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Capsicum species grown for pepper production suffer severely from thrips damage, urging the identification of natural resistance. Resistance levels are commonly assessed on leaves. However, Capsicum plants are flower-bearing during most of the production season, and thrips also feed on pollen and flower tissues. In order to obtain a comprehensive estimate of elements contributing to thrips resistance, flower tissues should be considered as well. Therefore, we assessed resistance to Frankliniella occidentalis in flowers, leaves, and whole plants of ten Capsicum accessions. Using choice assays, we found that thrips prefer flowers of certain accessions over others. The preference of adult thrips for flowers was positively correlated to trehalose and fructose concentration in anthers as well as to pollen quantity. Resistance measured on leaf discs and thrips population development on whole plants was significantly and positively correlated. Leaf-based resistance thus translates to reduced thrips population development. Results of the flower assays were not significantly correlated with resistance in leaves or on whole plants. This suggests that both leaves and flowers represent a different part of the resistance spectrum and should both be considered for understanding whole plant resistance and the identification of resistant Capsicum varieties.

Met Wageningse gentechnologie zijn aardappels te ontwikkelen die amper bestrijdingsmiddelen nodig hebben tegen de aardappelziekte Phytophthora. Toepassing zou binnen vijf jaar resistente aardappelrassen kunnen opleveren, maar genetische modificatie ligt gevoelig en iedereen wacht met smart op bericht uit Brussel. ‘We hebben nog vier maanden voordat het pootgoed begint te verrotten.’

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.

Disease resistance in crops is an important aspect of securing global food security. Resistant plants carry immune receptors that sense pathogen invasion often through the recognition of important pathogen virulence factors, known as effectors. Thus, identification and characterization of effectors is important for the fundamental understanding of virulence mechanisms and to aid in resistance breeding. In this thesis the VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1. Homologs were found in other plant pathogens and the role in virulence in these pathogens was analyzed. Ave1 homologs are differentially recognized by Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1. Knowledge of specific effector-receptor combinations and knowledge of effectors in general can be exploited to aid in breeding for durable resistance in crops.

Verwelkingsziekte bedreigt al jaren de boomkwekerij, m.n. in de teelt van laanbomen en rozen. Onderzoek naar deze bodemziekte is daarom hard nodig. Jelle Hiemstra en Bart van der Sluis geven in dit artikel een overview van de stand van zaken met betrekking tot onderzoek naar dit probleem.

De onderzoekers Henk Jalink en Rob van der Schoor van Wageningen UR hebben een innovatieve MIPS-LED camera ontwikkeld, waarmee collega-onderzoekers van de universiteit snel fotosynthese in planten kunnen vastleggen. Vivianne Vleeshouwers, is werkzaam bij Wageningen UR Plantenveredeling, en is een van de onderzoekers die de camera beproeft. Zij hoopt het apparaat te kunnen inzetten voor onderzoek naar phytophthoraresistentie in (nieuwe) aardappelrassen en wilde soorten.

Average potato yields in Dutch organic farming are rather low and variable, due to the disease known as late blight (Phytophthora infestans). Potato breeding companies, organic farmers and breeding scientists from the Louis Bolk Institute and Wageningen University, have joined forces in an umbrella program called Bioimpuls. Within this program there is a joint breeding project aimed at developing better adapted and late blight resistent varieties

Samenvatting van de inaugurele rede van Francine Govers op 11 juni 2009. Dit artikel beschrijft de stand van zaken in het onderzoek aan oömyceten en in het bijzonder aan Phytophthora infestans, de veroorzaker van de aardappelziekte. Er wordt ingegaan op ziektebestrijding en resistentieveredeling, de diversiteit van Phytophthora en zijn gastheren en de genetische blauwdruk: het DNA, de genen, de effectoren en de resistentiegenen.

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.

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.

Van 11-14 november 2012 vond in de Reehorst de conferentie ‘Next Generation Plant Breeding’ plaats. Tijdens deze bijeenkomst kwamen de grote uitdagingen van de toekomstige plantenveredeling aan de orde: de opkomst van nieuwe sequencing-technieken, de bijbehorende enorme hoeveelheid gegevens die geproduceerd wordt, inzicht in de genetica van de plant, de bijbehorende statistische methoden, geautomatiseerde waarneming van planten, en veel meer. Een impressie.

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.

Onder de noemer Green Challenge Meeldauw startte vorig jaar een door het Productschap Tuinbouw en bedrijfsleven gefinancierd en door LTO Glaskracht Nederland begeleid masterplan, dat meer inzicht en oplossingen moet bieden bij het voorkomen en bestrijden van echte meeldauw. De aanvankelijke focus op siergewassen is inmiddels verbreed met vruchtgroenten. Onderzoekster Jantineke Hofland-Zijlstra van Wageningen UR Glastuinbouw doet verslag van de voorlopige resultaten.

Met Wageningse gentechnologie zijn aardappels te ontwikkelen die amper bestrijdingsmiddelen nodig hebben tegen de aardappelziekte Phytophthora. Toepassing zou binnen vijf jaar resistente aardappelrassen kunnen opleveren, maar genetische modificatie ligt gevoelig en iedereen wacht met smart op bericht uit Brussel. ‘We hebben nog vier maanden voordat het pootgoed begint te verrotten.’

Grofweg lijken er twee stromingen te bestaan om de weerbaarheid van de bijenvolken te vergroten. De ene stroming selecteert op specifieke eigenschappen (vooral resistentie gericht). De andere stroming selecteert op verbeterde/natuurlijke aanpassing(en) aan de lokale ziekte/plaagdruk of zelfs de algemene lokale omstandigheden (zowel resistentie als tolerantie gericht). Sommige initiatieven passen heel duidelijk in een van deze stromingen, anderen hangen er meer tussen in. Als uitvloeisel van deze studie wordt eind 2015 een internationale workshop georganiseerd waar een doorsnede van deze initiatieven wordt uitgenodigd om gezamenlijk de state of the art vast te stellen en internationale afstemming te zoeken.

This brochure gives an overview of new plant breeding techniques. This overview is based on a more technical review of the scientific literature, published in a separate report. The overview presents the opportunities and limitations of these techniques from the point of view of potential applications in plant breeding with promising results for improving agricultural sustainability.

This brochure gives an overview of new plant breeding techniques. This overview is based on a more technical review of the scientific literature, published in a separate report. The overview presents the opportunities and limitations of these techniques from the point of view of potential applications in plant breeding with promising results for improving agricultural sustainability.

Er zijn zes nieuwe biologische aardappelrassen gepresenteerd die resistent zijn tegen phytophthora.

Grofweg lijken er twee stromingen te bestaan om de weerbaarheid van de bijenvolken te vergroten. De ene stroming selecteert op specifieke eigenschappen (vooral resistentie gericht). De andere stroming selecteert op verbeterde/natuurlijke aanpassing(en) aan de lokale ziekte/plaagdruk of zelfs de algemene lokale omstandigheden (zowel resistentie als tolerantie gericht). Sommige initiatieven passen heel duidelijk in een van deze stromingen, anderen hangen er meer tussen in. Als uitvloeisel van deze studie wordt eind 2015 een internationale workshop georganiseerd waar een doorsnede van deze initiatieven wordt uitgenodigd om gezamenlijk de state of the art vast te stellen en internationale afstemming te zoeken.

Resistentie tegen Botrytis cinerea is gevonden in wilde verwanten van tomaat en deze resistentie was meestal kwantitatief. Het doel van dit promotieonderzoek was om kwantitatieve loci (QTLs) te identificeren die bijdragen aan resistentie tegen B. cinerea. Met behulp van DNA-merkertechnologie en een populatie van introgressie-lijnen zijn tien QTLs geïdentificeerd. Geen van de afzonderlijke QTLs resulteerde in een niveau van resistentie dat overeen kwam de resistente ouder Solanum habrochaites LYC4. Dit betekent dat QTLs gecombineerd zullen moeten worden om Botrytis cinerea-resistente tomaten te verkrijgen. Dankzij de ontwikkelde DNA-merkers kunnen de geïdentificeerde chromosoomfragmenten met resistentiegenen nu gericht ingekruist worden.

Plantenveredeling staat tegenwoordig heel ver van de teler af. De reden is dat de moleculaire en genetische kennis enorme sprongen heeft gemaakt. Er is bovendien een hele geheimtaal ontstaan, waardoor de veredelaar vooral goed kan praten met vakgenoten en veel minder met de teler. Hier een aantal recente inzichten op een rij.

De iep, en dan met name de ‘Belgica’, was aan het begin van de 20ste eeuw in grote delen van Nederland de beeldbepalende boom. De opkomst van de iepziekte leidde tot het verdwijnen van zeer veel van deze Hollandse iepen. Door de voortdurende problemen met de iepziekte kreeg de iep in de tweede helft van de 20ste eeuw bovendien een slechte naam en nam het relatieve belang van deze boom sterk af. De iep is echter een ideale boom, zowel in de moderne stad als in het buitengebied, en kan slecht gemist worden. Onderzoek laat zien dat de iep nu echter weer volop toekomst heeft. De nieuwe iepen die inmiddels op de markt zijn hebben een zeer hoge mate van resistentie en zijn algemeen bruikbaar, ook in de huidige situatie met de nog alom aanwezige iepziekte.

Genetische modificatie blijft onderwerp van debat. Greenpeacecampagneleider Herman van Bekkem en onderzoeker Bert Lotz van Wageningen UR beantwoorden elkaars vragen over gentech.

De iep, en dan met name de ‘Belgica’, was aan het begin van de 20ste eeuw in grote delen van Nederland de beeldbepalende boom. De opkomst van de iepziekte leidde tot het verdwijnen van zeer veel van deze Hollandse iepen. Door de voortdurende problemen met de iepziekte kreeg de iep in de tweede helft van de 20ste eeuw bovendien een slechte naam en nam het relatieve belang van deze boom sterk af. De iep is echter een ideale boom, zowel in de moderne stad als in het buitengebied, en kan slecht gemist worden. Onderzoek laat zien dat de iep nu echter weer volop toekomst heeft. De nieuwe iepen die inmiddels op de markt zijn hebben een zeer hoge mate van resistentie en zijn algemeen bruikbaar, ook in de huidige situatie met de nog alom aanwezige iepziekte.

Door de iepziekte is de iep grotendeels uit het landschap verdwenen. Zonde, want het is voor veel moeilijke plekken de ideale boom. Onderzoek van PPO en Alterra laat nu echter zien dat er voldoende resistente iepen zijn om de boom weer volop te gaan gebruiken.

Verwelkingsziekte bedreigt al jaren de boomkwekerij, m.n. in de teelt van laanbomen en rozen. Onderzoek naar deze bodemziekte is daarom hard nodig. Jelle Hiemstra en Bart van der Sluis geven in dit artikel een overview van de stand van zaken met betrekking tot onderzoek naar dit probleem.

Key words: Capsicum, Bemisia tabaci, trichome density, cuticle thickness Whitefly is economically one of the most threatening pests of pepper worldwide, which is mainly caused by its ability to transmit many different viruses. In this research, we characterized pepper germplasm to identify whitefly-resistant accessions that will form the basis for future resistance breeding. Forty-four pepper accessions representing four species (Capsicum annuum, C. frutescens, C. chinense, C. baccatum) were screened for resistance to whiteflies. Screening parameters were adult survival (AS) and oviposition rate (OR) in a no-choice test and whitefly, egg and nymphal density in free-choice tests. To combine parameters in free-choice tests, a plant resistance value was calculated. The results show that AS and OR were significantly different among accessions and were positively correlated, which was also the case for the parameters in the free-choice tests. Accessions identified as highly resistant in no-choice and free-choice tests generally were C. annuum. Whitefly density and OR correlated positively with trichome density and negatively with cuticle thickness of leaves.

Het is een zwaar phytophthora seizoen geweest. Nu zijn telers weer helemaal gespitst op resistente rassen. Daarbij richten de ogen zich op het Bioimpuls veredelingsprogramma. Traditionele veredeling uit wilde rassen is een lange weg, maar levert wel meer op. En niet vergeten: smaak en uiterlijk tellen net zo goed. Tijd voor een update