Search

Chapter 1 of this thesis introduces the question of why some individuals possess higher cognitive abilities than others. The chapter discusses traditional strategies employed to study the neurobiology of intelligence: brain imaging to investigate brain structure and function and genome-wide association studies to identify genes associated with intelligence. However, there is little information on how neuronal properties relate to intelligence. We propose that single-cell transcriptomics combined with functional and morphological analysis of neurons in the human neocortex may provide an opportunity to understand how genes of intelligence can act on cortical structure and function contributing to human cognitive ability. In chapter 2 we investigated the association between human intelligence and the structural and physiological properties of neurons. The study utilized electrophysiological recordings of neurosurgically resected temporal cortex in combination with presurgical IQ scores. The results demonstrate that high IQ scores associate with larger temporal cortical thickness and larger, more complex dendrites of pyramidal neurons. In silico analysis reveals that these dendritic trees allow pyramidal neurons to track activity of synaptic inputs with higher temporal precision, due to fast action potential kinetics. The study concludes that human intelligence is associated with neuronal complexity, action potential kinetics, and efficient information transfer from inputs to output within cortical neurons. Chapter 3 investigates whether cortical laminar architecture and cellular properties of the left temporal lobe relate to verbal intelligence by extending our previous findings with histological data and more detailed analysis. The findings indicate that individuals with higher general and verbal IQ scores have thicker left temporal cortex due to the selective increase in layer 2 and 3 thickness, accompanied by lower neuron densities and larger dendrites and cell body size of pyramidal neurons in these layers. These neurons also maintain faster action potential kinetics under pressure, which improves information processing. The study concludes that verbal mental ability associates with selective adaptations of supragranular layers and their cellular micro-architecture and function in the left, but not right, temporal cortex. Chapter 4 explores the genetic basis of human intelligence and how it relates to human brain evolution. The study investigates whether genes associated with human cognition and human accelerated regions (HARs) implicated in human brain evolution are expressed in adult human cortical neurons and brain areas of cognition and how their expression relates to neuronal function and structure. The findings reveal that these gene sets are preferentially expressed in layer 3 excitatory neurons in the middle temporal gyrus (MTG). The study also identifies a subset of genes associated with dendritic length, with predominantly synaptic functions and high abundance of HARs. The results suggest that the mechanisms underlying human brain evolution and interindividual differences in intelligence might share genetic origin and manifest in specific neuronal types. In conclusion, this thesis provides novel insights into the neurobiological basis of intelligence. By investigating the cellular and molecular mechanisms underlying differences in cognitive ability, we have identified specific adaptations in neuronal structure and function that associate with higher IQ scores and verbal mental ability. Our findings suggest that genes implicated in human brain evolution and cognitive function may act on the same biological processes, manifesting in specific neuronal types. This work highlights the potential of integrating single-cell transcriptomics, functional and morphological analysis of neurons, and brain imaging to better understand the complex neurobiology of higher brain functions.

Well-being is a complex, and multifaceted construct that includes feeling good and functioning well. There is a growing global recognition of well-being as an important research topic and public policy goal. Well-being is related to less behavioral and emotional problems, and is associated with many positive aspects of daily life, including longevity, higher educational achievement, happier marriage, and more productivity at work. People differ in their levels of well-being, i.e., some people are in general happier or more satisfied with their lives than others. These individual differences in well-being can arise from many different factors, including biological (genetic) influences and environmental influences. To enhance the development of future mental health prevention and intervention strategies to increase well-being, more knowledge about these determinants and factors underlying well-being is needed. In this dissertation, I aimed to increase the understanding of the etiology in a series of studies using different methods, including systematic reviews, meta-analyses, twin designs, and molecular genetic designs. In part I, we brought together all published studies on the neural and physiological factors underlying well-being. This overview allowed us to critically investigate the claims made about the biology involved in well-being. The number of studies on the neural and physiological factors underlying well-being is increasing and the results point towards potential correlates of well-being. However, samples are often still small, and studies focus mostly on a single biomarker. Therefore, more well-powered, data-driven, and integrative studies across biological categories are needed to better understand the neural and physiological pathways that play a role in well-being. In part II, we investigated the overlap between well-being and a range of other phenotypes to learn more about the etiology of well-being. We report a large overlap with phenotypes including optimism, resilience, and depressive symptoms. Furthermore, when removing the genetic overlap between well-being and depressive symptoms, we showed that well-being has unique genetic associations with a range of phenotypes, independently from depressive symptoms. These results can be helpful in designing more effective interventions to increase well-being, taking into account the overlap and possible causality with other phenotypes. In part III, we used the extreme environmental change during the COVID-19 pandemic to investigate individual differences in the effects of such environmental changes on well-being. On average, we found a negative effect of the pandemic on different aspects of well-being, especially further into the pandemic. Whereas most previous studies only looked at this average negative effect of the pandemic on well-being, we focused on the individual differences as well. We reported large individual differences in the effects of the pandemic on well-being in both chapters. This indicates that one-size-fits-all preventions or interventions to maintain or increase well-being during the pandemic or lockdowns will not be successful for the whole population. Further research is needed for the identification of protective factors and resilience mechanisms to prevent further inequality during extreme environmental situations. In part IV, we looked at the real-time assessment of well-being, investigating the feasibility and results of previous studies. The real-time assessment of well-being, related variables, and the environment can lead to new insights about well-being, i.e., results that we cannot capture with traditional survey research. The real-time assessment of well-being is therefore a promising area for future research to unravel the dynamic nature of well-being fluctuations and the interaction with the environment in daily life. Integrating all results in this dissertation confirmed that well-being is a complex human trait that is influenced by many interrelated and interacting factors. Future directions to understand individual differences in well-being will be a data-driven approach to investigate the complex interplay of neural, physiological, genetic, and environmental factors in well-being.

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.

Eigenschappen van dieren zijn in meer of mindere mate erfelijk. Ze gaan over van ouders op nakomelingen. Maar ervaren fokkers weten dat in de fokkerij 1+1 geen 2 is. Welke wetmatigheden en welke toevalligheden spelen een rol in de erfelijkheid? Wat heeft het DNA-onderzoek ons daar recentelijk over geleerd en wat kunnen we daarmee?

Om te weten welke mechanismen ten grondslag liggen aan het verlies van resistentie van aardappelrassen tegen Phytophthora infestans is het van groot belang om inzicht te krijgen in effectoren die het pathogeen produceert. Bij Wageningen Universiteit is (promotie)onderzoek gedaan naar de identificatie van een P. infestans-Avr-gen, in het bijzonder de avirulentie- en effector-activiteit, de domeinstructuur en de subcellulaire lokalisatie van het Avr-eiwit, en de specificiteit van het bijbehorende aardappel-R-gen

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.

Dutch composer Peter Schat’s (1935-2003) pursuit of a compositional system that could generate and preserve intervallic relationships, while allowing the composer as much flexibility as possible to manipulate musical material, led him to develop the tone-clock system. Fundamentally comprised of the twelve possible trichords, the tone clock permits each to generate a complete twelve-tone series through the “steering” principle, a concept traced to Boulez’s technique of pitch-class set multiplication. This study serves as an overview of Schat’s tone-clock system and focuses primarily on the effects of the steering function in “Genen” (2000). Furthermore, I expand on the tone-clock system by combining transformational theory with Julian Hook’s uniform triadic transformations and my proposed STEER and STEERS functions, which express the procedures of the steering principle as a mathematical formula. Using a series of transformational networks, I illustrate the unifying effect steering has on different structural levels in “Genen,” a post-tonal composition.

The Dutch scrapie eradication programme aims at the exclusive use of homozygous ARR/ARR breeding rams by the end of 2004. As a consequence, breeds with a small population size and a low frequency of the ARR allele may suffer unacceptable losses of genetic diversity and high inbreeding levels. We simulated three breeding strategies to assess their efficacy in eliminating scrapie sensitive alleles and their effect on inbreeding levels. Under mild selection, both homozygous and heterozygous ARR rams were used indiscriminately. Under moderate selection, homozygous ARR rams were used preferably but they were supplemented with heterozygous rams when necessary. Under severe selection, only homozygous rams were used. Severe selection mimics then the proposed eradication programme. Simulations were carried out with allelic frequencies and population parameters of existent rare breeds in The Netherlands. With severe selection all simulated breeds showed unacceptably high inbreeding rates (>0.5% per year). For some breeds, moderate selection resulted in acceptable inbreeding rates, while for other breeds only mild selection resulted in acceptable rates. The frequency of the ARR allele after 5 years of selection was only slightly lower with moderate selection than with severe selection (0.8% lower on average), but it was clearly lower with mild selection. Based on these simulations, we propose a selection programme where with low frequencies of the ARR allele, mild selection is used initially. Once the ARR frequencies rise to a particular value, the switch can be made to moderate selection. The population size (e.g. below 750, 750 to 3750 and above 3750 ewes) determines the frequency of the ARR allele at which the switch can be made (33%, 25% and 10%, respectively). With even higher ARR frequencies (above 70%, 50% and 33%, respectively) the regime can be changed to severe selectionThe Dutch scrapie eradication programme aims at the exclusive use of homozygous ARR/ARR breeding rams by the end of 2004. As a consequence, breeds with a small population size and a low frequency of the ARR allele may suffer unacceptable losses of genetic diversity and high inbreeding levels. We simulated three breeding strategies to assess their efficacy in eliminating scrapie sensitive alleles and their effect on inbreeding levels. Under mild selection, both homozygous and heterozygous ARR rams were used indiscriminately. Under moderate selection, homozygous ARR rams were used preferably but they were supplemented with heterozygous rams when necessary. Under severe selection, only homozygous rams were used. Severe selection mimics then the proposed eradication programme. Simulations were carried out with allelic frequencies and population parameters of existent rare breeds in The Netherlands. With severe selection all simulated breeds showed unacceptably high inbreeding rates (> 0.5% per year). For some breeds, moderate selection resulted in acceptable inbreeding rates, while for other breeds only mild selection resulted in acceptable rates. The frequency of the ARR allele after 5 years of selection was only slightly lower with moderate selection than with severe selection (0.8% lower on average), but it was clearly lower with mild selection. Based on these simulations, we propose a selection programme where with low frequencies of the ARR allele, mild selection is used initially. Once the ARR frequencies rise to a particular value, the switch can be made to moderate selection. The population size ( e. g. below 750, 750 to 3750 and above 3750 ewes) determines the frequency of the ARR allele at which the switch can be made (33%, 25% and 10%, respectively). With even higher ARR frequencies (above 70%, 50% and 33%, respectively) the regime can be changed to severe selection.

Eén van de criteria voor het bewaren van rassen is de aanwezigheid van een uniek kenmerk in dat ras. Vaak is dat een unieke kleuraftekening, maar soms zijn dat ook kenmerken die een duidelijk effect hebben op het functioneren. Wereldwijd zijn er bij een aantal schapenrassen dit soort opmerkelijke genen gevonden.

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.

Los baculovirus se vienen utilizando desde hace tiempo como agentes de control biológico contra lepidópteros, dípteros e himenópteros debido a su eficacia, alta especificidad y seguridad frente a organismos que no son el objeto del tratamiento. El nuclepoliedrovirus múltiple de Spodoptera exigua (SeMNPV) tiene un estrecho rango de huésped y solo afecta a larvas de la rosquilla verde, Spodoptera exigua. Los aislados naturales del SeMNPV están compuestos de mezclas de genotipos que se creen que son importantes para la supervivencia del virus. La amplificación de SeMNPV en cultivo celular conduce a una rápida perdida de infectividad in vivo asociada con la selección preferente de genotipos con grandes delecciones. Esta tesis se centra en la caracterización biológica del baculovirus de Spodoptera exigua para mejorar nuestro conocimiento sobre el papel de la diversidad genotípica y sobre la implicación de genes individuales en patogenicidad, virulencia y transmisión. Avances en la tecnología de secuenciación de ADN y los más bajos costes de la misma han facilitado la identificación de genes virales que pueden jugar un papel importante en el proceso de infectividad del SeMNPV. En un estudio previo la secuenciación y comparación de 7 genotipos diferentes del SeMNPV con diferentes propiedades insecticidas, como la virulencia y la patogenicidad, permitieron la identificación de algunos ORFs como genes candidatos que pueden tener un efecto en esas propiedades insecticidas: se4, se5, se28, se76, se87 y se129 (Thézé et al., 2014). En el Capitulo 2 se construyó un sistema de recombinación basado en bacmidos utilizando el genotipo VT- SeAL1 para deleccionar individualmente los ORFs previamente identificados. La delección de se4, se5, se76 y se129 disminuyó la patogenicidad del virus comparado con el virus derivado del bacmido al insertar aislado silvestre, SeBacAL1. La delección del se87 no afectó la patogenicidad, mientras que la deleccion de se28 aumento ligeramente la patogenicidad. La delección individual de los genes se4, se28, se76, se87 y se129 no afectó la virulencia del virus, aunque la delección de se5 retrasó el tiempo de mortalidad alrededor de 7 horas. Interesantemente, el genotipo silvestre SeAL1 mostró ser ligeramente menos virulento que el bacmido SeBacAL1, lo cual puede ser debido a la inserción del plásmido de clonaje en la región intergénica del se27 y se28 del genoma de SeAL1. En conjunto, la deleccion de se5 mostró los efectos más significativos en las propiedades insecticidas estudiadas del SeMNPV, con un descenso en la patogenicidad de casi 10 veces y un retraso en el tiempo de mortalidad de 7 horas, comparado con el virus derivado del SeBacAL1., Un estudio previo sobre la ecología del SeMNPV reveló que el genotipo designado VT-SeAL1 produjo un 100% de infecciones encubiertas en adultos que habían sobrevivido a un tratamiento con OB en el estadio larvario. En cambio, otro genotipo designado HT-SeG25 solo produjo un 16% de infecciones encubiertas. Esto nos llevó a plantear la hipótesis de que algunos genotipos de SeMNPV pueden estar asociados con una ruta de transmisión vertical del virus, mientras otros genotipos pueden estar asociados a una ruta de transmisión horizontal. La comparación de genotipos de transmisión vertical con genotipos de transmisión horizontal, Thézé et al. (2014) permitió identificar tres ORFs que pueden estar envueltos en la transmisión vertical del virus: se5, se96 y se99. En el Capítulo 3 el sistema de recombinación de bacmidos desarrollado en el Capítulo 2 se utilizó para deleccionar estos ORFs y comprobar el efecto sobre la capacidad de transmisión vertical del virus. Larvas de Spodoptera exigua fueron infectadas sub-letalmente con una concentración equivalente a la CL40 y las infecciones sub-letales fueron detectadas en adultos mediante PCR cuantitativa (Q-PCR). El análisis no mostró diferencias significativas en el número de adultos que adquirieron una infección persistente con ninguno de los virus, lo que sugiere que dichos genes no están involucrados en la transmisión vertical del virus o, si lo están, lo hacen conjuntamente con otros genes. Las estructura genotípica de diferentes aislados silvestres de SeMNPV es muy similar a la composición genotípica de una población natural del SfMNPV. Las poblaciones de ambos baculovirus se caracterizan por la presencia de diferentes genotipos con delecciones en la misma región del genoma, la comprendida entre el se12 y el se40 en el caso de SeMNPV, y entre sf20 y sf36 en el caso de SfMNPV. En dichas regiones se localizan importantes genes: cathepsin, chitinase, gp37, ptp2, egt, pkip, arif1, pif1, pif2 y fgf, así como algunas ORFs de función desconocida. En el Capitulo 4 la estructura genotípica de los aislados SeUS2, SeUS1 y SfNIC fue comparada para determinar los mecanismos de acción evolutivos y ecológicos de estas estructuras genotípicas poblacionales tan similares., El alineamiento de las secuencias que flanquean a los puntos de delección de los diferentes genotipos indicó que se trata de un mecanismo de evolución independiente que genera y mantiene los genotipos en las respectivas poblaciones de estos virus. El aislamiento de un genotipo deleccionado y los estudios de complementación con diferentes mezclas de genotipo deleccionado y completo, claramente demostró una interacción entre los genotipos en la población viral. El genotipo deleccionado SeUS2-C tuvo una ventaja replicativa en cultivo celular, ya que estaba presente en un 81% de las placas aisladas en cultivo celular después de la inoculación de hemolinfa extraída de larvas infectadas con SeUS2-WT. Sin embargo, el genotipo completo SeUS2-A solo se encontró presente en un 19% de las placas. La presencia de una alta concentración de SeUS2-C en OBs co-ocluidos con el genotipo completo SeUS2-A comprometió severamente la patogenicidad del virus. Interesantemente, la prevalencia del genotipo SeUS2-C fue solo del 25% en el aislado silvestre SeUS2, comparado con el 81% de placas aisladas en cultivo celular. Se cree que los genotipos deleccionados se han generado debido a funciones importantes en la transmisibilidad del virus y que la diversidad genotípica del virus está estructurada para maximizar la probabilidad de transmisión. El aislado SeMNPV-US1 está también compuesto por varios genotipos, muchos de los cuales llevan delecciones de tamaño variable en la región del genoma comprendida entre el se15y el se41. En el Capítulo 5 se generaron bácmidos que contenían el genoma completo de SeMNPV (SeBac10) y un genotipo natural con una deleccion de 9.5Kb (SeBac72). El SeBac72 mostró una proliferación viral más eficiente en células de S. exigua que el genotipo completo SeBac10, lo cual concuerda con lo que se encontró el Capítulo 4. La secuenciación de SeBac72 puso de manifiesto que los genes se16- se28 estaban afectados por la deleccion. El análisis de diferentes delecciones de ORFs individuales del bácmido SeBac10, permitió identificar al se28 como el gen responsable de prevenir la proliferación de SeMNPV en cultivo celular. Sin embargo, la expresión de se28 en un bácmido de Autographa califórnica MNPV, un virus heterólogo que pertenece al grupo I de los Alphabaculovirus, no bloqueó la proliferación viral. Sorprendentemente, el silenciamiento de se28 mediante RNAi en el genotipo completo SeBac10 no condujo a un aumento de la propagación del virus en cultivo celular, sugiriendo que no es el transcrito ni la proteína, sino la secuencia de ADN y/o la secuencia topológica de se28 lo que determina la proliferación viral. La comparación de la secuencia de la región se27-se30 del genoma de SeMNPV con la región ac15-ac18 del genoma de AcMNPV mostró un 43% de homología a nivel de ADN, aunque la secuencia de aminoácidos de se28 y ac16 (DA26) no son homólogas. Ambos genes se28/ac16 son parte de regiones hipervariables asociadas con inserciones/delecciones. En conjunto, la región del gen se28 ha sido identificada como un regulador clave en la propagación del virus y se cree que puede ser responsable de la variación genotípica encontrada en los aislados naturales de baculovirus. En conclusión, los resultados presentados en esta tesis profundizan nuestro conocimiento en la diversidad genética y genotípica de SeMNPV, y de los baculovirus en general, y puede ayudar en el futuro a la mejora de las estrategias de control biológico basadas en baculovirus.

Eigenschappen van dieren zijn in meer of mindere mate erfelijk. Ze gaan over van ouders op nakomelingen. Maar ervaren fokkers weten dat in de fokkerij 1+1 geen 2 is. Welke wetmatigheden en welke toevalligheden spelen een rol in de erfelijkheid? Wat heeft het DNA-onderzoek ons daar recentelijk over geleerd en wat kunnen we daarmee?

The objective of this study was to develop an in-vitro bioassay using cultured Intestinal Porcine Epithelial Cells (IPEC-J2) and evaluate the capability of this assay to predict enterocyte-specific physiological and immunological processes induced by nutrients/additives in the intestines of farm animals. Responses to five nutrients/feed-additives, similar to those studied in animal trials, performed in the Feed4Foodure framework, were measured by gene expression analysis of IPEC-J2 cells either under stressed (Salmonella) or non-stressed conditions. Response genes were analysed using bioinformatics web-tools in order to identify dominant biological processes induced by these nutrients/feed-additives and to predict key-genes/proteins important for regulation of these biological proc

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.

The objective of this study was to develop an in-vitro bioassay using cultured Intestinal Porcine Epithelial Cells (IPEC-J2) and evaluate the capability of this assay to predict enterocyte-specific physiological and immunological processes induced by nutrients/additives in the intestines of farm animals. Responses to five nutrients/feed-additives, similar to those studied in animal trials, performed in the Feed4Foodure framework, were measured by gene expression analysis of IPEC-J2 cells either under stressed (Salmonella) or non-stressed conditions. Response genes were analysed using bioinformatics web-tools in order to identify dominant biological processes induced by these nutrients/feed-additives and to predict key-genes/proteins important for regulation of these biological proc

Copy-paste ruïneert graanoogst. Schimmels erven hun schadelijke genen van hun voorouders, zoals Darwin en graantelers al lang wisten, maar blijken ook gewoon genen met de buren uit te wisselen, zelfs als dat een heel andere soort is. Genen lenen is normaler dan biologen dachten. Zelfs de mens zelf is het resultaat van een onmogelijk geachte kruising van totaal verschillende soorten.

Als een spore is geland op het plantoppervlak, scheidt Botrytis cinerea enzymen en metabolieten uit waaronder pectine-afbrekende enzymen zoals de zes endopolygalacturonases (BcPGs). Elk van deze BcPGs breken pectine uit de plantencelwanden en middenlamel op hun eigen wijze af. Pectine zit verweven in de plantencelwanden en zorgt voor flexibiliteit van de cellen en de stabiliteit van de structuur van een weefsel. Op het moment dat pectine wordt afgebroken, wordt de verbinding tussen plantencellen verbroken, verliezen de celwanden hun flexibiliteit en het weefsel daarmee haar stabiliteit. Door middel van de BcPGs is Botrytis cinerea dus in staat het plantoppervlak van een gezonde plant binnen te dringen, door de middenlamel te groeien en plantenweefsel om te zetten in schimmelbiomassa en zodoende rot te veroorzaken.