and calamine soil (Zn/Cd enriched), and a non-metallicolous ecotype, growing on regular, non-metalliferous soils. The physiological differences that underlie variation in heavy metal accumulation and tolerance are well-understood, and the molecular basis of hyperaccumulation and tolerance has been explored by transcript profiling in the presence of metals and by comparative transcriptome analysis using N. caerulescens and non-hyperaccumulators such as Arabidopsis thaliana. The genetic variation which emerged during the evolution of metal hyperaccumulation has not yet been investigated. The work described in this thesis considers the identification of genetic variation under selection for Zn/Cd hyperaccumulation and tolerance by next generation resequencing of the wild metallicolous (calamine) and non-metallicolous populations and the generation of a mutant N. caerulescens library for functional analysis. The regulation of flowering time was also investigated, using early flowering mutants selected from the mutant library.

Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance. A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium. After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs. In Chapter 2 , we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role. Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function. In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species. In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein. In Chapter 5 , we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution. In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber. In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T a A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2. Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.

Powdery mildew (PM) is a major fungal disease that threatens thousands of plant species. PM is caused by Podosphaera leucotricha in apple and Erysiphe necator in grapevine. Powdery mildew is controlled by frequent applications of fungicides, having negative effects on the environment, and leading to additional costs for growers. To reduce the amount of chemicals required to control this pathogen, the development of resistant apple and grapevine varieties should become a priority. PM pathogenesis is associated with up-regulation of specific MLO genes during early stages of infection, causing down-regulation of plant defense pathways. These up-regulated genes are responsible for PM susceptibility (S-genes) and their knock-out causes durable and broad-spectrum resistance. All MLO S-genes of dicots belong to the phylogenetic clade V. In grapevine, four genes belong to clade V. VvMLO7, 11 and 13 are up-regulated during PM infection, while VvMLO6 is not. Chapter 2 reports the genome-wide characterization and sequence analysis of the MLO gene family in apple, peach and woodland strawberry, and the isolation of apricot MLO homologs. Twenty-one homologues were found in apple, 19 in peach and 17 in woodland strawberry. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Candidate genes for causing PM susceptibility were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen P. leucotricha. In apple, clade V genes MdMLO11 and 19 were up-regulated, whereas the two other members of clade V, MdMLO5 and 7, were not up-regulated. The clade VII gene MdMLO18 was also up-regulated upon P. leucotricha infection. Chapter 3 reports the knock-down, through RNA interference, of MdMLO11 and 19, as well as complementation of the mutant phenotype by expression of the MdMLO18 gene in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 resulted in a reduction of PM disease severity up to 75%, whereas the knock-down of MdMLO11, alone or combined with MdMLO19, did not cause any reduction or additional reduction of susceptibility compared to MdMLO19 alone. Complementation by MdMLO18 did not restore susceptibility. Cell wall appositions (papillae), a response to PM infection, were found in both susceptible plants and PM resistant plants where MdMLO19 was knocked-down, but were larger in resistant lines. The expression analysis of 17 genes related to plant defense, and quantification of phenolic metabolites in resistant lines revealed line-specific changes compared to the control. Chapter 4 evaluates the presence of non-functional alleles of the MdMLO19 S-gene in apple germplasm. The screening of the re-sequencing data of 63 apple genotypes led to the identification of 627 SNP in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18 and MdMLO19). Insertion T-1201 in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino-acids and the calmodulin-binding domain. The presence of the insertion was evaluated in a collection of 159 apple genotypes: it was homozygous in 53 genotypes, 45 of which were resistant or very resistant to PM, four partially susceptible and four not assessed. These results strongly suggest that this insertion is causative for the observed PM resistance. The absence of a clear fitness cost associated to the loss-of-function of MdMLO19, might have contributed to the high frequency of the mutation in breeding germplasm and cultivars. Among the genotypes containing the homozygous insertion, ‘McIntosh’ and ‘Fuji’ are commonly used in apple breeding. After barley and tomato, apple is the third species with a reported natural non-functional mlo allele in its germplasm, with the important difference that the allele is present in a relatively large number of apple genotypes, most of which not related to each other. Chapter 5 reports the knock-down through RNA interference of four grapevine MLO genes, all members of clade V. VvMLO7, 11 and 13 are up-regulated in early stages of infection, whereas VvMLO6 is not responsive to the pathogen. Knock-down of VvMLO6, 11 and 13, alone or combined, did not decrease PM severity, whereas the knock-down of VvMLO7, alone or in combination with VvMLO6 and VvMLO11, caused a reduction of severity of 77%. Cell wall appositions (papillae), a response to PM attack, were present in both resistant and susceptible lines, but were larger in resistant lines. Thirteen genes involved in defense were less up-regulated in resistant plants, highlighting the reduction of PM disease severity. In Chapter 6 we discuss the results presented in this thesis. The pivotal role of MLO genes in the interaction of PM pathogens with apple and grapevine is described and further experiments aimed at addressing open questions are proposed. The results described in this thesis open interesting avenues in MLO genes research, particularly the finding that a natural mlo mutation in apple appeared to be more common than expected. This mutation is directly applicable in marker assisted breeding for durable PM resistance in apple.

Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in many plant species. These non-zygotic embryos go through analogous developmental phases and are morphologically similar to the zygotic embryo. Despite its fundamental importance and enormous application potential, the mechanisms that alter cell fate from non-embryonic to embryonic are elusive. In the past decades, a variety of different model systems have been used to identify regulators of embryo induction, but it is unclear if these act in a common network. We recently found that inhibition of auxin response in the extra-embryonic suspensor cells cell-autonomously and predictably triggers a switch towards embryo identity. In my thesis I have used the suspensor-derived embryogenesis as a uniform model system to study the crucial first reprogramming step of embryo initiation process. Through genome-wide transcriptional profiling upon local (suspensor-specific) auxin response inhibition (Chapter 2) and through testing the ability of fifteen known embryogenesis inducers to promote embryo formation in suspensor cells (Chapter 3), we suggest that suspensor to embryo transformation requires a defined set of genetic regulators. The results obtained in my thesis provide essential tools and basis for further research and are a step forward to understanding the first step of embryo initiation process and to unravel the mystery of totipotency in plants.

Op 20 juni 2011 promoveerde Hendrik Rietman aan Wageningen University (WUR) op zijn proefschrift getiteld: “Putting the Phytophthora infestans genome sequence at work; multiple novel avirulence and potato resistance gene candidates revealed”. Zijn promotor, Prof. Dr. Richard G. F. Visser, en co-promotor, Dr. Ir. Vivianne G. A. A. Vleeshouwers, zijn beiden verbonden aan de leerstoelgroep Plantenveredeling van de WUR, waar ook het onderzoek werd uitgevoerd. Tevens is er samengewerkt met de leerstoelgroep Fytopathologie (WUR), het Sainsbury Lab en het James Hutton Instituut (beiden gevestigd in het Verenigd Koninkrijk). Financiering was afkomstig van het ‘Parapluplan Phytophthora’ en WUR Plantenveredeling.

Genomics 4.0 - Syntenic Gene and Genome Duplication Drives Diversification of Plant Secondary Metabolism and Innate Immunity in Flowering Plants Johannes A. Hofberger1, 2, 3 1 Biosystematics Group, Wageningen University & Research Center, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands (August 2012 – December 2013) 2 Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands (December 2010 – July 2012) 3 Chinese Academy of Sciences/Max Planck Partner Institute for Computational Biology, 320 Yueyang Road, Shanghai 200031, PR China (January 2014 – December 2014) TWO-SENTENCE SUMMARY Large-scale comparative analysis of Big Data from next generation sequencing provides powerful means to exploit the potential of nature in context of plant breeding and biotechnology. In this thesis, we combine various computational methods for genome-wide identification of gene families involved in (a) plant innate immunity and (a) biosynthesis of defense-related plant secondary metabolites across 21 species, assess dynamics that affected evolution of underlying traits during 250 Million Years of flowering plant radiation and provide data on more than 4500 loci that can underpin crop improvement for future food and live quality. GENERAL ABSTRACT As sessile organisms, plants are permanently exposed to a plethora of potentially harmful microbes and other pests. The surprising resilience to infections observed in successful lineages is due to a complex defense network fighting off invading pathogens. Within this network, a sophisticated plant innate immune system is accompanied by a multitude of specialized biosynthetic pathways that generate more than 200,000 secondary metabolites with ecological, agricultural, energy and medicinal importance. The rapid diversification of associated genes was accompanied by a series of duplication events in virtually all plant species, including local duplication of short sequences as well as multiplication of all chromosomes due to meiotic errors (plant polyploidy). In a comparative genomics approach, we combined several bioinformatics techniques for large-scale identification of multi-domain and multi-gene families that are involved in plant innate immunity or defense-related secondary metabolite pathways across 21 representative flowering plant genomes. We introduced a framework to trace back duplicate gene copies to distinct ancient duplication events, thereby unravelling a differential impact of gene and genome duplication to molecular evolution of target genes. Comparing the genomic context among homologs within and between species in a phylogenomics perspective, we discovered orthologs conserved within genomic regions that remained structurally immobile during flowering plant radiation. In summary, we described a complex interplay of gene and genome duplication that increased genetic versatility of disease resistance and secondary metabolite pathways, thereby expanding the playground for functional diversification and thus plant trait innovation and success. Our findings give fascinating insights to evolution across lineages and can underpin crop improvement for food, fiber and biofuels production

Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats. Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous fungi contain very large numbers of genes related to SM production. Yet these genes are typically silent under laboratory conditions. There are now many tools and strategies available to activate or clone silent SM genes. This thesis details our efforts to apply various methods to define and then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato containing many silent SM genes and gene clusters. In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and increase the chemical diversity of fungi are detailed. These include the deletion or over-expression of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation of growth conditions. Heterologous expression of silent SM genes in a production host is also discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10 polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to apply the aforementioned strategies to induce these silent genes and obtain new compounds. The importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential source of novel biosynthetic enzymes. In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic genes linked to SM production, such as those encoding decorating enzymes and regulators. Products of these SM genes or gene clusters were speculated, based on their similarity to those characterized in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other fungal species. Remarkably, two predicted functional gene clusters were linked to the production of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous findings, the majority of SM genes remained silent and only cladofulvin was detected. During growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle. In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim of discovering new compounds. Many Ascomycete-specific global regulators of SM production and morphological development in other fungi were identified in C. fulvum. We investigated three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin. This suggests that cladofulvin production is not required for asexual reproduction. The main finding of this chapter is that global regulator manipulation cannot considered to be a universal tool to discover new fungal natural products. In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that is produced by many fungi and plants. Once synonymous only with constipation, this former laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans. We predicted early acting cladofulvin genes and cloned them for heterologous expression in A. oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the point at which the pathways to cladofulvin and monodictyphenone production diverge. It was speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery of functionally similar genes and enzymes, providing a template to engineer enzymes capable of forming novel dimers from existing monomers. In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most active during early and late stages of infection of tomato, but are down-regulated during biotrophic growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this way, we aimed to test the effect of cladofulvin over-production on disease symptom development. The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post inoculation (dpi). At each time point the infections were inspected microscopically to detect any phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection. Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves. In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between 8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet (UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these results suggested that cladofulvin confers resistance to abiotic stress. In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly, the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might enable discovery of homologous genes encoding enzymes with different specificities. Combining bioinformatic and functional analyses should prove to be a powerful strategy for discovering compounds with new biological activities, or enzymes relevant to metabolic engineering.

Summary Current treatments of inflammatory disorders are often based on therapeutic proteins. These proteins, so-called biopharmaceuticals, are isolated from a natural resource or, more often, made using cell based fermentation systems. The most common production platforms are based on the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae or mammalian cell lines (mainly Chinese hamster ovarian (CHO) and murine myeloma (SP2/0) cells). Each platform has advantages and disadvantages and the protein to be produced largely dictates the choice of platform. Plants could provide a unique alternative production platform, as they combine the advantage of E. coli being economic with the advantages of mammalian cell lines being able to fold complex proteins, assemble heteromultimeric protein complex and, upon glyco-engineering, provide proteins with human type N-glycans. Furthermore, plant production is easily scaled up as the infrastructure is already in place due to our need for food and feed and plants have a limited risk of contamination with human pathogens. Transient transformation of N. benthamiana is a valuable plant production platform, as it is unmatched in terms of speed (matter of weeks). This thesis describes the production of a variety of proteins and protein complexes in planta that are or may be used as biopharmaceuticals. Since 1982, plants can be genetically manipulated, which has lead to the production of many proteins in a variety of plants. Initially, the plants greatest drawback was limited protein yield. However, significant increases in yield have been achieved in the last two decades, predominantly by increasing transformation efficiency and/or level of transcription. Nowadays several plant expression systems exist that facilitate high protein production levels. Most experience is based on the production of antibodies, mainly of the IgG isotype, as these are often used as biopharmaceuticals. In general, IgG antibodies are produced on a scale of several grams per kilogram fresh weight. However, production levels of antibodies have shown to be variable and production levels of particular proteins, such as cytokines, have lagged behind. Cytokines form a large group of immune-signalling molecules and several cytokines have promising therapeutic potential. Their short in vivo half-life suggests an inherent instability, which is regarded the major production bottleneck. In chapter 2 we describe the production of interleukin (IL)-10, a cytokine with immunosuppressive properties. We show that in contrast to mouse IL-10, human IL-10 multimerises extensively in planta. Both human and mouse IL-10 form homodimeric protein complexes through a mechanism referred to as 3D domain swapping. Only for human IL-10 3D domain swapping results in extensive multimerisation. By fusing IL-10 to green fluorescent protein (GFP) multimerisation was visualised, demonstrating that human IL-10 forms granules of organelle size. We discovered that for mouse IL-10 granule formation was prevented by N-glycosylation of the N-terminus. Introduction of this N-glycosylation site in human IL-10 partly prevented granule formation. The insertion of a glycine-serine linker between alpha helices D and E of human IL-10, allowing IL-10 to swap its own domains and form a stable monomer. This prevented granule formation completely and boosted protein yield 30-fold. However, the now comparable yields of human and mouse IL-10 were still low when compared to for instance IgG. In the next two chapters we shifted the focus to the production of antibodies. Thus far the IgA isotype received little attention as candidate biopharmaceutical. However, the unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. Therefore, we compared the plant-based expression of IgA to IgG (Chapter 3). The variable regions of three antibodies commonly used in the clinic to treat inflammatory disorders (Infliximab, Adalimumab and Ustekinumab) were grafted on either an IgG1κ or IgA1κ backbone. Surprisingly, we achieved comparable high expression levels for all antibodies. The large variation in antibody yield found in literature is therefore likely due to differences in expression systems and experimental conditions, but not to the antibody idiotype used. Evaluation of the secretion efficiency and N-glycosylation profiles revealed compelling differences between IgG and IgA antibodies when expressed in planta. Compared to IgG, IgA was poorly secreted. Poor secretion is most likely due to vacuolar targeting of IgA, as it was previously demonstrated that murine IgA contained a cryptic vacuolar targeting signal in the tailpiece. IgA also carried different N-glycans compared to IgG, which were never found to be a main fraction of a plant-produced protein. The predominant N-glycan on IgA lacked both the typical plant core α1,3-fucose and one terminal N-acetylglucosamine (GlcNAc). The core α1,3-fucose is most likely not added due to low accessibility of the core of the N-glycans. The lack of one GlcNAc may be due to inefficient addition of this sugar residue or (partial) β-hexosaminidase activity that may occur in both the vacuole and the apoplast. Whether the vacuolar targeting plays a role in the lack of the GlcNAc and what protein intrinsic properties influence partial GlcNAc addition or removal is unclear. We also showed that the N-glycosylation site in the IgA tailpiece does not always receive a N-glycan, which may be significant for secretory IgA formation as described in chapter 4. In chapter 4 we describe the expression of this large heteromultimeric protein complex. Secretory IgA consists of two IgA complexes that are connected via the joining chain and associate with a part of the polymeric-Ig-receptor (called the secretory component). The challenge for sIgA expression is that assembly of four polypeptides (the alpha heavy chain, the light chain, the joining chain and the secretory component) in a 4:4:1:1 ratio is required. Previous studies on stable and transient expression of sIgA demonstrated that sIgA formation in plants is possible. However, sIgA was always accompanied by a large proportion of monomeric IgA as well as other assembly intermediates. The presence of assembly intermediates may be caused be unequal expression and/or stability of the individual components. However, we demonstrated that not the expression strategy, but protein complex assembly through impaired incorporation of the joining chain was the limiting step for sIgA production. Because joining chain incorporation depends on N-glycosylation of the IgA tailpiece we hypothesise that the partial N-glycosylation of the IgA tailpiece is the cause of inefficient joining chain incorporation. The efficiency of N-glycosylation may be improved by mutation of the glycosylation site N-X-S to an N-X-T site, as the latter glycosylation motif was shown to be more often occupied in a large-scale glycosylation site analysis. Successful engineering of the plant glycosylation pathway to provide biopharmaceuticals with human N-glycan types has been achieved. Glyco-engineering also provides the opportunity to generate potentially interesting N-glycan types with regard to immunogenicity for vaccination purposes or protein trafficking for in vivo targeting of biopharmaceuticals. In chapter 5 we describe the expression of the Schistosoma mansoni egg antigen omega-1, an immunomodulatory protein with therapeutic potential. Its biological activity depends on its RNAse activity and its N-glycans that enable internalisation by human dendritic cells. Omega-1 cannot be isolated from natural resources in sufficient quantities to study its in vivo biology i.e. which N-glycan types facilitate full activity of omega-1. Therefore, we set up the plant-based production of this protein. S. mansoni-derived omega-1 predominantly carries diantennary N-glycans with a Lewis X motif on one or both antennae. Lewis X consists of a β1,4-linked galactose and α1,3-linked fucose residue attached to a terminal GlcNAc. This attachment is performed by β1,4-galactosyltransferase and α1,3-fucosyltransferase IXa that naturally do not occur in plants. Co-expression of these two glycosyltransferases with omega-1 resulted in a N-glycan profile comparable to S. mansoni-derived omega-1. However, it was necessary to control the expression of the β1,4-galactosyltransferase to contain this enzyme in the trans-Golgi compartment. Overexpression most likely leads to overflow of β1,4-galactosyltransferase to the medial-Golgi and galactosylation at this stage disturbs the activity of other glycosyltransferases resulting in hybrid N-glycan types. We also observed that more than 90% of omega-1 was secreted to the apoplast, which facilitated efficient purification. This demonstrated that plants, as previously suggested, are not poor protein secretors. However, the underlying protein properties controlling secretion efficiency are currently unknown. Both omega-1 and IgG are very efficiently secreted under natural circumstances. Yet, despite the fact that we used the same signal peptide to facilitate secretion of IgG and omega-1, IgG was only secreted from plant cells to a maximum of 28%. More knowledge on protein secretion efficiency in plants may overcome cumbersome purification, currently the greatest bottleneck in the downstream processing of plant produced proteins. Finally in chapter 6, we shifted focus from the expression of a particular protein to the effect of codon use on protein yield. We designed a codon optimisation strategy that, unlike other strategies, turned out to be surprisingly robust. This strategy is based on a general codon bias found in plants. Because this codon bias could be found among plant species, including monocots and dicots, and resulted in an increase in mRNA stability and translatability, we suspected that this codon bias arises from a selection pressure on the mRNA structure. We extended this general codon bias to representative species of other kingdoms of life and demonstrate that there is a selection pressure increasing mRNA stability and translatability (more protein per mRNA molecule). Stability was the result of an increase in the number of nucleotide bonds. However, there is a trade off between mRNA stability and translatability, and the nucleotide bonds in an mRNA should be well balanced over the entire molecule, making it ‘airy’, to ensure efficient translation. Altogether we conclude that transformation efficiency and level of transcription are no longer limiting factors for protein yield upon plant-based expression. However, an increase in translation initiation and translation rate dictated by codon use may still provide an increase in protein production. Furthermore, we show that many proteins demonstrated specific production bottlenecks. The yield of human IL-10 was hampered by its extensive multimerisation, sIgA assembly is most likely limited by inefficient N-glycosylation of the tailpiece of IgA, both IgG and IgA are inefficiently secreted compared to omega-1 and IgA displayed an aberrant N-glycosylation profile. Therefore, more knowledge on how protein intrinsic properties influence protein yield and/or quality of heterologous produced proteins in plants should now be generated. Transient expression in N. benthamiana is an ideal tool to study these protein intrinsic properties that limit protein yield in heterologous expression.

Understanding genetic variation for freezing tolerance is important for unraveling an adaptative strategy of species and for finding out an effective way to improve crop productivity to unfavorable winter environments. The aim of this thesis was to examine natural variation for components of freezing tolerance beyond what has been done using the model organism Arabidopsis thaliana. Experiments using B. stricta were carried out to identify potentially novel and beneficial traits, and loci related to cold acclimation, or using a number of related Brassicaceae species were performed to understand mechanism for cold deacclimation. Our results strongly indicate differential regulatory mechanisms are involved in cold acclimation as well as cold deacclimation.Although we are still far from understanding those mechanisms, we have shown that exploiting natural variation using wild species provides new perspectives on ecologically important adaptation to cold, and may contribute to improve tolerance in crucifer species.

Phytophthora pathogens are notorious for causing severe damage to many agriculturally and ornamentally important plants. Effective plant resistance depends largely on the capacity to perceive pathogens and to activate rapid defence. Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity upon recognition of matching effectors secreted by Phytophthora. However, Phytophthora pathogens are notoriously difficult to control due to their rapid adaptation to evade R protein-mediated recognition. Hence, exploring novel resistance components is instrumental for developing durable resistance. Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous and endogenous stimuli to initiate plant defence. One RLK that was previously identified as a novel Phytophthora resistance component is the Arabidopsis L-type lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of 45 members in Arabidopsis but whether or not the other members function in Phytophthora resistance was thus far unknown. The research described in this thesis was aimed at unravelling the role of LecRKs in plant immunity, in particular to Phytophthora pathogens. Chapter I describes various Phytophthora diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to Phytophthora pathogens. In Chapter II, we describe the development of a new Arabidopsis-Phytophthora pathosystem. We demonstrated that Phytophthora capsici is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and indole glucosinolates biosynthesis pathways are required for P. capsici resistance (Chapter IIa). The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem can be used as an additional tool to pinpoint essential components of Phytophthora resistance. We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of LecRKs in Phytophthora resistance. In Chapter III we describe a systematic phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other LecRKs function in Phytophthora resistance. We have also analysed whether the LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to bacterial or fungal pathogens, but none of the lines showed visible developmental changes under normal conditions or upon abiotic stress treatment. Combining these phenotypic data with LecRK expression profiles obtained from publicly available datasets revealed that LecRKs that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function display diverse expression patterns. Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora resistance was further investigated. In Chapter IV we describe that overexpression of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to Phytophthora, but also induced plant cell death. A mutation in the kinase domain abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce Phytophthora resistance as did deletion of the lectin domain. Cell death induction however, only required the kinase, not the lectin domain. Since transient expression of both LecRKs in Nicotiana benthamiana also resulted in increased Phytophthora resistance and induction of cell death, we used N. benthamiana to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for Phytophthora resistance. Collectively, these results illustrate that the Phytophthora resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death phenotype. By co-immunoprecipitation we identified putative interacting proteins, one of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis, the ABC transporter ABCG40, was found to interact in planta with both LecRK-IX.1 and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants showed compromised Phytophthora resistance, indicating that ABCG40 has a function in Phytophthora resistance. In Chapter V, we describe the generation of stable transgenic N. benthamiana plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of LecRK-IX.1 induced plant cell death. Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed increased resistance towards P. capsici or Phytophthora infestans. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora resistance upon interfamily transfer. Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in Phytophthora resistance. In Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both N. benthamiana and tomato lack LecRKs homologous to Arabidopsis LecRKs of clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional analysis of various Solanaceous LecRKs using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance. These results indicate that the role of clade IX LecRKs in Phytophthora resistance is conserved across plant species. In Chapter VII, the experimental data presented in this thesis are summarized and discussed in a broader context. We present an overview of the current understanding of LecRKs in plant immunity and discuss how LecRKs can be exploited to improve plant resistance.

Plants live in complex environments and are under constant threat of being attacked by herbivorous arthropods. Consequently plants possess an arsenal of sophisticated mechanisms in order to defend themselves against their ubiquitous attackers. Induced indirect defenses involve the attraction of natural enemies of herbivores, such as predators and parasitoids. Predators and parasitoids use odors emitted by damaged plants that serve as a “cry for help” to find their respective prey or host herbivore. The aim of this thesis was to use a multidisciplinary approach, with focus on molecular and chemical methods, combined with behavioral investigations, to elucidate the mechanisms of plant responses to multiple herbivory that affect a tritrophic system consisting of a plant, an herbivore and a natural enemy. Induced plant defenses are regulated by a network of defense signaling pathways in which phytohormones act as signaling molecules. Accordingly, simulation of herbivory by exogenous application of phytohormones and actual herbivory by the two-spotted spider mite Tetranychus urticae affected transcript levels of a defense gene involved in indirect defense in Lima bean. However, two other genes involved in defense were not affected at the time point investigated. Moreover, application of a low dose of JA followed by minor herbivory by T. urticae spider mites affected gene transcript levels and emissions of plant volatiles commonly associated with herbivory. Only endogenous phytohormone levels of jasmonic acid (JA), but not salicylic acid (SA), were affected by treatments. Nevertheless, the low-dose JA application resulted in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defense after herbivore infestation. Caterpillar feeding as well as application of caterpillar oral secretion on mechanically inflicted wounds are frequently used to induce plant defense against biting-chewing insects, which is JA-related. Feeding damage by two caterpillar species caused mostly identical induction of gene transcription, but combination of mechanical damage and oral secretions of caterpillars caused differential induction of the transcription of defense genes. Nevertheless, gene transcript levels for plants that subsequently experienced an infestation by T. urticae were only different for a gene potentially involved in direct defense of plants that experienced a single event of herbivory by T. urticae. Indirect defense was not affected. Also sequential induction of plant defense by caterpillar oral secretion and an infestation by T. urticae spider mites did not interfere with attraction of the specialist predatory mite P. persimilis in olfactometer assays. The predator did distinguish between plants induced by spider mites and plants induced by the combination of mechanical damage and caterpillar oral secretion but not between plants with single spider mite infestation and plants induced by caterpillar oral secretion prior to spider mite infestation. The composition of the volatile blends emitted by plants induced by spider mites only or by the sequential induction treatment of caterpillar oral secretion followed by spider mite infestation were similar. Consequently, the induction of plant indirect defense as applied in these experiments was not affected by previous treatment with oral secretion of caterpillars. Moreover, herbivory by conspecific T. urticae mites did not affect gene transcript levels or emission of volatiles of plants that experienced two bouts of herbivore attack by conspecific spider mites compared to plants that experienced only one bout of spider mite attack. This suggests that Lima bean plants do no increase defense in response to sequential herbivory by T. urticae. In conclusion, using a multidisciplinary approach new insights were obtained in the mechanisms of induction of indirect plant defense and tritrophic interactions in a multiple herbivore context, providing helpful leads for future research on plant responses to multiple stresses.

Summary Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating potato diseases worldwide. To successfully colonize its host, P. infestans secretes a plethora of RXLR effectors that translocate into host cells to modulate plant defense. The RXLR effectors form the largest and most diverse effector family in oomycete plant pathogens, and include several that were demonstrated to trigger host resistance mediated by intracellular host immune receptors. Chapter 1 is a summary focussing on the molecular mechanisms underlying host–pathogen interactions. It introduces the multi-layered innate immune system of plants, as well as the strategies that pathogens exploit to circumvent and suppress host defense. Furthermore, it highlights the importance of vesicle-trafficking during plant defense. The central subject of this thesis is AVR1, one of the race-specific avirulence (AVR) factors of P. infestans. AVR1 triggers plant resistance mediated by its corresponding potato Nucleotide-binding Leucine-rich repeat (NLR) resistance protein R1. P. infestans isolates that are avirulent on R1-containing potato cultivars always contain AVR1, while virulent isolates lack AVR1 but contain a related gene that we baptized as AVR1-like. AVR1 has all hallmarks of a typical RXLR effector; it contains a signal peptide, an RXLR domain and a C-terminal effector domain that contains two W motifs and one Y motif. In addition, it has, at the very end a stretch of 38 amino acids in length that we named the Tail (T)-region. AVR1-like, or in short A-L, shares high sequence similarity with AVR1. However, due to a premature stop codon the 38 amino acid T-region is missing. Chapter 2 explores the conserved motifs and regions in the C-terminal effector domain of AVR1 that are required to trigger R1-mediated hypersensitive response (HR). Various truncated and chimeric constructs of AVR1 and A-L were generated and assayed for their ability to elicit R1-mediated HR. Results show that the T-region of AVR1 plays an important role in HR activation. Furthermore, we revealed that R1 recognizes two epitopes in AVR1, one located in the C-terminal region containing the conserved W and Y motifs, and one comprised by the T region. In Chapter 3 the subcellular localization of AVR1 and R1 was investigated. Both were demonstrated to be nucleocytoplasmic proteins. We artificially modified the nucleocytoplasmic partitioning of AVR1 and R1 using nuclear localization and export signals (NLS/NES), and studied the effect on R1-AVR1 recognition. This revealed that nuclear localization of both AVR1 and R1 is important to induce R1-mediated immunity. In addition, we showed that AVR1-mediated suppression of CRN2-induced cell death is dependent on cytosolic localization of AVR1. In Chapter 4, we investigated how AVR1 modulates host defense. In a yeast two-hybrid screening we identified the exocyst subunit Sec5 as a host target for AVR1. Interaction between AVR1 and Sec5 was confirmed in planta by co-immunoprecipitation and bimolecular fluorescent complementation. Although A-L shares high sequence similarity with AVR1, we found that it is not able to interact with Sec5. Sec5 was shown to be required for proper plant defense against P. infestans. The role of Sec5 in plant response upon pathogen attack was further supported by its role in callose deposition and in secretion of the pathogenesis-related protein PR-1, which indicates that Sec5 plays a crucial role in vesicle trafficking during host defense. AVR1 is able to suppress callose deposition while A-L is not, which suggests that P. infestans manipulates host vesicle trafficking by secretion of AVR1 to target Sec5. Overall, our findings unravelled a novel strategy that oomycete pathogens exploit in order to modulate host defense. In Chapter 5 we further analysed the potential virulence activities of AVR1 and A-L. Both AVR1 and A-L were able to promote P. infestans colonization, indicating that both are genuine P. infestans virulence factors. Moreover, AVR1 was found to suppress not only callose deposition, but also Sec5-dependent cell death induced by the P. infestans elicitors INF1 and CRN2. In contrast, A-L was neither able to suppress Sec5-dependent nor Sec5-independent cell death. The conserved C-terminal motifs and regions required for virulence activity of AVR1 were investigated using AVR1 truncated constructs. In addition, the conserved C-terminal motifs and regions of AVR1 required for Sec5 interaction were studied by Y2H assays. Although the T-region of AVR1 was found to be sufficient to facilitate P. infestans colonization and suppression of CRN2-induced cell death, it could not fully accommodate the interaction of AVR1 with Sec5. Instead, both the Y motif and the T-region of AVR1 appear to be required for Sec5 targeting. Next to Sec5, the role of other exocyst subunits in Phytophthora resistance was studied (Chapter 6). The evolutionary relationships of exocyst subunits from three Solanaceous plants, i.e. Nicotiana benthamiana, tomato and potato, were investigated in comparison to their Arabidopsis orthologs. Virus-induced gene silencing in N. benthamiana of the majority of the exocyst subunit genes (exo84s were not yet included) showed that, except for some Exo70 members, all other tested exocyst subunits are required for plant defense against P. infestans and callose deposition. In addition, all of the analysed exocyst subunit gene-silenced tomato plants showed gain of susceptibility to both P. infestans and Phytophthora capsici. In Chapter 7, our findings obtained in this thesis on the mechanisms of AVR1-triggered host immunity and susceptibility are discussed in a broader perspective with emphasis on the current developments in the field of effector biology.

Summary Current treatments of inflammatory disorders are often based on therapeutic proteins. These proteins, so-called biopharmaceuticals, are isolated from a natural resource or, more often, made using cell based fermentation systems. The most common production platforms are based on the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae or mammalian cell lines (mainly Chinese hamster ovarian (CHO) and murine myeloma (SP2/0) cells). Each platform has advantages and disadvantages and the protein to be produced largely dictates the choice of platform. Plants could provide a unique alternative production platform, as they combine the advantage of E. coli being economic with the advantages of mammalian cell lines being able to fold complex proteins, assemble heteromultimeric protein complex and, upon glyco-engineering, provide proteins with human type N-glycans. Furthermore, plant production is easily scaled up as the infrastructure is already in place due to our need for food and feed and plants have a limited risk of contamination with human pathogens. Transient transformation of N. benthamiana is a valuable plant production platform, as it is unmatched in terms of speed (matter of weeks). This thesis describes the production of a variety of proteins and protein complexes in planta that are or may be used as biopharmaceuticals. Since 1982, plants can be genetically manipulated, which has lead to the production of many proteins in a variety of plants. Initially, the plants greatest drawback was limited protein yield. However, significant increases in yield have been achieved in the last two decades, predominantly by increasing transformation efficiency and/or level of transcription. Nowadays several plant expression systems exist that facilitate high protein production levels. Most experience is based on the production of antibodies, mainly of the IgG isotype, as these are often used as biopharmaceuticals. In general, IgG antibodies are produced on a scale of several grams per kilogram fresh weight. However, production levels of antibodies have shown to be variable and production levels of particular proteins, such as cytokines, have lagged behind. Cytokines form a large group of immune-signalling molecules and several cytokines have promising therapeutic potential. Their short in vivo half-life suggests an inherent instability, which is regarded the major production bottleneck. In chapter 2 we describe the production of interleukin (IL)-10, a cytokine with immunosuppressive properties. We show that in contrast to mouse IL-10, human IL-10 multimerises extensively in planta. Both human and mouse IL-10 form homodimeric protein complexes through a mechanism referred to as 3D domain swapping. Only for human IL-10 3D domain swapping results in extensive multimerisation. By fusing IL-10 to green fluorescent protein (GFP) multimerisation was visualised, demonstrating that human IL-10 forms granules of organelle size. We discovered that for mouse IL-10 granule formation was prevented by N-glycosylation of the N-terminus. Introduction of this N-glycosylation site in human IL-10 partly prevented granule formation. The insertion of a glycine-serine linker between alpha helices D and E of human IL-10, allowing IL-10 to swap its own domains and form a stable monomer. This prevented granule formation completely and boosted protein yield 30-fold. However, the now comparable yields of human and mouse IL-10 were still low when compared to for instance IgG. In the next two chapters we shifted the focus to the production of antibodies. Thus far the IgA isotype received little attention as candidate biopharmaceutical. However, the unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. Therefore, we compared the plant-based expression of IgA to IgG (Chapter 3). The variable regions of three antibodies commonly used in the clinic to treat inflammatory disorders (Infliximab, Adalimumab and Ustekinumab) were grafted on either an IgG1κ or IgA1κ backbone. Surprisingly, we achieved comparable high expression levels for all antibodies. The large variation in antibody yield found in literature is therefore likely due to differences in expression systems and experimental conditions, but not to the antibody idiotype used. Evaluation of the secretion efficiency and N-glycosylation profiles revealed compelling differences between IgG and IgA antibodies when expressed in planta. Compared to IgG, IgA was poorly secreted. Poor secretion is most likely due to vacuolar targeting of IgA, as it was previously demonstrated that murine IgA contained a cryptic vacuolar targeting signal in the tailpiece. IgA also carried different N-glycans compared to IgG, which were never found to be a main fraction of a plant-produced protein. The predominant N-glycan on IgA lacked both the typical plant core α1,3-fucose and one terminal N-acetylglucosamine (GlcNAc). The core α1,3-fucose is most likely not added due to low accessibility of the core of the N-glycans. The lack of one GlcNAc may be due to inefficient addition of this sugar residue or (partial) β-hexosaminidase activity that may occur in both the vacuole and the apoplast. Whether the vacuolar targeting plays a role in the lack of the GlcNAc and what protein intrinsic properties influence partial GlcNAc addition or removal is unclear. We also showed that the N-glycosylation site in the IgA tailpiece does not always receive a N-glycan, which may be significant for secretory IgA formation as described in chapter 4. In chapter 4 we describe the expression of this large heteromultimeric protein complex. Secretory IgA consists of two IgA complexes that are connected via the joining chain and associate with a part of the polymeric-Ig-receptor (called the secretory component). The challenge for sIgA expression is that assembly of four polypeptides (the alpha heavy chain, the light chain, the joining chain and the secretory component) in a 4:4:1:1 ratio is required. Previous studies on stable and transient expression of sIgA demonstrated that sIgA formation in plants is possible. However, sIgA was always accompanied by a large proportion of monomeric IgA as well as other assembly intermediates. The presence of assembly intermediates may be caused be unequal expression and/or stability of the individual components. However, we demonstrated that not the expression strategy, but protein complex assembly through impaired incorporation of the joining chain was the limiting step for sIgA production. Because joining chain incorporation depends on N-glycosylation of the IgA tailpiece we hypothesise that the partial N-glycosylation of the IgA tailpiece is the cause of inefficient joining chain incorporation. The efficiency of N-glycosylation may be improved by mutation of the glycosylation site N-X-S to an N-X-T site, as the latter glycosylation motif was shown to be more often occupied in a large-scale glycosylation site analysis. Successful engineering of the plant glycosylation pathway to provide biopharmaceuticals with human N-glycan types has been achieved. Glyco-engineering also provides the opportunity to generate potentially interesting N-glycan types with regard to immunogenicity for vaccination purposes or protein trafficking for in vivo targeting of biopharmaceuticals. In chapter 5 we describe the expression of the Schistosoma mansoni egg antigen omega-1, an immunomodulatory protein with therapeutic potential. Its biological activity depends on its RNAse activity and its N-glycans that enable internalisation by human dendritic cells. Omega-1 cannot be isolated from natural resources in sufficient quantities to study its in vivo biology i.e. which N-glycan types facilitate full activity of omega-1. Therefore, we set up the plant-based production of this protein. S. mansoni-derived omega-1 predominantly carries diantennary N-glycans with a Lewis X motif on one or both antennae. Lewis X consists of a β1,4-linked galactose and α1,3-linked fucose residue attached to a terminal GlcNAc. This attachment is performed by β1,4-galactosyltransferase and α1,3-fucosyltransferase IXa that naturally do not occur in plants. Co-expression of these two glycosyltransferases with omega-1 resulted in a N-glycan profile comparable to S. mansoni-derived omega-1. However, it was necessary to control the expression of the β1,4-galactosyltransferase to contain this enzyme in the trans-Golgi compartment. Overexpression most likely leads to overflow of β1,4-galactosyltransferase to the medial-Golgi and galactosylation at this stage disturbs the activity of other glycosyltransferases resulting in hybrid N-glycan types. We also observed that more than 90% of omega-1 was secreted to the apoplast, which facilitated efficient purification. This demonstrated that plants, as previously suggested, are not poor protein secretors. However, the underlying protein properties controlling secretion efficiency are currently unknown. Both omega-1 and IgG are very efficiently secreted under natural circumstances. Yet, despite the fact that we used the same signal peptide to facilitate secretion of IgG and omega-1, IgG was only secreted from plant cells to a maximum of 28%. More knowledge on protein secretion efficiency in plants may overcome cumbersome purification, currently the greatest bottleneck in the downstream processing of plant produced proteins. Finally in chapter 6, we shifted focus from the expression of a particular protein to the effect of codon use on protein yield. We designed a codon optimisation strategy that, unlike other strategies, turned out to be surprisingly robust. This strategy is based on a general codon bias found in plants. Because this codon bias could be found among plant species, including monocots and dicots, and resulted in an increase in mRNA stability and translatability, we suspected that this codon bias arises from a selection pressure on the mRNA structure. We extended this general codon bias to representative species of other kingdoms of life and demonstrate that there is a selection pressure increasing mRNA stability and translatability (more protein per mRNA molecule). Stability was the result of an increase in the number of nucleotide bonds. However, there is a trade off between mRNA stability and translatability, and the nucleotide bonds in an mRNA should be well balanced over the entire molecule, making it ‘airy’, to ensure efficient translation. Altogether we conclude that transformation efficiency and level of transcription are no longer limiting factors for protein yield upon plant-based expression. However, an increase in translation initiation and translation rate dictated by codon use may still provide an increase in protein production. Furthermore, we show that many proteins demonstrated specific production bottlenecks. The yield of human IL-10 was hampered by its extensive multimerisation, sIgA assembly is most likely limited by inefficient N-glycosylation of the tailpiece of IgA, both IgG and IgA are inefficiently secreted compared to omega-1 and IgA displayed an aberrant N-glycosylation profile. Therefore, more knowledge on how protein intrinsic properties influence protein yield and/or quality of heterologous produced proteins in plants should now be generated. Transient expression in N. benthamiana is an ideal tool to study these protein intrinsic properties that limit protein yield in heterologous expression.

Summary Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating potato diseases worldwide. To successfully colonize its host, P. infestans secretes a plethora of RXLR effectors that translocate into host cells to modulate plant defense. The RXLR effectors form the largest and most diverse effector family in oomycete plant pathogens, and include several that were demonstrated to trigger host resistance mediated by intracellular host immune receptors. Chapter 1 is a summary focussing on the molecular mechanisms underlying host–pathogen interactions. It introduces the multi-layered innate immune system of plants, as well as the strategies that pathogens exploit to circumvent and suppress host defense. Furthermore, it highlights the importance of vesicle-trafficking during plant defense. The central subject of this thesis is AVR1, one of the race-specific avirulence (AVR) factors of P. infestans. AVR1 triggers plant resistance mediated by its corresponding potato Nucleotide-binding Leucine-rich repeat (NLR) resistance protein R1. P. infestans isolates that are avirulent on R1-containing potato cultivars always contain AVR1, while virulent isolates lack AVR1 but contain a related gene that we baptized as AVR1-like. AVR1 has all hallmarks of a typical RXLR effector; it contains a signal peptide, an RXLR domain and a C-terminal effector domain that contains two W motifs and one Y motif. In addition, it has, at the very end a stretch of 38 amino acids in length that we named the Tail (T)-region. AVR1-like, or in short A-L, shares high sequence similarity with AVR1. However, due to a premature stop codon the 38 amino acid T-region is missing. Chapter 2 explores the conserved motifs and regions in the C-terminal effector domain of AVR1 that are required to trigger R1-mediated hypersensitive response (HR). Various truncated and chimeric constructs of AVR1 and A-L were generated and assayed for their ability to elicit R1-mediated HR. Results show that the T-region of AVR1 plays an important role in HR activation. Furthermore, we revealed that R1 recognizes two epitopes in AVR1, one located in the C-terminal region containing the conserved W and Y motifs, and one comprised by the T region. In Chapter 3 the subcellular localization of AVR1 and R1 was investigated. Both were demonstrated to be nucleocytoplasmic proteins. We artificially modified the nucleocytoplasmic partitioning of AVR1 and R1 using nuclear localization and export signals (NLS/NES), and studied the effect on R1-AVR1 recognition. This revealed that nuclear localization of both AVR1 and R1 is important to induce R1-mediated immunity. In addition, we showed that AVR1-mediated suppression of CRN2-induced cell death is dependent on cytosolic localization of AVR1. In Chapter 4, we investigated how AVR1 modulates host defense. In a yeast two-hybrid screening we identified the exocyst subunit Sec5 as a host target for AVR1. Interaction between AVR1 and Sec5 was confirmed in planta by co-immunoprecipitation and bimolecular fluorescent complementation. Although A-L shares high sequence similarity with AVR1, we found that it is not able to interact with Sec5. Sec5 was shown to be required for proper plant defense against P. infestans. The role of Sec5 in plant response upon pathogen attack was further supported by its role in callose deposition and in secretion of the pathogenesis-related protein PR-1, which indicates that Sec5 plays a crucial role in vesicle trafficking during host defense. AVR1 is able to suppress callose deposition while A-L is not, which suggests that P. infestans manipulates host vesicle trafficking by secretion of AVR1 to target Sec5. Overall, our findings unravelled a novel strategy that oomycete pathogens exploit in order to modulate host defense. In Chapter 5 we further analysed the potential virulence activities of AVR1 and A-L. Both AVR1 and A-L were able to promote P. infestans colonization, indicating that both are genuine P. infestans virulence factors. Moreover, AVR1 was found to suppress not only callose deposition, but also Sec5-dependent cell death induced by the P. infestans elicitors INF1 and CRN2. In contrast, A-L was neither able to suppress Sec5-dependent nor Sec5-independent cell death. The conserved C-terminal motifs and regions required for virulence activity of AVR1 were investigated using AVR1 truncated constructs. In addition, the conserved C-terminal motifs and regions of AVR1 required for Sec5 interaction were studied by Y2H assays. Although the T-region of AVR1 was found to be sufficient to facilitate P. infestans colonization and suppression of CRN2-induced cell death, it could not fully accommodate the interaction of AVR1 with Sec5. Instead, both the Y motif and the T-region of AVR1 appear to be required for Sec5 targeting. Next to Sec5, the role of other exocyst subunits in Phytophthora resistance was studied (Chapter 6). The evolutionary relationships of exocyst subunits from three Solanaceous plants, i.e. Nicotiana benthamiana, tomato and potato, were investigated in comparison to their Arabidopsis orthologs. Virus-induced gene silencing in N. benthamiana of the majority of the exocyst subunit genes (exo84s were not yet included) showed that, except for some Exo70 members, all other tested exocyst subunits are required for plant defense against P. infestans and callose deposition. In addition, all of the analysed exocyst subunit gene-silenced tomato plants showed gain of susceptibility to both P. infestans and Phytophthora capsici. In Chapter 7, our findings obtained in this thesis on the mechanisms of AVR1-triggered host immunity and susceptibility are discussed in a broader perspective with emphasis on the current developments in the field of effector biology.

Phytophthora pathogens are notorious for causing severe damage to many agriculturally and ornamentally important plants. Effective plant resistance depends largely on the capacity to perceive pathogens and to activate rapid defence. Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity upon recognition of matching effectors secreted by Phytophthora. However, Phytophthora pathogens are notoriously difficult to control due to their rapid adaptation to evade R protein-mediated recognition. Hence, exploring novel resistance components is instrumental for developing durable resistance. Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous and endogenous stimuli to initiate plant defence. One RLK that was previously identified as a novel Phytophthora resistance component is the Arabidopsis L-type lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of 45 members in Arabidopsis but whether or not the other members function in Phytophthora resistance was thus far unknown. The research described in this thesis was aimed at unravelling the role of LecRKs in plant immunity, in particular to Phytophthora pathogens. Chapter I describes various Phytophthora diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to Phytophthora pathogens. In Chapter II, we describe the development of a new Arabidopsis-Phytophthora pathosystem. We demonstrated that Phytophthora capsici is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and indole glucosinolates biosynthesis pathways are required for P. capsici resistance (Chapter IIa). The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem can be used as an additional tool to pinpoint essential components of Phytophthora resistance. We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of LecRKs in Phytophthora resistance. In Chapter III we describe a systematic phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other LecRKs function in Phytophthora resistance. We have also analysed whether the LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to bacterial or fungal pathogens, but none of the lines showed visible developmental changes under normal conditions or upon abiotic stress treatment. Combining these phenotypic data with LecRK expression profiles obtained from publicly available datasets revealed that LecRKs that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function display diverse expression patterns. Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora resistance was further investigated. In Chapter IV we describe that overexpression of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to Phytophthora, but also induced plant cell death. A mutation in the kinase domain abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce Phytophthora resistance as did deletion of the lectin domain. Cell death induction however, only required the kinase, not the lectin domain. Since transient expression of both LecRKs in Nicotiana benthamiana also resulted in increased Phytophthora resistance and induction of cell death, we used N. benthamiana to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for Phytophthora resistance. Collectively, these results illustrate that the Phytophthora resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death phenotype. By co-immunoprecipitation we identified putative interacting proteins, one of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis, the ABC transporter ABCG40, was found to interact in planta with both LecRK-IX.1 and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants showed compromised Phytophthora resistance, indicating that ABCG40 has a function in Phytophthora resistance. In Chapter V, we describe the generation of stable transgenic N. benthamiana plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of LecRK-IX.1 induced plant cell death. Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed increased resistance towards P. capsici or Phytophthora infestans. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora resistance upon interfamily transfer. Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in Phytophthora resistance. In Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both N. benthamiana and tomato lack LecRKs homologous to Arabidopsis LecRKs of clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional analysis of various Solanaceous LecRKs using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance. These results indicate that the role of clade IX LecRKs in Phytophthora resistance is conserved across plant species. In Chapter VII, the experimental data presented in this thesis are summarized and discussed in a broader context. We present an overview of the current understanding of LecRKs in plant immunity and discuss how LecRKs can be exploited to improve plant resistance.

Plants live in complex environments and are under constant threat of being attacked by herbivorous arthropods. Consequently plants possess an arsenal of sophisticated mechanisms in order to defend themselves against their ubiquitous attackers. Induced indirect defenses involve the attraction of natural enemies of herbivores, such as predators and parasitoids. Predators and parasitoids use odors emitted by damaged plants that serve as a “cry for help” to find their respective prey or host herbivore. The aim of this thesis was to use a multidisciplinary approach, with focus on molecular and chemical methods, combined with behavioral investigations, to elucidate the mechanisms of plant responses to multiple herbivory that affect a tritrophic system consisting of a plant, an herbivore and a natural enemy. Induced plant defenses are regulated by a network of defense signaling pathways in which phytohormones act as signaling molecules. Accordingly, simulation of herbivory by exogenous application of phytohormones and actual herbivory by the two-spotted spider mite Tetranychus urticae affected transcript levels of a defense gene involved in indirect defense in Lima bean. However, two other genes involved in defense were not affected at the time point investigated. Moreover, application of a low dose of JA followed by minor herbivory by T. urticae spider mites affected gene transcript levels and emissions of plant volatiles commonly associated with herbivory. Only endogenous phytohormone levels of jasmonic acid (JA), but not salicylic acid (SA), were affected by treatments. Nevertheless, the low-dose JA application resulted in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defense after herbivore infestation. Caterpillar feeding as well as application of caterpillar oral secretion on mechanically inflicted wounds are frequently used to induce plant defense against biting-chewing insects, which is JA-related. Feeding damage by two caterpillar species caused mostly identical induction of gene transcription, but combination of mechanical damage and oral secretions of caterpillars caused differential induction of the transcription of defense genes. Nevertheless, gene transcript levels for plants that subsequently experienced an infestation by T. urticae were only different for a gene potentially involved in direct defense of plants that experienced a single event of herbivory by T. urticae. Indirect defense was not affected. Also sequential induction of plant defense by caterpillar oral secretion and an infestation by T. urticae spider mites did not interfere with attraction of the specialist predatory mite P. persimilis in olfactometer assays. The predator did distinguish between plants induced by spider mites and plants induced by the combination of mechanical damage and caterpillar oral secretion but not between plants with single spider mite infestation and plants induced by caterpillar oral secretion prior to spider mite infestation. The composition of the volatile blends emitted by plants induced by spider mites only or by the sequential induction treatment of caterpillar oral secretion followed by spider mite infestation were similar. Consequently, the induction of plant indirect defense as applied in these experiments was not affected by previous treatment with oral secretion of caterpillars. Moreover, herbivory by conspecific T. urticae mites did not affect gene transcript levels or emission of volatiles of plants that experienced two bouts of herbivore attack by conspecific spider mites compared to plants that experienced only one bout of spider mite attack. This suggests that Lima bean plants do no increase defense in response to sequential herbivory by T. urticae. In conclusion, using a multidisciplinary approach new insights were obtained in the mechanisms of induction of indirect plant defense and tritrophic interactions in a multiple herbivore context, providing helpful leads for future research on plant responses to multiple stresses.

Zinc (Zn) and cadmium (Cd) are toxic metals that can cause serious soil contamination when present in excess. Especially Cd exposure is a threat to human health. Plants can potentially be used to clean-up the Zn- and/or Cd-contaminated soils in a technology called “Phytoremediation”. Two major requirements for this technology are the availability of plants that are accumulating Zn/Cd to high levels in their leaves and that are producing high biomass. Noccaea caerulescens is a natural model Zn/Cd/Ni hyperaccumulator species that can accumulate up to 3% of Zn and 1% of Cd. Unfortunately it does not make a lot of biomass, and is thus unsuitable for phytoremediation. To overcome this limitation, higher biomass producing crops like tobacco can be genetically modified with genes from N. caerulescens to induce metal tolerance and hyperaccumulation. NcZNT1 and NcMTP1 are metal transporter genes in N. caerulescens. Zeshan Hassan has engineered tobacco to express both these genes, separately and in combination, to investigate their role in Zn, Cd accumulation and tolerance and to explore their potential in improving the phytoremediation capacity of plants. Transgenic tobacco lines were more tolerant to high Zn and Cd, leading to higher biomass compared to non-transformed controls, and they accumulated 2-4 fold higher Zn and 2-3.5 fold higher Cd when grown in artificial nutrient solution systems. More importantly they also did so when grown in soil collected from a metal-contaminated site. One of the conclusions of Zeshan Hassan’s PhD thesis is that both NcZNT1 and NcMTP1 genes are important for Zn and Cd tolerance and accumulation and that transgenic tobacco expressing these genes have enhanced phytoremediation capacity, which could lead to interesting future applications.

Zinc (Zn) and cadmium (Cd) are toxic metals that can cause serious soil contamination when present in excess. Especially Cd exposure is a threat to human health. Plants can potentially be used to clean-up the Zn- and/or Cd-contaminated soils in a technology called “Phytoremediation”. Two major requirements for this technology are the availability of plants that are accumulating Zn/Cd to high levels in their leaves and that are producing high biomass. Noccaea caerulescens is a natural model Zn/Cd/Ni hyperaccumulator species that can accumulate up to 3% of Zn and 1% of Cd. Unfortunately it does not make a lot of biomass, and is thus unsuitable for phytoremediation. To overcome this limitation, higher biomass producing crops like tobacco can be genetically modified with genes from N. caerulescens to induce metal tolerance and hyperaccumulation. NcZNT1 and NcMTP1 are metal transporter genes in N. caerulescens. Zeshan Hassan has engineered tobacco to express both these genes, separately and in combination, to investigate their role in Zn, Cd accumulation and tolerance and to explore their potential in improving the phytoremediation capacity of plants. Transgenic tobacco lines were more tolerant to high Zn and Cd, leading to higher biomass compared to non-transformed controls, and they accumulated 2-4 fold higher Zn and 2-3.5 fold higher Cd when grown in artificial nutrient solution systems. More importantly they also did so when grown in soil collected from a metal-contaminated site. One of the conclusions of Zeshan Hassan’s PhD thesis is that both NcZNT1 and NcMTP1 genes are important for Zn and Cd tolerance and accumulation and that transgenic tobacco expressing these genes have enhanced phytoremediation capacity, which could lead to interesting future applications.

Plant secondary metabolites (PSM) such as terpenes and phenolic compounds are known to have numerous ecological roles, notably in defence against herbivores, pathogens and abiotic stresses and in interactions with competitors and mutualists. This book reviews recent developments in the field to provide a synthesis of the function, ecology and evolution of PSM, revealing our increased awareness of their integrative role in connecting natural systems. It emphasises the multiple roles of secondary metabolites in mediating the interactions between organisms and their environment at a range of scales of ecological organisation, demonstrating how genes encoding for PSM biosynthetic enzymes can have effects from the cellular scale within individual plants all the way to global environmental processes. A range of recent methodological advances, including molecular, transgenic and metabolomic techniques, are illustrated and promising directions for future studies are identified, making this a valuable reference for researchers and graduate students in the field.

Plant secondary metabolites (PSM) such as terpenes and phenolic compounds are known to have numerous ecological roles, notably in defence against herbivores, pathogens and abiotic stresses and in interactions with competitors and mutualists. This book reviews recent developments in the field to provide a synthesis of the function, ecology and evolution of PSM, revealing our increased awareness of their integrative role in connecting natural systems. It emphasises the multiple roles of secondary metabolites in mediating the interactions between organisms and their environment at a range of scales of ecological organisation, demonstrating how genes encoding for PSM biosynthetic enzymes can have effects from the cellular scale within individual plants all the way to global environmental processes. A range of recent methodological advances, including molecular, transgenic and metabolomic techniques, are illustrated and promising directions for future studies are identified, making this a valuable reference for researchers and graduate students in the field.

Op 20 juni 2011 promoveerde Hendrik Rietman aan Wageningen University (WUR) op zijn proefschrift getiteld: “Putting the Phytophthora infestans genome sequence at work; multiple novel avirulence and potato resistance gene candidates revealed”. Zijn promotor, Prof. Dr. Richard G. F. Visser, en co-promotor, Dr. Ir. Vivianne G. A. A. Vleeshouwers, zijn beiden verbonden aan de leerstoelgroep Plantenveredeling van de WUR, waar ook het onderzoek werd uitgevoerd. Tevens is er samengewerkt met de leerstoelgroep Fytopathologie (WUR), het Sainsbury Lab en het James Hutton Instituut (beiden gevestigd in het Verenigd Koninkrijk). Financiering was afkomstig van het ‘Parapluplan Phytophthora’ en WUR Plantenveredeling.

In the past two decades genome sequencing has developed from a laborious and costly technology employed by large international consortia to a widely used, automated and affordable tool used worldwide by many individual research groups. Genome sequences of many food animals and crop plants have been deciphered and are being exploited for fundamental research and applied to improve their breeding programs. The developments in sequencing technologies have also impacted the associated bioinformatics strategies and tools, both those that are required for data processing, management, and quality control, and those used for interpretation of the data. This thesis focuses on the application of genome sequencing, assembly and annotation to two members of the Solanaceae family, tomato and potato. Potato is the economically most important species within the Solanaceae, and its tubers contribute to dietary intake of starch, protein, antioxidants, and vitamins. Tomato fruits are the second most consumed vegetable after potato, and are a globally important dietary source of lycopene, beta-carotene, vitamin C, and fiber. The chapters in this thesis document the generation, exploitation and interpretation of genomic sequence resources for these two species and shed light on the contents, structure and evolution of their genomes. Chapter 1introduces the concepts of genome sequencing, assembly and annotation, and explains the novel genome sequencing technologies that have been developed in the past decade. These so-called Next Generation Sequencing platforms display considerable variation in chemistry and workflow, and as a consequence the throughput and data quality differs by orders of magnitude between the platforms. The currently available sequencing platforms produce a vast variety of read lengths and facilitate the generation of paired sequences with an approximately fixed distance between them. The choice of sequencing chemistry and platform combined with the type of sequencing template demands specifically adapted bioinformatics for data processing and interpretation. Irrespective of the sequencing and assembly strategy that is chosen, the resulting genome sequence, often represented by a collection of long linear strings of nucleotides, is of limited interest by itself. Interpretation of the genome can only be achieved through sequence annotation – that is, identification and classification of all functional elements in a genome sequence. Once these elements have been annotated, sequence alignments between multiple genomes of related accessions or species can be utilized to reveal the genetic variation on both the nucleotide and the structural level that underlies the difference between these species or accessions. Chapter 2describes BlastIf, a novel software tool that exploits sequence similarity searches with BLAST to provide a straightforward annotation of long nucleotide sequences. Generally, two problems are associated with the alignment of a long nucleotide sequence to a database of short gene or protein sequences: (i) the large number of similar hits that can be generated due to database redundancy; and (ii) the relationships implied between aligned segments within a hit that in fact correspond to distinct elements on the sequence such as genes. BlastIf generates a comprehensible BLAST output for long nucleotide sequences by reducing the number of similar hits while revealing most of the variation present between hits. It is a valuable tool for molecular biologists who wish to get a quick overview of the genetic elements present in a newly sequenced segment of DNA, prior to more elaborate efforts of gene structure prediction and annotation. In Chapter 3 a first genome-wide comparison between the emerging genomic sequence resources of tomato and potato is presented. Large collections of BAC end sequences from both species were annotated through repeat searches, transcript alignments and protein domain identification. In-depth comparisons of the annotated sequences revealed remarkable differences in both gene and repeat content between these closely related genomes. The tomato genome was found to be more repetitive than the potato genome, and substantial differences in the distribution of Gypsy and Copia retrotransposable elements as well as microsatellites were observed between the two genomes. A higher gene content was identified in the potato sequences, and in particular several large gene families including cytochrome P450 mono-oxygenases and serine-threonine protein kinases were significantly overrepresented in potato compared to tomato. Moreover, the cytochrome P450 gene family was found to be expanded in both tomato and potato when compared to Arabidopsis thaliana, suggesting an expanded network of secondary metabolic pathways in the Solanaceae. Together these findings present a first glimpse into the evolution of Solanaceous genomes, both within the family and relative to other plant species. Chapter 4explores the physical and genetic organization of tomato chromosome 6 through integration of BAC sequence analysis, High Information Content Fingerprinting, genetic analysis, and BAC-FISH mapping data. A collection of BACs spanning substantial parts of the short and long arm euchromatin and several dispersed regions of the pericentrometric heterochromatin were sequenced and assembled into several tiling paths spanning approximately 11 Mb. Overall, the cytogenetic order of BACs was in agreement with the order of BACs anchored to the Tomato EXPEN 2000 genetic map, although a few striking discrepancies were observed. The integration of BAC-FISH, sequence and genetic mapping data furthermore provided a clear picture of the borders between eu- and heterochromatin on chromosome 6. Annotation of the BAC sequences revealed that, although the majority of protein-coding genes were located in the euchromatin, the highly repetitive pericentromeric heterochromatin displayed an unexpectedly high gene content. Moreover, the short arm euchromatin was relatively rich in repeats, but the ratio of Gypsy and Copia retrotransposons across the different domains of the chromosome clearly distinguished euchromatin from heterochromatin. The ongoing whole-genome sequencing effort will reveal if these properties are unique for tomato chromosome 6, or a more general property of the tomato genome. Chapter 5presents the potato genome, the first genome sequence of an Asterid. To overcome the problems associated with genome assembly due tothe high level of heterozygosity that is observed in commercial tetraploid potato varieties, a homozygous doubled-monoploid potato clone was exploited to sequence and assemble 86% of the 844 Mb genome. This potato reference genome sequence was complemented with re-sequencing of aheterozygous diploid clone, revealing the form and extent of sequence polymorphism both between different genotypes and within a single heterozygous genotype. Gene presence/absence variants and other potentially deleterious mutations were found to occur frequently in potato and are a likely cause of inbreeding depression. Annotation of the genome was supported by deep transcriptome sequencing of both the doubled-monoploid and the heterozygous potato, resulting in the prediction of more than 39,000 protein coding genes. Transcriptome analysis provided evidence for the contribution of gene family expansion, tissue specific expression, and recruitment of genes to new pathways to the evolution of tuber development. The sequence of the potato genome has provided new insights into Eudicot genome evolution and has provided a solid basis for the elucidation of the evolution of tuberisation. Many traits of interest to plant breeders are quantitative in nature and the potato sequence will simplify both their characterization and deployment to generate novel cultivars. The outstanding challenges in plant genome sequencing are addressed in Chapter 6. The high concentration of repetitive elements and the heterozygosity and polyploidy of many interesting crop plant species currently pose a barrier for the efficient reconstruction of their genome sequences. Nonetheless, the completion of a large number of new genome sequences in recent years and the ongoing advances in sequencing technology provide many excitingopportunities for plant breeding and genome research. Current sequencing platforms are being continuously updated and improved, and novel technologies are being developed and implemented in third-generation sequencing platforms that sequence individual molecules without need for amplification. While these technologies create exciting opportunities for new sequencing applications, they also require robust software tools to process the data produced through them efficiently. The ever increasing amount of available genome sequences creates the need for an intuitive platform for the automated and reproducible interrogation of these data in order to formulate new biologically relevant questions on datasets spanning hundreds or thousands of genome sequences.

Individual variation in milk yield response to concentrate intake and milking interval length During the last century in the Netherlands milk production per cow has almost tripled. Accordingly, the amount of concentrates yearly fed per cow strongly increased. Furthermore, automation and robotisation has changed dairy management, especially by the introduction of automatic concentrate feeders and milking systems. A new management concept, emerging in the last decades, is Precision Livestock Farming (PLF). The objective of PLF is to optimize livestock production, by on-line monitoring and control of the production process, utilizing the technical possibilities of automation and robotisation. Nowadays, individual settings for daily concentrate supply and milking frequency are based on standards, ignoring individual variation in milk yield response on concentrate intake and milking frequency. This leads to the main hypothesis for this thesis research that profitability of dairy farming can be improved by utilizing information on individual variation in response. The first objective of this research was to quantify the individual variation in milk yield response to concentrate intake and milking interval length, in order to assess the economic prospects of applying individual optimal settings for concentrate supply and milking frequency. In the first observational study (Ch. 2), data from 299 cows on four farms in the first 3 weeks of the lactation were collected. Individual response in daily milk yield to concentrate intake was analysed by a random coefficient model. During the first three weeks of lactation, considerable variation in individual milk yield response to concentrate intake was found on all four farms. An economic simulation was carried out, based on the estimated parameter values in the observational study. Individual economically optimized settings for concentrate supply were compared with conventional strategies for concentrate supply based on averaged population response parameters. Applying individual economic optimal settings for concentrate supply during early lactation, potential economic gain ranges from 0.20 to 2.03 €/cow/day. In a second observational study (Ch.3), data of normal uninterrupted milkings during one week from 311 cows kept in 5 separate herds on one farm were collected. The data set consisted of 4,915 records and random coefficient models were fitted to estimate the individual effects of milking interval on daily milk yield and milking duration. Between individuals, considerable variation in milk yield and milking duration was found in response to milking interval. Based on the estimated individual response, a simulation was carried out in order to optimize the utilization of an AMS for different herd sizes and occupation rates. Applying optimal individual milking intervals for a herd of 60 cows and an AMS operating at an occupation rate of 64%, the average milking interval reduced from 0.421 day to 0.400 day, the daily milk yield at the herd level increased from 1,883 to 1,909 kg/day, and milk revenues increased from 498 to 507 €/day. In the actual situation, the herd consisted of 60 cows. A further increase of daily milk revenues per AMS was possible by increasing the operation rate and/or herd size. The conclusion is that between dairy cows there is a considerable variation in effects of concentrate intake and milking interval length on milk yield and, consequently, milking duration. A marked increase in economic profits of dairy production is possible by improvement of the concentrate allocation and/or the utilisation of an AMS, applying optimal individual settings based on the actual individual response in milk yield. Development of adaptive models The second objective was the development and testing of adaptive models for on-line estimation of the actual individual response in milk yield to concentrate intake and milking interval length. In Ch. 4 adaptive dynamic models for on-line estimation of the actual individual milk yield response to concentrate intake and milking interval length were evaluated. The parameters in these models may change over time and are updated through a Bayesian approach for on-line analysis of time series. Time series data of daily milk yield during the first 200 days of lactation from 17 cows were analysed with different adaptive dynamic models. Three models were evaluated: a model with linear terms for concentrate intake and length of milking interval, a model with linear and quadratic terms, and an enhanced model in order to obtain more stable parameter estimates. The linear model was only useful for forecasting milk production and the estimated parameters of the quadratic model turned out to be unstable. The parsimony of the enhanced model lead to far more stable parameter estimates. In Ch. 5 an adaptive dynamic model was used for time series analysis of herd mean daily milk yield, in order to quantify the impact of heat stress and to assess the potential for monitoring and control of milk production. Time series data of daily milk yield from 2003 to 2006 were collected on six experimental research farms in The Netherlands. The impact of heat stress was quantified in terms of critical temperature, duration and loss in milk yield. The estimated critical temperature was 17.8 oC, the duration was 5.5 days, and loss in milk yield 31.4 kg milk/cow/year, averaged over farms. Besides estimation of the impact of heat stress, level and trend, including a weekly cyclical pattern were estimated to evaluate the production process. The Bayesian approach for on-line analysis of time series comprises also a procedure for the detection of potential outliers and other deteriorations that might be promising for monitoring the production process. Outliers and other process deteriorations are adequately detected by this monitoring procedure. The conclusion is that on-line estimation of the actual individual response in milk yield and milking duration is possible following a Bayesian approach for time series using an adaptive dynamic model. Besides estimation of the actual response the Bayesian approach adequately detects process deteriorations. Therefore, adaptive dynamic models provide a useful tool for control and monitoring of the dairy production process.

In the past two decades genome sequencing has developed from a laborious and costly technology employed by large international consortia to a widely used, automated and affordable tool used worldwide by many individual research groups. Genome sequences of many food animals and crop plants have been deciphered and are being exploited for fundamental research and applied to improve their breeding programs. The developments in sequencing technologies have also impacted the associated bioinformatics strategies and tools, both those that are required for data processing, management, and quality control, and those used for interpretation of the data. This thesis focuses on the application of genome sequencing, assembly and annotation to two members of the Solanaceae family, tomato and potato. Potato is the economically most important species within the Solanaceae, and its tubers contribute to dietary intake of starch, protein, antioxidants, and vitamins. Tomato fruits are the second most consumed vegetable after potato, and are a globally important dietary source of lycopene, beta-carotene, vitamin C, and fiber. The chapters in this thesis document the generation, exploitation and interpretation of genomic sequence resources for these two species and shed light on the contents, structure and evolution of their genomes. Chapter 1introduces the concepts of genome sequencing, assembly and annotation, and explains the novel genome sequencing technologies that have been developed in the past decade. These so-called Next Generation Sequencing platforms display considerable variation in chemistry and workflow, and as a consequence the throughput and data quality differs by orders of magnitude between the platforms. The currently available sequencing platforms produce a vast variety of read lengths and facilitate the generation of paired sequences with an approximately fixed distance between them. The choice of sequencing chemistry and platform combined with the type of sequencing template demands specifically adapted bioinformatics for data processing and interpretation. Irrespective of the sequencing and assembly strategy that is chosen, the resulting genome sequence, often represented by a collection of long linear strings of nucleotides, is of limited interest by itself. Interpretation of the genome can only be achieved through sequence annotation – that is, identification and classification of all functional elements in a genome sequence. Once these elements have been annotated, sequence alignments between multiple genomes of related accessions or species can be utilized to reveal the genetic variation on both the nucleotide and the structural level that underlies the difference between these species or accessions. Chapter 2describes BlastIf, a novel software tool that exploits sequence similarity searches with BLAST to provide a straightforward annotation of long nucleotide sequences. Generally, two problems are associated with the alignment of a long nucleotide sequence to a database of short gene or protein sequences: (i) the large number of similar hits that can be generated due to database redundancy; and (ii) the relationships implied between aligned segments within a hit that in fact correspond to distinct elements on the sequence such as genes. BlastIf generates a comprehensible BLAST output for long nucleotide sequences by reducing the number of similar hits while revealing most of the variation present between hits. It is a valuable tool for molecular biologists who wish to get a quick overview of the genetic elements present in a newly sequenced segment of DNA, prior to more elaborate efforts of gene structure prediction and annotation. In Chapter 3 a first genome-wide comparison between the emerging genomic sequence resources of tomato and potato is presented. Large collections of BAC end sequences from both species were annotated through repeat searches, transcript alignments and protein domain identification. In-depth comparisons of the annotated sequences revealed remarkable differences in both gene and repeat content between these closely related genomes. The tomato genome was found to be more repetitive than the potato genome, and substantial differences in the distribution of Gypsy and Copia retrotransposable elements as well as microsatellites were observed between the two genomes. A higher gene content was identified in the potato sequences, and in particular several large gene families including cytochrome P450 mono-oxygenases and serine-threonine protein kinases were significantly overrepresented in potato compared to tomato. Moreover, the cytochrome P450 gene family was found to be expanded in both tomato and potato when compared to Arabidopsis thaliana, suggesting an expanded network of secondary metabolic pathways in the Solanaceae. Together these findings present a first glimpse into the evolution of Solanaceous genomes, both within the family and relative to other plant species. Chapter 4explores the physical and genetic organization of tomato chromosome 6 through integration of BAC sequence analysis, High Information Content Fingerprinting, genetic analysis, and BAC-FISH mapping data. A collection of BACs spanning substantial parts of the short and long arm euchromatin and several dispersed regions of the pericentrometric heterochromatin were sequenced and assembled into several tiling paths spanning approximately 11 Mb. Overall, the cytogenetic order of BACs was in agreement with the order of BACs anchored to the Tomato EXPEN 2000 genetic map, although a few striking discrepancies were observed. The integration of BAC-FISH, sequence and genetic mapping data furthermore provided a clear picture of the borders between eu- and heterochromatin on chromosome 6. Annotation of the BAC sequences revealed that, although the majority of protein-coding genes were located in the euchromatin, the highly repetitive pericentromeric heterochromatin displayed an unexpectedly high gene content. Moreover, the short arm euchromatin was relatively rich in repeats, but the ratio of Gypsy and Copia retrotransposons across the different domains of the chromosome clearly distinguished euchromatin from heterochromatin. The ongoing whole-genome sequencing effort will reveal if these properties are unique for tomato chromosome 6, or a more general property of the tomato genome. Chapter 5presents the potato genome, the first genome sequence of an Asterid. To overcome the problems associated with genome assembly due tothe high level of heterozygosity that is observed in commercial tetraploid potato varieties, a homozygous doubled-monoploid potato clone was exploited to sequence and assemble 86% of the 844 Mb genome. This potato reference genome sequence was complemented with re-sequencing of aheterozygous diploid clone, revealing the form and extent of sequence polymorphism both between different genotypes and within a single heterozygous genotype. Gene presence/absence variants and other potentially deleterious mutations were found to occur frequently in potato and are a likely cause of inbreeding depression. Annotation of the genome was supported by deep transcriptome sequencing of both the doubled-monoploid and the heterozygous potato, resulting in the prediction of more than 39,000 protein coding genes. Transcriptome analysis provided evidence for the contribution of gene family expansion, tissue specific expression, and recruitment of genes to new pathways to the evolution of tuber development. The sequence of the potato genome has provided new insights into Eudicot genome evolution and has provided a solid basis for the elucidation of the evolution of tuberisation. Many traits of interest to plant breeders are quantitative in nature and the potato sequence will simplify both their characterization and deployment to generate novel cultivars. The outstanding challenges in plant genome sequencing are addressed in Chapter 6. The high concentration of repetitive elements and the heterozygosity and polyploidy of many interesting crop plant species currently pose a barrier for the efficient reconstruction of their genome sequences. Nonetheless, the completion of a large number of new genome sequences in recent years and the ongoing advances in sequencing technology provide many excitingopportunities for plant breeding and genome research. Current sequencing platforms are being continuously updated and improved, and novel technologies are being developed and implemented in third-generation sequencing platforms that sequence individual molecules without need for amplification. While these technologies create exciting opportunities for new sequencing applications, they also require robust software tools to process the data produced through them efficiently. The ever increasing amount of available genome sequences creates the need for an intuitive platform for the automated and reproducible interrogation of these data in order to formulate new biologically relevant questions on datasets spanning hundreds or thousands of genome sequences.

Individual variation in milk yield response to concentrate intake and milking interval length During the last century in the Netherlands milk production per cow has almost tripled. Accordingly, the amount of concentrates yearly fed per cow strongly increased. Furthermore, automation and robotisation has changed dairy management, especially by the introduction of automatic concentrate feeders and milking systems. A new management concept, emerging in the last decades, is Precision Livestock Farming (PLF). The objective of PLF is to optimize livestock production, by on-line monitoring and control of the production process, utilizing the technical possibilities of automation and robotisation. Nowadays, individual settings for daily concentrate supply and milking frequency are based on standards, ignoring individual variation in milk yield response on concentrate intake and milking frequency. This leads to the main hypothesis for this thesis research that profitability of dairy farming can be improved by utilizing information on individual variation in response. The first objective of this research was to quantify the individual variation in milk yield response to concentrate intake and milking interval length, in order to assess the economic prospects of applying individual optimal settings for concentrate supply and milking frequency. In the first observational study (Ch. 2), data from 299 cows on four farms in the first 3 weeks of the lactation were collected. Individual response in daily milk yield to concentrate intake was analysed by a random coefficient model. During the first three weeks of lactation, considerable variation in individual milk yield response to concentrate intake was found on all four farms. An economic simulation was carried out, based on the estimated parameter values in the observational study. Individual economically optimized settings for concentrate supply were compared with conventional strategies for concentrate supply based on averaged population response parameters. Applying individual economic optimal settings for concentrate supply during early lactation, potential economic gain ranges from 0.20 to 2.03 €/cow/day. In a second observational study (Ch.3), data of normal uninterrupted milkings during one week from 311 cows kept in 5 separate herds on one farm were collected. The data set consisted of 4,915 records and random coefficient models were fitted to estimate the individual effects of milking interval on daily milk yield and milking duration. Between individuals, considerable variation in milk yield and milking duration was found in response to milking interval. Based on the estimated individual response, a simulation was carried out in order to optimize the utilization of an AMS for different herd sizes and occupation rates. Applying optimal individual milking intervals for a herd of 60 cows and an AMS operating at an occupation rate of 64%, the average milking interval reduced from 0.421 day to 0.400 day, the daily milk yield at the herd level increased from 1,883 to 1,909 kg/day, and milk revenues increased from 498 to 507 €/day. In the actual situation, the herd consisted of 60 cows. A further increase of daily milk revenues per AMS was possible by increasing the operation rate and/or herd size. The conclusion is that between dairy cows there is a considerable variation in effects of concentrate intake and milking interval length on milk yield and, consequently, milking duration. A marked increase in economic profits of dairy production is possible by improvement of the concentrate allocation and/or the utilisation of an AMS, applying optimal individual settings based on the actual individual response in milk yield. Development of adaptive models The second objective was the development and testing of adaptive models for on-line estimation of the actual individual response in milk yield to concentrate intake and milking interval length. In Ch. 4 adaptive dynamic models for on-line estimation of the actual individual milk yield response to concentrate intake and milking interval length were evaluated. The parameters in these models may change over time and are updated through a Bayesian approach for on-line analysis of time series. Time series data of daily milk yield during the first 200 days of lactation from 17 cows were analysed with different adaptive dynamic models. Three models were evaluated: a model with linear terms for concentrate intake and length of milking interval, a model with linear and quadratic terms, and an enhanced model in order to obtain more stable parameter estimates. The linear model was only useful for forecasting milk production and the estimated parameters of the quadratic model turned out to be unstable. The parsimony of the enhanced model lead to far more stable parameter estimates. In Ch. 5 an adaptive dynamic model was used for time series analysis of herd mean daily milk yield, in order to quantify the impact of heat stress and to assess the potential for monitoring and control of milk production. Time series data of daily milk yield from 2003 to 2006 were collected on six experimental research farms in The Netherlands. The impact of heat stress was quantified in terms of critical temperature, duration and loss in milk yield. The estimated critical temperature was 17.8 oC, the duration was 5.5 days, and loss in milk yield 31.4 kg milk/cow/year, averaged over farms. Besides estimation of the impact of heat stress, level and trend, including a weekly cyclical pattern were estimated to evaluate the production process. The Bayesian approach for on-line analysis of time series comprises also a procedure for the detection of potential outliers and other deteriorations that might be promising for monitoring the production process. Outliers and other process deteriorations are adequately detected by this monitoring procedure. The conclusion is that on-line estimation of the actual individual response in milk yield and milking duration is possible following a Bayesian approach for time series using an adaptive dynamic model. Besides estimation of the actual response the Bayesian approach adequately detects process deteriorations. Therefore, adaptive dynamic models provide a useful tool for control and monitoring of the dairy production process.

Plants are not able to move or escape and have to confront environmental challenges like nutrient and water deprivation, low and high temperatures, and biotic stress imposed by pathogens like viruses, bacteria, fungi, nematodes and insects that all compete for plant nutrient sources. The outcome of a plant-pathogen interaction can vary from mild symptoms that are hardly harmful to the host to complete destruction of the host plant. Plants have evolved various mechanisms to counter-attack infections by pathogens. Mechanisms of evasion or suppression of basal host defense by pathogens on the one hand, and specific recognition of a pathogen by its host and activation of downstream defense signaling on the other hand, are complex and both organisms have to come up with sophisticated strategies to survive their encounters. In principle these encounters have two possible outcomes: (i) a pathogen successfully infects the host plant, which is also referred to as a compatible interaction (the pathogen is virulent and the host plant is susceptible), or (ii) the pathogen cannot successfully infect the host plant which stays healthy, also referred to as an incompatible interaction (the pathogen is avirulent and the host plant is resistant). Nearly 70 years ago, Harold Flor (1942) studied the genetics of the interaction between the flax rust fungus Melampsora lini and flax, Linum usitatissimum. Based on these studies he postulated the so-called gene-for-gene hypothesis (Flor 1942) which states that for each dominant resistance (R) gene in the host there is a matching dominant avirulence (Avr) gene in the pathogen. Co-occurrence and expression of both genes leads to an incompatible interaction that is often associated with a hypersensitive response (HR). The interaction between the fungus Cladosporium fulvum (syn. Passalora fulva) and the host tomato (Solanum lycopersicum) is an excellent model to study plant-pathogen interactions and obeys to the gene-for-gene hypothesis. C. fulvum is a biotrophic pathogen that causes leaf mold of tomato, avoids breaching the cell wall and exclusively colonizes the tomato leaf apoplast while establishing a long-term feeding relationship with the living cells of the host. During the infection process, the fungus secretes several effector molecules, relatively small, cysteine–rich proteins. They are likely to contribute to pathogen fitness and play a role in pathogen virulence. According to the 'Zig-Zag' model that explains the evolutionary development of plant-pathogen interactions, effectors are required for ETS (effector -triggered susceptibility). Tomato plants that carry cognate Cf resistance genes recognize the effector and elicit a defense response known as the hypersensitive response (HR), nowadays known as effector-triggered immunity (ETI). In this thesis I have focused on several molecular and biochemical aspects of the Avr2 and Cf-2 gene pair and on an additional gene, Rcr3 (required for Cladosporium resistance), that is required for Cf-2-mediated resistance with an emphasis on the role of Avr2 in ETS and ETI in the C. fulvum-tomato interaction. The gene-for-gene hypothesis postulated by Harrold Flor has inspired many plant pathologists and initiated numerous plant-pathogen studies as discussed in chapter 1. This hypothesis has lead to the characterization of various host plant R genes and cognate pathogen Avr genes from fungi, bacteria and oomycetes. Plant resistance proteins are the basic molecules that mediate a defense reaction, triggered by cognate effectors directed against the pathogens, are found extracellularly as well as intracellularly and are divided in classes based on the composition of different subdomains that may have various functions. Particularly the LRR domain(s) are involved in recognition, regulating protein activation and signal transduction and are highly adjustable in diverse binding specificities to self and non-self molecules. The nucleotide binding (NB) domain acts a switch for activation of downstream host defenses, often resulting in HR. Inappropriate R protein folding and activation is controlled by intramolecular interactions between the various domains and by hetero-multimeric protein complexes. Studies on interactions of plants, especially Arabidopsis thaliana, with prokaryotic pathogens have resulted in major scientific breakthroughs with respect to the gene-for-gene hypothesis. Research on the bacterial Type Three Secretion System and the delivery of the effectors has indentified sophisticated mechanisms for perception and recognition of pathogens and regulation of host resistance. The functions of effectors of eukaryotic plant pathogens remain largely unknown so far. Oomycete pathogens such as Phytophthora infestans produce various types of effectors during infection of their hosts. One class of oomycete effectors localizes to, and operates in, the extracellular matrix while the other class acts inside the host plant cell. Recent studies on the interaction of the flax rust fungus Melampsora lini with flax (Linum usitatissimum) has revealed a number of direct Avr-R protein interactions in vitro. These interactions are expected to occur in the haustorial matrix which is produced by the fungus during host infection. Secreted Avr proteins of C. fulvum interact exclusively with the corresponding extracellular Cf proteins of tomato. The C. fulvum-tomato pathosystem is one of the most well-studied plant pathogen interactions and revealed important insights in perception and recognition of Avr proteins. For many years it was assumed that the interactions beween C. fulvum Avrs and tomato R proteins occurred in a direct manner, but proof for such interactions has never been obtained. Indirect interactions were more likely and obeyed to the guard hypothesis wherein the Avr protein interacts with a host target and this interaction is monitored, or guarded, by the Cf- protein. Chapter 2 reports on the avirulence function of Avr2 in the Cf-2-mediated resistance that also requires the extracellular tomato cysteine protease Rcr3. The interaction between Avr2, Cf-2 and Rcr3 obeys to the guard hypothesis. Purified heterologously expressed and affinity-tagged Rcr3 and Avr2 were applied in co-immunoprecipitation assays and revealed a physical interaction between Avr2 and Rcr3 independent of additional plant and or fungal factors. It is shown that Avr2 binds and inhibits Rcr3, and blocking of the active site of Rcr3 by the irreversible cysteine protease inhibitor E-64 eliminates this interaction. The interaction with and the inhibition of Rcr3 by Avr2 occurs in a pH-dependent fashion and the pH optimum for Rcr3 activity and its inhibition by Avr2 coincides with the pH of the tomato apoplast. Cysteine protease activity profiling showed that, in addition to Rcr3, Avr2 inhibits several other apoplastic cysteine proteases in tomato, but this inhibition did not lead to Cf-2-mediated HR. Infiltration of purified active Rcr3, or E-64-inactivated Rcr3, in combination with Avr2 in Cf-2/rcr3 tomato leaves revealed that only the Avr2-Rcr3 inhibition complex triggers Cf-2-dependent HR. It is proposed that Avr2 modifies Rcr3 which is recognized by Cf-2 and initiates the HR. This study represents the first indirect fungus-plant gene-for-gene interaction that obeys to the guard hypothesis. In chapter 3 the focus is on the virulence function of Avr2, and it is demonstrated that Avr2 has an indisputable intrinsic biological virulence function for C. fulvum during infection of tomato. Silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. Heterologous expression of Avr2 in tomato resulted in enhanced susceptibility towards natural Avr2-defective C.fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. In A. thaliana, Avr2 expression resulted in enhanced susceptibility to various extracellular fungal pathogens including Botrytis cinerea and Verticillium dahliae. Microarray analysis of unchallenged A. thaliana plants showed that Avr2 expression induced a global transcription profile that is comparable to the profile upon pathogen challenge. Cysteine protease activity profiling and LC-MS/MS analyses showed that Avr2 inhibits multiple extracellular A. thaliana cysteine proteases. Similar results were obtained for tomato, showing that Avr2 inhibits multiple cysteine proteases including Rcr3 and its close relative Pip1. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases that are required for basal host defense. In chapter 4 the emphasis is on Avr2 protein features and the mode of inhibition of Rcr3. Like many other Avr genes, Avr2 lacks homology with sequences deposited in public databases. The mature Avr2 protein contains 8 cysteine residues and biochemical analyses revealed that all of these are involved in disulphide bridging, showing a unique disulphide bridge pattern. Based on a bioinformatics analysis, site-specific mutations were made in the Avr2 protein and affinity-tagged wild-type and mutant proteins were produced by heterologous expression in the yeast Pichia pastoris. After affinity purification, all proteins were infiltrated in tomato Cf-2 plants, and proteins with altered HR inducing activity were tested for their ability to inhibit Rcr3. From these assays it became evident that especially the C-terminal six amino acids that also include one disulphide bridge are essential for the interaction with and inhibition of Rcr3. All these data show that Avr2 is a novel type of cysteine protease inhibitor. Chapter 5 is a general discussion about the role of plant cysteine proteases and cysteine protease inhibitors in plant-pathogen interactions. Microbial pathogens and host plants both employ cysteine proteases and cysteine protease inhibitors as weapons for attack and defence. This so-called arms race has led to multiple attacks and counter-attacks that have shaped co-evolution between pathogens and their host plants. Examples of some prokaryotic plant pathogens that employ cysteine proteases as effector proteins to suppress plant defense will be discussed, in addition to some eukaryotic pathogens that use cysteine protease inhibitors for the same purpose. Examples of plant cysteine proteases will be discussed that are involved in multiple processes including plant development, plant defense and processes in programmed cell death.

Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management. Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature. LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora. In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments. This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object.

Onlangs is de DNA-sequentie van het genoom van Cladosporium fulvum bepaald. Het voornaamste doel daarvan is de identificatie en karakterisering van nieuwe effectors.

Potato is consumed worldwide and represents the fourth most important staple food crop after rice and wheat. Potato cultivars display a large variety of color, shape, taste, cooking properties and starch content but are all derived from the same species; Solanum tuberosum. Potato breeding is an economic important activity for international breeding companies, but also plays an important role in breaking the circle of poverty for small farmers. In the Andean region, most farmers use many different potato genotypes combined with farming practices transmitted orally over thousands of years. The most prominent menace to potato production is Late Blight caused by the oomycete Phytophthora infestans which destroys leaves, stems and tubers. Differences of breeding methods between the potato grown in South America and in the rest of the world is related to differences in the consequences of Late Blight infection. In the 19th, century, entire potato fields in Ireland were devastated while in South America P. infestans proliferation was readily inhibited. This difference is found in the biodiversity reserve such as that of the Chiloé archipelago in Chile where local people cultivate about 200 varieties of native potato. Obviously, the genetic diversity of cultivated native potato acts as a shield against this versatile pathogen. Inspired by this model to solve the problems raised by the extensive use of potato monoculture, growers and breeders need to maintain genetic diversity in the European staple food crops. In exploring the South American native potato collection, Solanum demissum and later on Solanum bulbocastanum appeared to be a source of resistance genes (Rpi) to P. infestans. The S. demissum Rpi genes were transmitted to potato breeding clones by traditional introgression breeding. However the fading of their ability in providing effective resistance against Late Blight infection was witnessed within a decade. In the pursuit to provide a hopefully more durable protection in existing potato cultivars, plant breeding scientists proposed to directly introduce South American native potato Rpi genes in modern potato varieties by using a so-called cisgenic approach. This in contrast to transgenic plants which can contain genes which have originated from non related genera or even different kingdoms. Breeding of cisgenic plants is on its way to public acceptance because of its inherent resemblance to natural crossing and because efforts are made by the scientific community to explain the principles of cisgenesis. Lessons were learned from the flexibility of P. infestans to overcome the effect of newly introduced Rpi genes and, therefore, efforts are still ongoing to discover and clone new Rpi genes from native potatoes. With this in mind, a new family of Rpi genes represented by Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 were characterized in clones derived from S. bulbocastanum, S. demissum, S. edinense and S. microdontum. We accomplished in this research the physical isolation of these genes, the molecular characterization of their functionality and the allelic distribution in the Petota collection. Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 belong to the potato linkage group IV and all contain signature sequences characteristic of LZ-NBS-LRR proteins. The closest known R gene so far is RPP13 from Arabidopsis thaliana which shares an amino-acid sequence similarity of 35%. The LRR domains of Rpi-blb3, Rpi-abpt, R2 and R2-like proteins are highly homologous, whilst LZ and NBS domains are more polymorphic with those of R2 being the most divergent. All four Rpi genes recognize the recently identified RXLR effector protein PiAVR2 which is secreted by P.infestans in the cytoplasm of plant cells during the infection process. Unlike Rpi-blb3, Rpi-abpt, R2 and R2-like , the S. microdontum resistance gene Rpi-mcd1.1 does not interact with PiAVR2 and provides a different resistance spectrum. Rpi-mcd1.1 shares 90% nucleotide identity with Rpi-blb3 and polymorphic nucleotides are mainly located in the LRR region. The S. bulbocastanum haplotypes of Rpi-blb1, Rpi-blb2 and Rpi-blb3 were discovered in several Mexican diploid as well as polyploid species closely related to S. bulbocastanum. These three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their distribution is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2 and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2 and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach some accessions could be identified that contain novel, yet unknown, Late Blight resistance factors in addition to the Rpi-blb1, Rpi-blb2 and Rpi-blb3 genes Analysis of the sequences obtained in different allele mining strategies suggests an evolution of the major late blight locus on linkage group IV through recombination and point mutations. By making use of the sequence information provided by the alleles, we identified the repeats and amino acids in the LRR domain which are specific for PiAVR2 recognition. Finally, we discussed the results described in this thesis in a potato/ P. infestans co-evolution context.

Een tomaat die niet meer vatbaar is voor meeldauw, bij welke ziektedruk dan ook. Het kan! Wageningse onderzoekers zijn daarin geslaagd door een bepaald gen stil te leggen. Dat gen ‘helpt’ de schimmel en na stilleggen is die zijn hulpje kwijt. Genen uitzetten is een alternatief voor resistentieveredeling. Het kan bij alle tuinbouwgewassen een oplossing bieden voor ziekteproblemen. Daarvoor moet wel de Europese wetgeving veranderen, maar daar zit beweging in.

Een tomaat die niet meer vatbaar is voor meeldauw, bij welke ziektedruk dan ook. Het kan! Wageningse onderzoekers zijn daarin geslaagd door een bepaald gen stil te leggen. Dat gen ‘helpt’ de schimmel en na stilleggen is die zijn hulpje kwijt. Genen uitzetten is een alternatief voor resistentieveredeling. Het kan bij alle tuinbouwgewassen een oplossing bieden voor ziekteproblemen. Daarvoor moet wel de Europese wetgeving veranderen, maar daar zit beweging in.

Plants are not able to move or escape and have to confront environmental challenges like nutrient and water deprivation, low and high temperatures, and biotic stress imposed by pathogens like viruses, bacteria, fungi, nematodes and insects that all compete for plant nutrient sources. The outcome of a plant-pathogen interaction can vary from mild symptoms that are hardly harmful to the host to complete destruction of the host plant. Plants have evolved various mechanisms to counter-attack infections by pathogens. Mechanisms of evasion or suppression of basal host defense by pathogens on the one hand, and specific recognition of a pathogen by its host and activation of downstream defense signaling on the other hand, are complex and both organisms have to come up with sophisticated strategies to survive their encounters. In principle these encounters have two possible outcomes: (i) a pathogen successfully infects the host plant, which is also referred to as a compatible interaction (the pathogen is virulent and the host plant is susceptible), or (ii) the pathogen cannot successfully infect the host plant which stays healthy, also referred to as an incompatible interaction (the pathogen is avirulent and the host plant is resistant). Nearly 70 years ago, Harold Flor (1942) studied the genetics of the interaction between the flax rust fungus Melampsora lini and flax, Linum usitatissimum. Based on these studies he postulated the so-called gene-for-gene hypothesis (Flor 1942) which states that for each dominant resistance (R) gene in the host there is a matching dominant avirulence (Avr) gene in the pathogen. Co-occurrence and expression of both genes leads to an incompatible interaction that is often associated with a hypersensitive response (HR). The interaction between the fungus Cladosporium fulvum (syn. Passalora fulva) and the host tomato (Solanum lycopersicum) is an excellent model to study plant-pathogen interactions and obeys to the gene-for-gene hypothesis. C. fulvum is a biotrophic pathogen that causes leaf mold of tomato, avoids breaching the cell wall and exclusively colonizes the tomato leaf apoplast while establishing a long-term feeding relationship with the living cells of the host. During the infection process, the fungus secretes several effector molecules, relatively small, cysteine–rich proteins. They are likely to contribute to pathogen fitness and play a role in pathogen virulence. According to the 'Zig-Zag' model that explains the evolutionary development of plant-pathogen interactions, effectors are required for ETS (effector -triggered susceptibility). Tomato plants that carry cognate Cf resistance genes recognize the effector and elicit a defense response known as the hypersensitive response (HR), nowadays known as effector-triggered immunity (ETI). In this thesis I have focused on several molecular and biochemical aspects of the Avr2 and Cf-2 gene pair and on an additional gene, Rcr3 (required for Cladosporium resistance), that is required for Cf-2-mediated resistance with an emphasis on the role of Avr2 in ETS and ETI in the C. fulvum-tomato interaction. The gene-for-gene hypothesis postulated by Harrold Flor has inspired many plant pathologists and initiated numerous plant-pathogen studies as discussed in chapter 1. This hypothesis has lead to the characterization of various host plant R genes and cognate pathogen Avr genes from fungi, bacteria and oomycetes. Plant resistance proteins are the basic molecules that mediate a defense reaction, triggered by cognate effectors directed against the pathogens, are found extracellularly as well as intracellularly and are divided in classes based on the composition of different subdomains that may have various functions. Particularly the LRR domain(s) are involved in recognition, regulating protein activation and signal transduction and are highly adjustable in diverse binding specificities to self and non-self molecules. The nucleotide binding (NB) domain acts a switch for activation of downstream host defenses, often resulting in HR. Inappropriate R protein folding and activation is controlled by intramolecular interactions between the various domains and by hetero-multimeric protein complexes. Studies on interactions of plants, especially Arabidopsis thaliana, with prokaryotic pathogens have resulted in major scientific breakthroughs with respect to the gene-for-gene hypothesis. Research on the bacterial Type Three Secretion System and the delivery of the effectors has indentified sophisticated mechanisms for perception and recognition of pathogens and regulation of host resistance. The functions of effectors of eukaryotic plant pathogens remain largely unknown so far. Oomycete pathogens such as Phytophthora infestans produce various types of effectors during infection of their hosts. One class of oomycete effectors localizes to, and operates in, the extracellular matrix while the other class acts inside the host plant cell. Recent studies on the interaction of the flax rust fungus Melampsora lini with flax (Linum usitatissimum) has revealed a number of direct Avr-R protein interactions in vitro. These interactions are expected to occur in the haustorial matrix which is produced by the fungus during host infection. Secreted Avr proteins of C. fulvum interact exclusively with the corresponding extracellular Cf proteins of tomato. The C. fulvum-tomato pathosystem is one of the most well-studied plant pathogen interactions and revealed important insights in perception and recognition of Avr proteins. For many years it was assumed that the interactions beween C. fulvum Avrs and tomato R proteins occurred in a direct manner, but proof for such interactions has never been obtained. Indirect interactions were more likely and obeyed to the guard hypothesis wherein the Avr protein interacts with a host target and this interaction is monitored, or guarded, by the Cf- protein. Chapter 2 reports on the avirulence function of Avr2 in the Cf-2-mediated resistance that also requires the extracellular tomato cysteine protease Rcr3. The interaction between Avr2, Cf-2 and Rcr3 obeys to the guard hypothesis. Purified heterologously expressed and affinity-tagged Rcr3 and Avr2 were applied in co-immunoprecipitation assays and revealed a physical interaction between Avr2 and Rcr3 independent of additional plant and or fungal factors. It is shown that Avr2 binds and inhibits Rcr3, and blocking of the active site of Rcr3 by the irreversible cysteine protease inhibitor E-64 eliminates this interaction. The interaction with and the inhibition of Rcr3 by Avr2 occurs in a pH-dependent fashion and the pH optimum for Rcr3 activity and its inhibition by Avr2 coincides with the pH of the tomato apoplast. Cysteine protease activity profiling showed that, in addition to Rcr3, Avr2 inhibits several other apoplastic cysteine proteases in tomato, but this inhibition did not lead to Cf-2-mediated HR. Infiltration of purified active Rcr3, or E-64-inactivated Rcr3, in combination with Avr2 in Cf-2/rcr3 tomato leaves revealed that only the Avr2-Rcr3 inhibition complex triggers Cf-2-dependent HR. It is proposed that Avr2 modifies Rcr3 which is recognized by Cf-2 and initiates the HR. This study represents the first indirect fungus-plant gene-for-gene interaction that obeys to the guard hypothesis. In chapter 3 the focus is on the virulence function of Avr2, and it is demonstrated that Avr2 has an indisputable intrinsic biological virulence function for C. fulvum during infection of tomato. Silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. Heterologous expression of Avr2 in tomato resulted in enhanced susceptibility towards natural Avr2-defective C.fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. In A. thaliana, Avr2 expression resulted in enhanced susceptibility to various extracellular fungal pathogens including Botrytis cinerea and Verticillium dahliae. Microarray analysis of unchallenged A. thaliana plants showed that Avr2 expression induced a global transcription profile that is comparable to the profile upon pathogen challenge. Cysteine protease activity profiling and LC-MS/MS analyses showed that Avr2 inhibits multiple extracellular A. thaliana cysteine proteases. Similar results were obtained for tomato, showing that Avr2 inhibits multiple cysteine proteases including Rcr3 and its close relative Pip1. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases that are required for basal host defense. In chapter 4 the emphasis is on Avr2 protein features and the mode of inhibition of Rcr3. Like many other Avr genes, Avr2 lacks homology with sequences deposited in public databases. The mature Avr2 protein contains 8 cysteine residues and biochemical analyses revealed that all of these are involved in disulphide bridging, showing a unique disulphide bridge pattern. Based on a bioinformatics analysis, site-specific mutations were made in the Avr2 protein and affinity-tagged wild-type and mutant proteins were produced by heterologous expression in the yeast Pichia pastoris. After affinity purification, all proteins were infiltrated in tomato Cf-2 plants, and proteins with altered HR inducing activity were tested for their ability to inhibit Rcr3. From these assays it became evident that especially the C-terminal six amino acids that also include one disulphide bridge are essential for the interaction with and inhibition of Rcr3. All these data show that Avr2 is a novel type of cysteine protease inhibitor. Chapter 5 is a general discussion about the role of plant cysteine proteases and cysteine protease inhibitors in plant-pathogen interactions. Microbial pathogens and host plants both employ cysteine proteases and cysteine protease inhibitors as weapons for attack and defence. This so-called arms race has led to multiple attacks and counter-attacks that have shaped co-evolution between pathogens and their host plants. Examples of some prokaryotic plant pathogens that employ cysteine proteases as effector proteins to suppress plant defense will be discussed, in addition to some eukaryotic pathogens that use cysteine protease inhibitors for the same purpose. Examples of plant cysteine proteases will be discussed that are involved in multiple processes including plant development, plant defense and processes in programmed cell death.

Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management. Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature. LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora. In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments. This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object.

Potato is consumed worldwide and represents the fourth most important staple food crop after rice and wheat. Potato cultivars display a large variety of color, shape, taste, cooking properties and starch content but are all derived from the same species; Solanum tuberosum. Potato breeding is an economic important activity for international breeding companies, but also plays an important role in breaking the circle of poverty for small farmers. In the Andean region, most farmers use many different potato genotypes combined with farming practices transmitted orally over thousands of years. The most prominent menace to potato production is Late Blight caused by the oomycete Phytophthora infestans which destroys leaves, stems and tubers. Differences of breeding methods between the potato grown in South America and in the rest of the world is related to differences in the consequences of Late Blight infection. In the 19th, century, entire potato fields in Ireland were devastated while in South America P. infestans proliferation was readily inhibited. This difference is found in the biodiversity reserve such as that of the Chiloé archipelago in Chile where local people cultivate about 200 varieties of native potato. Obviously, the genetic diversity of cultivated native potato acts as a shield against this versatile pathogen. Inspired by this model to solve the problems raised by the extensive use of potato monoculture, growers and breeders need to maintain genetic diversity in the European staple food crops. In exploring the South American native potato collection, Solanum demissum and later on Solanum bulbocastanum appeared to be a source of resistance genes (Rpi) to P. infestans. The S. demissum Rpi genes were transmitted to potato breeding clones by traditional introgression breeding. However the fading of their ability in providing effective resistance against Late Blight infection was witnessed within a decade. In the pursuit to provide a hopefully more durable protection in existing potato cultivars, plant breeding scientists proposed to directly introduce South American native potato Rpi genes in modern potato varieties by using a so-called cisgenic approach. This in contrast to transgenic plants which can contain genes which have originated from non related genera or even different kingdoms. Breeding of cisgenic plants is on its way to public acceptance because of its inherent resemblance to natural crossing and because efforts are made by the scientific community to explain the principles of cisgenesis. Lessons were learned from the flexibility of P. infestans to overcome the effect of newly introduced Rpi genes and, therefore, efforts are still ongoing to discover and clone new Rpi genes from native potatoes. With this in mind, a new family of Rpi genes represented by Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 were characterized in clones derived from S. bulbocastanum, S. demissum, S. edinense and S. microdontum. We accomplished in this research the physical isolation of these genes, the molecular characterization of their functionality and the allelic distribution in the Petota collection. Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 belong to the potato linkage group IV and all contain signature sequences characteristic of LZ-NBS-LRR proteins. The closest known R gene so far is RPP13 from Arabidopsis thaliana which shares an amino-acid sequence similarity of 35%. The LRR domains of Rpi-blb3, Rpi-abpt, R2 and R2-like proteins are highly homologous, whilst LZ and NBS domains are more polymorphic with those of R2 being the most divergent. All four Rpi genes recognize the recently identified RXLR effector protein PiAVR2 which is secreted by P.infestans in the cytoplasm of plant cells during the infection process. Unlike Rpi-blb3, Rpi-abpt, R2 and R2-like , the S. microdontum resistance gene Rpi-mcd1.1 does not interact with PiAVR2 and provides a different resistance spectrum. Rpi-mcd1.1 shares 90% nucleotide identity with Rpi-blb3 and polymorphic nucleotides are mainly located in the LRR region. The S. bulbocastanum haplotypes of Rpi-blb1, Rpi-blb2 and Rpi-blb3 were discovered in several Mexican diploid as well as polyploid species closely related to S. bulbocastanum. These three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their distribution is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2 and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2 and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach some accessions could be identified that contain novel, yet unknown, Late Blight resistance factors in addition to the Rpi-blb1, Rpi-blb2 and Rpi-blb3 genes Analysis of the sequences obtained in different allele mining strategies suggests an evolution of the major late blight locus on linkage group IV through recombination and point mutations. By making use of the sequence information provided by the alleles, we identified the repeats and amino acids in the LRR domain which are specific for PiAVR2 recognition. Finally, we discussed the results described in this thesis in a potato/ P. infestans co-evolution context.

Changes in the genotype influence changes in morphology during evolution, giving rise to the vast diversity of morphological features that we observe. The ability to describe how genetic change causes morphological transformation is key for a mechanistic understanding of evolutionary change. This thesis examines morphological and genetic aspects of the origin of the amnioserosa, a unique tissue of higher flies (Cyclorrhapha), which combines different aspects of the amnion and the serosa, two protective extraembryonic epithelia commonly found in lower flies and other insects.Typically, extraembryonic epithelia develop from an amnio-serosal fold, which closes about the ventral side of the gastrulating embryo. The outer cell layer of the amnio-serosal fold becomes the serosa. This epithelium disjoins from the amnion and encloses the embryo. The inner cell layer of the amnio-serosal fold becomes the amnion and retains continuity with the dorsal epidermis of the embryo. Higher flies of the taxon Schizophora differ: they develop a single extraembryonic epithelium on the dorsal side called the amnioserosa. Here, a third type is reported in close relatives of Schizophora (lower Cyclorrhapha), in which the amnion is dorsal and the serosa complete. Based on the occurrence of these three extraembryonic tissue organizations in flies two major morphological transitions in the organization of the extraembryonic epithelia of flies are proposed, one that occurred before the cretaceous radiation of flies some 150 million years ago in which the amnion got relocated from the ventral to the dorsal side of the embryo, and another that occurred before the tertiary radiation of flies some 100 million years ago in which the dorsal amnion and the serosa combined into a single epithelium, the amnioserosa.The genetic analysis focuses on the homeobox gene zerknüllt (zen). Most insects express zen in their extraembryonic tissues. In the milkweed bug Oncopeltus, zen controls the fusion of the amnion with the serosa. In the flour beetle Tribolium, zen controls not only the fusion of the amnion with the serosa but also the distinction of serosal from amniotic cells. In the fruitfly Drosophila, zen controls the distinction of amnioserosal from embryonic cells. Here, it is shown that in lower cyclorrhaphan flies, zen controls serosal cell-fate, like in Tribolium. Furthermore, spatial and temporal differences in the extraembryonic expression of zen are reported between Drosophila and lower cyclorrhaphan flies. Extraembryonic zen expression in Drosophila abuts the posterior and lateral germ rudiment, unlike in lower cyclorrhaphans in which the prospective amnion is interspersed. Ectopic expression of zen in Megaselia through injection of capped mRNA had no effect on development, suggesting that spatial expression difference may not explain the morphological reorganization into an amnioserosa-like tissue in evolution. Temporally, extraembryonic zen expression in Drosophila is downregulated during the late phase of germband extension, unlike in lower cyclorrhaphans in which it persists. A new transfection-based technique for delivery of double-stranded RNA to embryonic cells was developed and used to mimic the time course of zen expression of Drosophila in Megaselia. This procedure was sufficient to create an amnioserosa-like tissue in Megaselia. In addition, differences in extraembryonic zen expression were observed between lower cyclorrhaphan species. In one species (Megaselia) it was restricted to dorsal balstoderm, like in Drosophila. In another species (Episyrphus), it extended to the anterior tip of the blastoderm egg. This difference cannot explain the origin of the amnioserosa but may reflect differences in embryonic pattern formation through bicoid, which determines the position of the head by means of localized mRNA. To test this prediction, ca. 60 kb upstream of zen (spanning the expected genomic position of bicoid) were sequenced in Episyrphus. This region did not contain a bicoid homologue.The research described in this thesis demonstrates that temporal change in the expression pattern of a single gene can explain the origin of the amnioserosa. This result shows that even at a macroevolutionary scale, i.e. over large phylogenetic distances, evo-devo studies can provide insight into the causal relationship between gene expression and morphology. In addition, the unique zen expression in Episyrphus and zen locus data from this species point to a possible difference in embryonic patterning in this taxon.

Changes in the genotype influence changes in morphology during evolution, giving rise to the vast diversity of morphological features that we observe. The ability to describe how genetic change causes morphological transformation is key for a mechanistic understanding of evolutionary change. This thesis examines morphological and genetic aspects of the origin of the amnioserosa, a unique tissue of higher flies (Cyclorrhapha), which combines different aspects of the amnion and the serosa, two protective extraembryonic epithelia commonly found in lower flies and other insects.Typically, extraembryonic epithelia develop from an amnio-serosal fold, which closes about the ventral side of the gastrulating embryo. The outer cell layer of the amnio-serosal fold becomes the serosa. This epithelium disjoins from the amnion and encloses the embryo. The inner cell layer of the amnio-serosal fold becomes the amnion and retains continuity with the dorsal epidermis of the embryo. Higher flies of the taxon Schizophora differ: they develop a single extraembryonic epithelium on the dorsal side called the amnioserosa. Here, a third type is reported in close relatives of Schizophora (lower Cyclorrhapha), in which the amnion is dorsal and the serosa complete. Based on the occurrence of these three extraembryonic tissue organizations in flies two major morphological transitions in the organization of the extraembryonic epithelia of flies are proposed, one that occurred before the cretaceous radiation of flies some 150 million years ago in which the amnion got relocated from the ventral to the dorsal side of the embryo, and another that occurred before the tertiary radiation of flies some 100 million years ago in which the dorsal amnion and the serosa combined into a single epithelium, the amnioserosa.The genetic analysis focuses on the homeobox gene zerknüllt (zen). Most insects express zen in their extraembryonic tissues. In the milkweed bug Oncopeltus, zen controls the fusion of the amnion with the serosa. In the flour beetle Tribolium, zen controls not only the fusion of the amnion with the serosa but also the distinction of serosal from amniotic cells. In the fruitfly Drosophila, zen controls the distinction of amnioserosal from embryonic cells. Here, it is shown that in lower cyclorrhaphan flies, zen controls serosal cell-fate, like in Tribolium. Furthermore, spatial and temporal differences in the extraembryonic expression of zen are reported between Drosophila and lower cyclorrhaphan flies. Extraembryonic zen expression in Drosophila abuts the posterior and lateral germ rudiment, unlike in lower cyclorrhaphans in which the prospective amnion is interspersed. Ectopic expression of zen in Megaselia through injection of capped mRNA had no effect on development, suggesting that spatial expression difference may not explain the morphological reorganization into an amnioserosa-like tissue in evolution. Temporally, extraembryonic zen expression in Drosophila is downregulated during the late phase of germband extension, unlike in lower cyclorrhaphans in which it persists. A new transfection-based technique for delivery of double-stranded RNA to embryonic cells was developed and used to mimic the time course of zen expression of Drosophila in Megaselia. This procedure was sufficient to create an amnioserosa-like tissue in Megaselia. In addition, differences in extraembryonic zen expression were observed between lower cyclorrhaphan species. In one species (Megaselia) it was restricted to dorsal balstoderm, like in Drosophila. In another species (Episyrphus), it extended to the anterior tip of the blastoderm egg. This difference cannot explain the origin of the amnioserosa but may reflect differences in embryonic pattern formation through bicoid, which determines the position of the head by means of localized mRNA. To test this prediction, ca. 60 kb upstream of zen (spanning the expected genomic position of bicoid) were sequenced in Episyrphus. This region did not contain a bicoid homologue.The research described in this thesis demonstrates that temporal change in the expression pattern of a single gene can explain the origin of the amnioserosa. This result shows that even at a macroevolutionary scale, i.e. over large phylogenetic distances, evo-devo studies can provide insight into the causal relationship between gene expression and morphology. In addition, the unique zen expression in Episyrphus and zen locus data from this species point to a possible difference in embryonic patterning in this taxon.

Apple is generally considered as a healthy food, but 2-3% European people can not eat this fruit because it provokes allergy reaction. Four classes of apple allergen genes have been identified, they are Mal d 1, Mal d 2, Mal d 3 and Mal d 4 . This thesis focuses on the genomic characterization of these candidate genes on the apple genome by linkage mapping through sequence-specific DNA markers.In total, 26 putative Mal d genes, 25 of them were mapped on 8 linkage groups:17 for Mal d 1 , two for Mal d 2 and Mal d 3 , and four for Mal d 4 .As for the patients in northern and central Europe, the major apple allergen is Mal d 1. Genetic analysis demonstrated the Mal d 1 genes on linkage group (Chromosomes) 16 are relevant. Furthermore, a molecular marker linked to low-Mal d 1 allergenic trait was identified.

Apple is generally considered as a healthy food, but 2-3% European people can not eat this fruit because it provokes allergy reaction. Four classes of apple allergen genes have been identified, they are Mal d 1, Mal d 2, Mal d 3 and Mal d 4 . This thesis focuses on the genomic characterization of these candidate genes on the apple genome by linkage mapping through sequence-specific DNA markers.In total, 26 putative Mal d genes, 25 of them were mapped on 8 linkage groups:17 for Mal d 1 , two for Mal d 2 and Mal d 3 , and four for Mal d 4 .As for the patients in northern and central Europe, the major apple allergen is Mal d 1. Genetic analysis demonstrated the Mal d 1 genes on linkage group (Chromosomes) 16 are relevant. Furthermore, a molecular marker linked to low-Mal d 1 allergenic trait was identified.