Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Worse comes to worst: bananas and Panama disease—when plant and pathogen clones meet
    Ordonez Roman, N.I. ; Seidl, M.F. ; Waalwijk, C. ; Drenth, A. ; Kilian, A. ; Thomma, B.P.H.J. ; Ploetz, R.C. ; Kema, G.H.J. - \ 2015
    PLoS Pathogens 11 (2015)11. - ISSN 1553-7366 - 7
    bananas - tropical small fruits - agricultural research - fungal diseases - fusarium oxysporum f.sp. cubense - soil fungi - pathogenicity - food production - genetic diversity - plant protection - bananen - tropisch kleinfruit - landbouwkundig onderzoek - schimmelziekten - fusarium oxysporum f.sp. cubense - bodemschimmels - pathogeniteit - voedselproductie - genetische diversiteit - gewasbescherming
    This article deals with: Bananas: their origin and global rollout; genetic diversity of Fusarium oxysporum f.sp. cubense, the causal agent of Panama Disease; Panama Disease: history repeats itself; tropical race 4, a single pathogen clone, threatens global banana production; strategies for sustainable Panama Disease management.
    Genetic baculovirus determinants for pathogenicity, virulence and transmission
    Serrano, A. - \ 2014
    Wageningen University. Promotor(en): Just Vlak; P. Caballero, co-promotor(en): Gorben Pijlman; D. Munoz. - Wageningen : Wageningen University - ISBN 9789462571358 - 160
    baculovirus - spodoptera exigua multiple nucleopolyhedrovirus - genetische analyse - genotypische variatie - pathogeniteit - virulentie - genen - biologische bestrijding - insectenplagen - baculovirus - spodoptera exigua multiple nucleopolyhedrovirus - genetic analysis - genetic variance - pathogenicity - virulence - genes - biological control - insect pests
    Risk of poultry compartments for transmission of High Pathogenic Avian Influenza
    Boender, G.J. ; Hagenaars, T.H.J. ; Backer, J.A. ; Nodelijk, G. ; Asseldonk, M.A.P.M. van; Bergevoet, R.H.M. ; Roermund, H.J.W. van - \ 2014
    Lelystad : CVI en LEI (Report number CVI: 14/I00028 ) - 28
    aviaire influenza - pluimveeziekten - pluimveehouderij - compartimenten - ziekteoverdracht - pathogeniteit - eu regelingen - wetgeving - diergezondheid - dierenwelzijn - pluimvee - avian influenza - poultry diseases - poultry farming - compartments - disease transmission - pathogenicity - eu regulations - legislation - animal health - animal welfare - poultry
    The application for a poultry compartment by VPI and the prospect of further ones motivates the Dutch Ministry of Economic Affairs and the Netherlands Food and Consumer Product Safety Authority to raise the following three questions: 1) What are the additional transmission risks that the (specific) VPI compartment poses during an HPAI epidemic, compared to a situation without compartment?; 2) What are the additional transmission risks that a compartment in general poses during an HPAI epidemic, depending on its characteristics?; 3) What are relevant evaluation criteria for granting the compartment status? In this study we addressed these questions by quantitatively assessing the veterinary risks based on mathematical model calculations, and by qualitatively discussing the (socio) economic aspects.
    Functional analysis of LysM effectors secreted by fungal plant pathogens
    Kombrink, A. - \ 2014
    Wageningen University. Promotor(en): Bart Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789461738578 - 119
    plantenziekteverwekkende schimmels - secretie - celwanden - chitine - bindende eiwitten - virulentie - pathogeniteit - hyfen - ziekteresistentie - verdedigingsmechanismen - plant pathogenic fungi - secretion - cell walls - chitin - binding proteins - virulence - pathogenicity - hyphae - disease resistance - defence mechanisms

    Chitin is a homopolymer of N-acetyl-d-glucosamine (GlcNAc)that is abundantly present in nature and found as a major structural component in the fungal cell wall. In Chapter 1,the role of chitin as an important factor in the interaction between fungal pathogens and their plant hosts is discussed. As plants do not produce chitin, they evolved to recognize fungal chitin as a non-self molecule by plasma membrane receptors that can activate host immune responses to stop fungal growth.To overcome those host immune responses, fungal pathogens secrete effector molecules that manipulate host physiology, including immune responses, to support colonization. The chitin-binding Lysin motif (LysM) effector Ecp6 from the fungal tomato pathogen Cladosporium fulvumwas previously demonstrated to contribute to virulence through interfering with the activation of chitin-induced host immune responses. Subsequently, LysM effector genes were found in the genomes of many fungal species.

    In Chapter 2 we describe the functional characterization of LysM effectors of the plant pathogenic fungi Mycosphaerella graminicola, Magnaporthe oryzae and Colletotrichum higginsianum, which cause leaf blotch disease of wheat, rice blast disease and anthracnose disease on Brassicaceae, respectively. This functional analysis revealed that the ability to perturb chitin-induced immunity is conserved among LysM effectors of these fungal plant pathogens. In addition, two LysM effectors that are secreted by M. graminicolawere found to protect fungal hyphae against cell wall hydrolytic enzymes from plants, demonstrating that LysM effectors can contribute to virulence of fungal plant pathogens in multiple ways.

    The M. graminicola LysM effector Mg3LysM and C. fulvum Ecp6 both contain three LysM domains and show a high overall similarity. However, whereas Mg3LysM can protect fungal hyphae against plant-derived cell wall hydrolytic enzymes, Ecp6 does not have this capacity. Chapter 3describes a functional analysis of the contribution of LysM domains of Mg3LysM to its protection ability. To this end a series of chimeric proteins were produced in whichLysM domains of Mg3LysM were swapped with the corresponding LysM domain of Ecp6.Analysis of these chimeras indicated that protection against the hydrolytic activity of plant enzymes is mediated by the concerted activity of LysM1 and LysM3 in Mg3LysM.

    LysM effectors do not only occur in foliar fungal plant pathogens, but also in soil-borne pathogens that infect their host through the roots. In Chapter 4, LysM effectors of the fungal soil-borne vascular wilt pathogen Verticillium dahliaeare described. Comparative genomics of eleven V. dahliae strains revealed that four LysM effectors are found in the core genome, which are referred to as core VdLysM effectors. Intriguingly, for none of the core LysM effector genes expression could be monitored during host colonization, and targeted deletion could not reveal a role in virulence, suggesting that the core LysM effectors do not act as virulence factors during host colonization. In addition to the core genome, V. dahliaestrains generally carry lineage-specific (LS) genomic regions. Interestingly, an additional LysM effector gene (Vd2LysM) was found in an LS region of V. dahliaestrain VdLs17 that is absent in all other sequenced V. dahliaestrains. Remarkably, the LS effector Vd2LysM was found to contribute to virulence of strain VdLs17. Like the previously characterized plant pathogen LysM effectors, also Vd2LysM was found to bind chitin and suppress chitin-induced immune responses. These results indicate that Vd2LysM interferes with chitin-induced immunity during host colonization by V. dahliaestrain VdLs17.

    Thus far, LysM effectors were demonstrated to contribute to virulence of various fungal plant pathogens through their ability to interfere with host immune responses. However, the presence of LysM effector genes in the genomes of non-pathogenic fungi and fungi with a saprophytic lifestyle suggests that LysM effectors contribute to fungal physiology in other manners as well. In Chapter 5we investigated the hypothesis that LysM effectors play a role in the interaction of fungi with other microbes in the environment, which could even be relevant for plant pathogenic fungi that encounter other microbes at the site of host infection. To investigate this hypothesis, assays were developed that allow to assess the attachment and antagonistic effects of particular bacterial species on fungi by employing the fungus Trichoderma viride, as this species is known to have accessible cell wall chitin upon growth in vitro. Assays to assess bacterial attachment and antagonistic activity in the absence or presence of LysM effectors indicate that LysM effectors play a role in the protection of fungi against bacterial competitors.

    In Chapter6, the major results described in this thesis are discussed and a perspective on the (potential) roles of LysM effectors in fungi with different lifestyles, including pathogenic as well as non-pathogenic fungi, is presented.

    Insertional mutagenesis in the vascular wilt pathogen Verticillium dahliae
    Santhanam, P. - \ 2014
    Wageningen University. Promotor(en): Bart Thomma, co-promotor(en): Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789461738257 - 124
    plantenziekteverwekkende schimmels - verticillium dahliae - verwelkingsziekten - insertiemutagenese - solanum lycopersicum - tomaten - modellen - pathogeniteit - genkartering - genomica - plant pathogenic fungi - verticillium dahliae - wilts - insertional mutagenesis - solanum lycopersicum - tomatoes - models - pathogenicity - gene mapping - genomics

    Vascular wilt diseases caused by soil-borne pathogens are among the most

    devastating plant diseases worldwide. The ascomycete fungus Verticillium dahliae

    causes vascular wilt diseases in hundreds of dicotyledonous plant species, including

    important crops such as eggplant, lettuce, olive, spinach and tomato. The resting

    structures, microsclerotia, are triggered by root exudates to germinate and penetrate

    the roots after which the fungus grows into the xylem vessels. The fungus colonizes

    these vessels and interferes with the transportation of water and nutrients, resulting in

    the development of symptoms such as stunting, wilting, chlorosis and vascular

    browning. Verticillium wilt diseases are difficult to control due to the longevity of the

    microsclerotia, the broad host range of the pathogen, the inability of fungicides to kill

    the fungus once it has colonized the xylem vessels and the lack of natural resistance in

    many plant species.

    Chapter 1 is the introduction to this thesis that describes the identified

    pathogenicity and virulence factors of V. dahliae and strategies to identify these

    components. In spite of the economic importance of V. dahliae, relatively few

    pathogenicity genes have been identified in this species. With the availability of

    whole genome sequences and the development of functional genomics tools such as

    random mutagenesis, targeted mutagenesis, transcriptomics, RNA interference

    (RNAi) and comparative genomics, more strategies have become available to identify

    novel pathogenicity and virulence genes.

    Chapter 2 focuses on the identification of virulence and pathogenicity genes

    of V. dahliae by screening of a library of random T-DNA insertion mutants. Using

    Agrobacterium tumefaciens-mediated transformation, 900 T-DNA transformants with

    random insertions were generated and screened for altered virulence on susceptible

    tomato plants. This screening, followed by inverse PCR on selected transformants,

    resulted in the identification of 55 potential pathogenicity and virulence genes. One of

    the potential pathogenicity genes, VdNRS/ER, is a homolog of a nucleotide-rhamnose

    synthase/epimerase-reductase (NRS/ER), which is presumably involved in the

    biosynthesis of UDP-rhamnose. Using targeted mutagenesis, VdNRS/ER was deleted

    from wild-type V. dahliae and the resulting deletion mutants were characterized.

    VdNRS/ER deletion mutants exhibit unaltered vegetative growth and sporulation, but

    the deletion mutants were no longer pathogenic on tomato and N. benthamiana and

    showed impaired root attachment on tomato seedlings. These data suggest that UDPrhamnose

    is required for pathogenesis of V. dahliae.

    Chapter 3 describes the role of the V. dahliae homolog of Sge1, a

    transcriptional regulator that was shown to play a role in pathogenicity and regulate

    effector gene expression in Fusarium oxysporum. In this chapter it is demonstrated

    that V. dahliae Sge1 (VdSge1) is required for radial growth and production of asexual

    conidiospores. It is furthermore shown that VdSge1 deletion strains have lost

    pathogenicity on tomato. Since the VdSge1 deletion mutants are not able to infect and

    colonize tomato plants, a tomato cell suspension culture was used to the study the

    expression of Ave1, as well as nine other genes of which the expression is highly

    induced in planta. This assay revealed that VdSge1 is not required for the induction of

    the Ave1 effector that activates resistance mediated by Ve1 in tomato. Furthermore,

    the expression of one other putative effector gene was not affected by VdSge1

    deletion. However, VdSge1 was shown to be required for the expression of six

    putative effector genes, whereas expression of the remaining two putative effectors

    genes was negatively regulated. Thus, the data show that VdSge1 is required for V.

    dahliae pathogenicity and differentially regulates effector gene expression.

    Chapter 4 describes the functional characterization of the gene family

    encoding necrosis- and ethylene-inducing-like proteins (NLPs) of V. dahliae. The

    cytotoxic activity of NLP family members was determined using agroinfiltration into

    tobacco leaves. This resulted in the identification of two out of the seven NLPs,

    VdNLP1 and VdNLP2, that induced plant cell death. The genes encoding these

    cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato.

    Targeted deletion of VdNLP1 and VdNLP2 significantly reduced the virulence of V.

    dahliae on tomato and Arabidopsis plants. In contrast, only deletion of VdNLP1

    affected virulence on N. benthamiana whereas deletion of NLP2 did not. However,

    subsequent transcriptional analysis revealed that VdNLP2 was not expressed in V.

    dahliae during colonization of N. benthamiana. Moreover, VdNLP2 also affects

    vegetative growth and conidiospore production. In conclusion, the expanded V.

    dahliae NLP family shows differential cytotoxic activity between family members

    and in planta induction of the cytotoxic NLP genes varies between plant hosts. In

    addition, VdNLP2 plays a role in vegetative growth and conidiospore production in

    addition to its contribution to virulence. Thus, evidence is provided for functional

    diversification within the V. dahliae NLP family.

    Finally in Chapter 5, the major findings of this thesis are discussed and

    placed in a broader perspective.

    Identification and characterization of novel effectors of Cladosporium fulvum
    Ökmen, B. - \ 2013
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare. - S.l. : s.n. - ISBN 9789461736383 - 190
    solanum lycopersicum - tomaten - plantenziekteverwekkende schimmels - passalora fulva - plant-microbe interacties - verdedigingsmechanismen - genomica - dna-sequencing - pathogeniteit - solanum lycopersicum - tomatoes - plant pathogenic fungi - passalora fulva - plant-microbe interactions - defence mechanisms - genomics - dna sequencing - pathogenicity

    In order to establish disease, plant pathogenic fungi deliver effectors in the apoplastic space surrounding host cells as well as into host cells themselves to manipulate host physiology in favour of their own growth. Cladosporium fulvum is a non-obligate biotrophic fungus causing leaf mould disease of tomato. For decades, this fungus has been a model to study the molecular basis of plant-pathogen interactions involving effector proteins. Characterization of these effectors revealed their roles in both virulence and avirulence as they facilitate colonization of the host in the absence of cognate tomato Cf resistance genes, but also trigger Cf-mediated resistance in the presence of these genes. The availability of the genome sequence of C. fulvum is a great resource allowing us to dissect and better understand the molecular interaction between this fungus and tomato, particularly with regards to identification of new effectors. Such knowledge is of important to improve current strategies not only for disease resistance breeding of tomato against C. fulvum, but also for other host plants that are attacked by pathogenic fungi with similar infection strategies and lifestyles.

    In chapter 1 we give an introduction to the C. fulvum-tomato pathosystem. In a compatible interaction, C. fulvum secretes small cysteine-rich effectors that positively contribute to fungal virulence. Two of these effectors are chitin-binding proteins including Avr4, which protects fungal cell walls against hydrolysis by plant chitinases, and Ecp6, which sequesters released small chitin fragments, thereby preventing induction of basal defense responses associated with their recognition by plant receptors. Another effector, Avr2, is an inhibitor of four tomato cysteine proteases that are also important for basal plant defense. However, in an incompatible interaction, these effectors are directly or indirectly perceived by corresponding resistance proteins (encoded by Cf resistance genes that belong to the class of receptor-like proteins; RLPs) mediating race-specific plant defense responses also known as effector-triggered immunity.

    In chapter 2 we exploit the availability of the genome sequence of C. fulvum to identify novel effectors involved in virulence and avirulence of this fungus. An in silico search was performed using common features of characterized C. fulvum effectors: they (i) contain a signal peptide, (ii) are small (<300 amino acids) and (iii) contain at least four cysteine residues (SSCPs). This search identified 271 SSCPs in the C. fulvum genome. A subset of 60 of these predicted effectors was heterologously expressed in tomato lines carrying different R-traits, including Cf-1, Cf-3, Cf-5, Cf-9B, Cf-11 and Cf-Ecp3 in order to identify the corresponding effectors that are recognized by the RLPs. Although the screen of this subset of SSCPs did not result in identification of a new avirulence gene, two non-specific necrosis-inducing proteins were identified. In addition, a homology search identified CfNLP1, a gene encoding a functional NEP1-like protein that triggers non-specific necrosis in plants. However, quantitative PCR showed that these three genes are lowly or not expressed during tomato infection, which was also true for the in planta expression of some of the effector candidates that were tested for recognition by Cf proteins. In contrast, all genes from C. fulvum encoding the effectors that have been reported so far are highly up-regulated during infection where they play an important role in establishing disease. Like Avr2, Avr4, Ecp2 and Ecp6, we report that Ecp4 and Ecp5 also are involved in virulence of C. fulvum on tomato. Finally, we discuss the limitations of only using bioinformatics approaches to identify novel effectors involved in virulence.

    Inchapter 3 we describe the identification and characterization of a novel effector secreted by C. fulvum. CfTom1 encodes a functional tomatinase enzyme, which belongs to family 10 of glycoside hydrolases (GH10). Bacterial and fungal pathogens of tomato secrete this enzyme to detoxify the toxic saponin, α-tomatine, into the less toxic compounds tomatidine and lycotetraose. Similarly, CfTom1 is responsible for α-tomatine deoxification by C. fulvum both in vitro and during infection of tomato. Accordingly, ∆cftom1 mutants are more sensitive to α-tomatine because they can no longer detoxify α-tomatine. They are less virulent on tomato plants than wild-type as reflected by a delay in disease symptom development and reduced fungal biomass production. In addition, tomatidine appears to be more toxic to tomato cells than α-tomatine, but it does not suppress plant defense responses as previously suggested in literature. Altogether, our results clearly indicate that CfTom1, the major or possibly only tomatinase enzyme produced by C. fulvum, contributes to full virulence of this fungus on tomato by detoxifying α-tomatine.

    Hardly anything is known about in planta regulation of effector genes. In chapter 4 we describe the functional characterization of CfWor1, a homologue of FoSge1, a conserved transcriptional regulator of effectors in Fusarium oxysporum f. sp. lycopersici. CfWor1 is also homologous to Wor1/Ryp1/Mit1 proteins, which are involved in morphological switches in Candida albicans, Histoplasma capsulatum and Saccharomyces cerevisiae, respectively. In contrast to FoSge1, CfWor1 is unlikely a positive regulator of effector genes because it is weakly expressed during infection of tomato. Compared to wild-type, ∆cfwor1 mutants show strong developmental and morphological defects. ∆cfwor1 mutants do not produce any conidia, but differentiate sclerotium-like structures and secrete an extracellular matrix that covers fungal hyphae.∆cfwor1 mutants are no longer virulent on tomato, likely because of developmental defects. Although constitutive expression of CfWor1 in C. fulvum did not cause any obvious developmental defects, except reduced conidia production, the transformants showed reduced virulence. Quantitative PCR on known effector and secondary metabolism genes in both ∆cfwor1 mutants and constitutive expression transformant revealed that the effect of CfWor1 on the expression of these genes is likely due to developmental defects rather than direct regulation. Complementationof a non-virulent ∆fosge1 mutant of F. oxysporum f. sp. lycopersici with full length CfWor1 or chimera of CfWor1 and FoSge1 restored expression of SIX effector genes, but not virulence, indicating that reduced virulence observed for the ∆fosge1 mutant is not solely due to loss of expression ofthese effector genes. Altogether, our study suggests that CfWor1 is a major regulator of development in C. fulvum which indirectly affects virulence.

    Chapter 5 provides a general discussion of the present work on C. fulvum effectors, with particular emphasis on comparative genomics and transcriptomics approaches to identify novel effectors involved in fungal virulence and avirulence. Our findings are put in a broader perspective including a discussion on how identification of effectors will improve our understanding of molecular interactions between plants and pathogenic fungi and how we can use this knowledge to develop new strategies for sustainable disease resistance breeding.

    Genome structure and pathogenicity of the fungal wheat pathogen Mycosphaerella graminicola
    M'Barek, S. Ben - \ 2011
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema. - [S.l.] : S.n. - ISBN 9789085859970 - 229
    triticum aestivum - triticum turgidum - tarwe - mycosphaerella graminicola - plantenziekteverwekkende schimmels - genoomstructuur - eiwitexpressieanalyse - pathogeniteit - pathogenesis-gerelateerde eiwitten - genomen - plasticiteit - plant-microbe interacties - triticum aestivum - triticum turgidum - wheat - mycosphaerella graminicola - plant pathogenic fungi - genomic structure - proteomics - pathogenicity - pathogenesis-related proteins - genomes - plasticity - plant-microbe interactions

    The phytopathogenic fungus Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn (asexual stage: Zymoseptoria tritici (Desm.) Quaedvlieg & Crous) causes septoria tritici leaf blotch (STB) in wheat and is one of the most important diseases of this crop worldwide. However, STB control, mainly based on the use of resistant cultivars and fungicides, is significantly hampered by the limited understanding of the genetic and biochemical bases of pathogenicity, and mechanisms of infection and resistance in the host. M. graminicola has a very active sexual cycle under field conditions, which is an important driver of STB epidemics. Moreover, it results in high genetic diversity of field populations that causes a major challenge for the development and sustainable management of resistant cultivars and the discovery of new antifungal compounds. Understanding the role of the sexual and asexual life cycles on genome composition of this versatile pathogen and its infection strategy is crucial in order to develop novel control methods.

    Chapter 1 is an introduction to the biology and pathogenicity of M. graminicola. In addition, it shortly describes the impact of improved and novel technologies on the speed, scope and scale of comparative genomics research.

    Chapter 2 provides detailed genetic analyses of two M. graminicola mapping populations, using mainly DArT markers, and the analysis of the meiotic transmission of unequal chromosome numbers. Polymorphisms in chromosome length and number were frequently observed in progeny isolates, of which 15–20% lacked one or more chromosomes despite their presence in one or both parents, but these had no apparent effect on sexual and pathogenic fitness. M. graminicola has up to eight so called dispensable chromosomes that can be easily lost - collectively called the dispensome - which is, so far, the highest number of dispensable chromosomes reported in filamentous fungi. They represent small-sized chromosomes and make up 38% of the chromosome complement of this pathogen. Much of the observed genome plasticity is generated during meiosis and could explain the high adaptability of M. graminicola in the field. The generated linkage map was crucial for finishing the M. graminicola genome sequence.

    Chapter 3 describes the M. graminicola genome sequence with highlights on genome structure and organization including the eight dispensable chromosomes. The genome comprises a core set of 13 chromosomes and a dispensome, consisting of eight chromosomes that are distinct from the core chromosomes in structure, gene and repeat content. The dispensome contains a higher frequency of transposons and the genes have a different codon use. Most of the genes present one the dispensome are also present on the core chromosomes but little synteny is observed neither between the M. graminicola dispensome and the core chromosomes nor with the chromosomes of other related Dothideomycetes. The dispensome likely originates from ancient horizontal transfer(s) from (an) unknown donor(s).

    Chapter 4 shows a global analysis of proteins secreted by M. graminicola in apoplastic fluids during infection. It focuses mainly on fungal proteins secreted in a compatible interaction. The study showed that many of the annotated secreted proteins have putative functions in fungal pathogenicity, such as cell wall degrading enzymes and proteases, but the function of a substantial number of the identified proteins is unknown. During compatible interactions proteins are primarily secreted during the later stages. However, many pathogenesis-related host proteins, such as PR-2, PR-3 and PR-9, accumulated earlier and at higher concentrations during incompatible interactions, indicating that fungal effectors are recognized by resistant plants and trigger resistant gene-mediated defence responses, though without a visible hypersensitive response.

    Chapter 5 further details the initial identification and characterization of necrosis-inducing proteins that are produced in culture filtrates (CFs) of M. graminicola. The necrosis-inducing activity of CFs is light dependent and inactivated by proteinase K and heat treatment (100C). This is reminiscent of the necrosis-inducing properties of host selective toxins of other Dothideomycete pathogens such as Stagonospora nodorum and Pyrenophora tritici-repentis. Subsequent purifications of CFs and mass spectrometry identified several candidate proteins with necrosis-inducing activity. Heterologous expression of the two most prominent proteins in Pichia pastoris produced sufficient quantities for infiltration assays in a panel of wheat cultivars that showed differential responses, suggesting specific recognition.

    Chapter 6 provides a general discussion of the thesis and puts the results obtained in a broader perspective with a focus on the genome structure of M. graminicola and its function. In addition, aspects of the hemi-biotrophic lifestyle, the relevance of secreted proteins for the wheat-M. graminicola pathosystem in relation to gene-for-gene models and the potential implications for resistance breeding strategies are discussed.

    Molecular characterization of beet necrotic yellow vein virus in Greece and transgenic approaches towards enhancing rhizomania disease resistance
    Pavli, O.I. - \ 2010
    Wageningen University. Promotor(en): Just Vlak; G.N. Skaracis, co-promotor(en): M. Prins; N.J. Panopoulos. - [S.l. : S.n. - ISBN 9789085855477 - 166
    beta vulgaris - suikerbieten - bietenrhizomanievirus - stammen (biologisch) - karakteristieken - pathogeniteit - plantenvirussen - ziekteresistentie - resistentie van variëteiten - transgene planten - genexpressie - griekenland - beta vulgaris - sugarbeet - beet necrotic yellow vein virus - strains - characteristics - pathogenicity - plant viruses - disease resistance - varietal resistance - transgenic plants - gene expression - greece
    Rhizomania disease of sugar beet, caused by Beet necrotic yellow vein virus (BNYVV), is responsible for severe economic losses. Due to the widespread occurrence of BNYVV and the absence of other practical and efficient control measures, economic viability of the crop is to the largest extent dependent on the use of varieties genetically resistant to the disease. Recent reports on the emergence of virus strains capable of compromising the Rz1-based resistance as well as on the spread of highly pathogenic RNA 5-containing BNYVV isolates have necessitated a detailed investigation of the situation as it evolves in Greece. The study revealed the widespread occurrence of BNYVV throughout the country as well as the prevalence of pathotype A isolates in all sugar beet growing regions. Sequence determination of the p25 protein, responsible for symptom development, pointed to the amino acid motifs ACHG/VCHG in the hypervariable amino acid region 67-70. However, the presence of valine (V) in position 67 was not associated with increased pathogenicity and resistance breaking properties. Disease severity appeared mostly dependent on agroclimatic conditions influencing the progress of the disease. A survey for a possible occurrence of Beet Soilborne Virus (BSBV) and Beet Virus Q (BVQ) in rhizomania infested fields revealed the co-existence of both viruses, with BVQ being systematically found in co-infections with BNYVV, while BSBV was in all cases only found in triple infections. Towards the exploitation of the antiviral properties of RNA silencing, three intron hairpin constructs carrying parts of the BNYVV replicase gene, were evaluated for their potential to confer rhizomania resistance in Ri T-DNA-transformed sugar beet roots. The results show that transgenic hairy roots were effectively protected against the virus disease and further indicate that the developed methodology for Agrobacterium rhizogenes-mediated transformation can be employed as a suitable platform to study transgene expression in sugar beet and other transformation recalcitrant crop species. In parallel, the potential to exploit the HrpZPsph protein from Pseudomonas syringae pv. phaseolicola for engineering rhizomania resistance in sugar beet against BNYVV was demonstrated by the successful engineering and protection against BNYVV in the model plant Nicotiana benthamiana
    Signaling pathways involved in pathogenicity and development of the fungal wheat pathogen Mycosphaerella graminicola
    Mehrabi, R. - \ 2006
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema; M.A. de Waard. - [S.l.] : S.n. - ISBN 9789085044093 - 176
    mycosphaerella graminicola - signaaltransductie - pathogeniteit - genetische analyse - regulator-genen - genexpressie - triticum aestivum - tarwe - septoria - triticum aestivum - wheat - mycosphaerella graminicola - septoria - signal transduction - pathogenicity - genetic analysis - regulatory genes - gene expression
    Mycosphaerella graminicola(Fuckel) J. Schröt is the causal agent of septoria tritici leaf blotch, which is the major foliar wheat disease in most temperate areas.Direct losses and the cost of control strategies contribute to the huge economical importance of this disease. M. graminicolais a dimorphic fungal pathogen that grows in yeast-like or filamentous fashion depending on the environmental conditions. Filamentation is required for pathogenicity of this non-appressorium forming pathogen, which has a hemibiotrophic lifestyle characterized by an initial biotrophic phase (about 10 days) that is followed by a necrotrophic phase during which numerous asexual and eventually sexual fructifications are produced. 

    This thesis is part of an ongoing research program aimed at understanding the genetic and molecular control of pathogenicity. The research was initiated with the generation of EST and genomic sequence data of the pathogen. This thesis describes the functional analyses of 10 genes encoding proteins involved in signal transduction pathways operating in M. graminicola when grown in vitro and in planta . Chapter one gives an introduction to the research topic. In chapter two, the role of the mitogen-activated protein kinase (MAPK)-encoding gene, MgFus3 , the ortholog of Fus3 controlling mating in Saccharomyces cerevisiae , is described. Disruption of this gene prevented melanization of mycelia and formation of pycnidia in vitro. MgFus3 mutants are non-pathogenic. This is ascribed to impaired penetration of stomata, possibly due to inability of the mutants to recognize stomata. 

    Chapter threedescribes the MAPK-encoding gene, MgSlt2 . This is the ortholog of Slt2 in S. cerevisiae that regulates cell wall biosynthesis. In M. graminicola , MgSlt2 plays a role in cell wall integrity since MgSlt2 mutants were affected in polarized growth and showedprogressive autolysis during aging. They were also hypersensitive to glucanase and several fungicides and did not produce aerial mycelium or melanin on potato dextrose agar (PDA). Pathogenicity assays revealed that virulence of MgSlt2 mutants was severely reduced. Electron microscopy and histopathological analyses showed that MgSlt2 mutants penetrated wheat stomata regularly, but were unable to establish invasive growth and did not produce asexual fructifications. BecauseMgSlt2 is involved in cell wall integrity, MgSlt2 mutants are probably more sensitive to hitherto unknown plant defense compounds, which might explain the compromised colonization of mesophyll tissue.

    Chapter fourdescribes characterization of MAPK-encoding gene, MgHog1 , and the p21-activated kinase (PAK)-encoding gene, MgSte20 .MgHog1 mutants were osmosensitive, highly resistant to the fungicides fludioxonil, fenpiclonil and iprodione did not melanize and were unable to switch from yeast-like to filamentous growth. As a result of the impaired dimorphic switch, MgHog1 mutants were unable to establish infectious germ tubes and therefore failed to penetrate wheat leaves. This demonstrates that dimorphic transition is a key factor in pathogenicity of M. graminicola. Phenotypes of MgSte20 mutants were identical to the wild-type isolate in all tested conditions.

    Fructification of M. graminicola is a complex process requiring differentiation of the infectious hyphae in the substomatal cavities during the latter stages of infection. In Chapter five, functional analyses of genes encoding the catalytic ( MgTpk2 ) and the regulatory ( MgBcy1 ) subunits of PKA showed that these genes are essential for asexual fructification. MgTpk2 and MgBcy1 mutants were able to germinate, penetrate and colonize mesophyll tissue, but were unable to differentiate pycnidia. Our data provide evidence that the cAMP pathway regulates filamentation through MgTpk2 and MgBcy1 . Disruption of MgTpk2 impaired filamentation. In addition, the MgTpk2 mutants became melanized faster and secreted a dark-brown pigment into yeast glucose broth medium (YGB), whereas MgBcy1 mutants showed delayed melanization on PDA and were osmosensitive. Overall, the divergent functions of the regulatory and the catalytic subunits of PKA indicate that proper regulation of PKA activity is required for various physiological processes including differentiation, filamentation, osmoregulation and melanization. 

    Chapter sixdescribes the characterization of three Gaprotein-encoding genes ( MgGpa1, MgGpa2 and MgGpa3 ) and one Gbprotein-encoding gene ( MgGpb1 ) in M. graminicola . Phylogenetic comparisons and sequence analyses of Gaproteins of M. graminicola revealed that MgGPA1 and MgGPA3 can be categorized as homologs of the mammalian Gaiand Gasfamilies, respectively, whereas MgGPA2 is unrelated to mammalian Gaproteins. MgGpa1 , MgGpa3 and MgGpb1 mutants exhibited different phenotypes when grown on PDA at 20 °C that were not observed when the temperature was raised to 28 °C. Melanization did not occur in the MgGpa1 and MgGpb1 mutants, and the former formed fluffy mycelia in YGB and hardly produced spores. MgGpb1 mutants showed a nested type of growth on PDA that resulted from hampered filamentation, numerous cell fusions and increased anastomosis. Therefore, we concluded that MgGpa1 negatively regulates filamentation, which is positively regulated by MgGpa3 and MgGpb1 . Interestingly, unlike the response of the MgGpa1 mutants, exogenous cAMP restored the phenotype of the wild-type in the MgGpb1 and MgGpa3 mutants, indicating a stimulating function for MgGPB1 and MgGPA3 and an inhibitory function for MgGPA1 in the regulation of the cAMP pathway. Pathogenicity assays revealed that MgGpa1 , MgGpa3 and MgGpb1 are required for virulence of M. graminicola whereas MgGpa2 is dispensable.

    Finally, in chapterseven the results described in this thesis are discussed in a broader perspective. The roles of the various kinases as well as G proteins during development in vitro and in planta for fungal pathogenesis in general and for M. graminicola in particular are highlighted. Also, the potential of M. graminicola as a new model fungus is addressed. The power of comparative genomics can be fully exploited once the genomic sequence of the fungus is available.
    Aspects of sexual reproduction in Mycosphaerella species on wheat and barley : genetic studies on specificity, mapping, and fungicide resistance
    Ware, S.B. - \ 2006
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema; M.A. de Waard. - [S.l.] : S.n. - ISBN 9789085045274 - 190
    triticum aestivum - tarwe - hordeum vulgare - gerst - mycosphaerella graminicola - geslachtelijke voortplanting - plantenziekteverwekkende schimmels - septoria - gastheerspecificiteit - genetische kartering - resistentie tegen pesticiden - virulentie - pathogeniteit - ziekteresistentie - overleving - triticum aestivum - wheat - hordeum vulgare - barley - mycosphaerella graminicola - septoria - sexual reproduction - survival - plant pathogenic fungi - host specificity - virulence - pathogenicity - genetic mapping - pesticide resistance - disease resistance

    Mycosphaerella species are haploid ascomycetes that cause major economic losses in crops that include cereals, citrus fruits, and bananas, among others. Two organisms in this genus are Mycosphaerella graminicola (Fuckel) .I. Schröt (anamorph Sepioria tritici) and Septoriapasserinii. M graminicola is the causal agent of septoria tritici blotch of both bread wheat and durum wheat species, and S. passerinii causes septoria speck!ed 1eaf blotch of barley. M. graminicola is a heterothaliic fungus with a very active sexual cycle, while no sexual cycle has been reported for S. passerinii.

    This thesis inciudes studies on mating and genetics of both M. graminicola and S. passerinii. Chapter 1 gives an introduction to these pathogens and an overview of the research topics. In Chapter 2. we studied the possibility of in planta generation of sexual progeny of the fungal wheat pathogen M graminicola when one of the parents was avirulent on a resistant host. We found that avirulent isolates are able to survive and even increase in biomass after inoculation onto resistant wheat cultivars and can complete sexual cycles on resistant cultivars to yield viable ascospores as long as the other parent is virulent. To our knowledge, this is the first time such a phenomenon has been described, and the possibility to generate such crosses opened the door for studies in Chapters 3 and 4.

    Chapter 3 describes the construction of two high-density genetic linkage maps of M graminicola using Diversity Arrays Technology (DArT) and the integration of these into a core map with common markers due to a common parental isolate. One of the maps was constructed based on segregations of progeny of two bread wheat-derived isolates, IPO323 and IPO94269, and the other was constructed from segregations of progeny of IPO323 and the durum wheat-derived isolate IPO95052. In total, 1,144 markers made up the integrated core map. Analyses from this study revealed that progeny had translocations, diploid and partial diploid linkage groups, and loss of entire linkage groups.

    Although M. graminicola causes disease on both bread wheat and durum wheat, isolates within the population show clear distinctions in either virulence on bread wheat or on durum wheat (host specificity)- In Chapter 4, we studied the genetic basis of host specificity in M. graminicola using 163 progeny from crosses between the Dutch bread wheat-derived 1PO323 and the Algerian durum wheat-derived TPO95052. Phenotyping of progeny was performed on a set of seven differential cultivars, and progeny crossed on either bread wheat or durum wheat could infect cultivars of bread wheat, durum wheat, both, or neither. These results were used to map nine quantitative trait loci (QTLs) on seven linkage groups in the high-densiry genetic linkage map from Chapter 3. One of these loci was previously mapped for cultivar specificity of IPO323in bread wheat, and the same locus was now mapped for host specificity of IPO323 to durum wheat. Our results show that the reported host specificity is probably the result of combinations of a number of independently inherited avirulence factors.

    In addition to avirulence genes, fungi can inherit other traits for survival. One such heritable trait is a point mutation in the mitochondrial genome that conveys resistance to strobilurin fungicides. Chapter 5 describes a study on the inheritance of strobilurin resistance. Resistant and sensitive isolates of M. graminicola were crossed on wheat seedlings that were both untreated and preventively treated with various concentrations of azoxystrobin (Amistar™), and progeny were analyzed to determine the rate of inheritance of the aforementioned mutation. Preventive rates from 3.125-200% Amistar™ resulted in completely resistant progeny populations despite the fact that the segregation of nuclear genes confirmed regular meiotic behavior. We conclude that sensitive isolates overcome the disruption of mitochondrial respiration and participate in sexual reproduction even under high fungicide pressure and that fungicide stress induces or results in preferential mating in M. graminicola.

    The barley pathogen S. passerinii clusters closely to M. graminicola in phylogenetic studies based on ITS sequences, and a high degree of genetic variation among isolates is found in nature. However, no teleomorph has been reported for this S. passerinii, and hence, it was considered to be asexual. Nevertheless, mating type idiomorphs were recently detected and isolated. In Chapter 6, we studied the possibility of a Mycosphaerella teleomorph associated with S. passerinii. Isolates with opposite mating types were co-inoculated onto barley cultivars, and leaves were monitored for the discharge of ascospores. Characterization of a segregating population by both molecular and phenotypic analyses confirmed that we successfully generated the hitherto unknown Mycosphaerella teleomorph of S. passerinii.

    Finally, the results of this thesis are discussed in a broader perspective in Chapter 7 in relation to epidemiology, co-evolution, and durability of resistance in the wheat-M. graminicola pathosystem. The proven ability of avirulent isolates of M. graminicola to generate sexual progeny on resistant cultivars represents a new dynamic in population genetics that has not'previously been considered in epidemiology. Resu!ts from this thesis emphasize the complex ways in which the sexual cycle contributes to the overall success of M. graminicola on wheat.

    Verticillium-verwelkingsziekte in bomen: detectie, voorspelling van aantasting en beheersing van de ziekte
    Goud, J.C. - \ 2004
    Gewasbescherming 35 (2004)2. - ISSN 0166-6495 - p. 78 - 80.
    bosbomen - plantenziekteverwekkende schimmels - verticillium dahliae - virulentie - pathogeniteit - bodembiologie - epidemiologie - boomteelt - biologische bestrijding - straatbomen - acer - catalpa bignonioides - forest trees - plant pathogenic fungi - verticillium dahliae - virulence - pathogenicity - soil biology - epidemiology - arboriculture - biological control - street trees - acer - catalpa bignonioides
    Resultaten van promotie-onderzoek, waarbij in twee veldproeven de relatie werd vastgesteld tussen verwelkingsziekte in Noorse esdoorn (Acer platanoides) en trompetboom (Catalpa bignonioides) en besmettingsgraad van de bodem. Tevens werd de effectiviteit van biologische grondontsmetting onderzocht als bestrijdingsmethode voor Verticillium dahliae in de boomkwekerij
    Verticillium wilt in trees. Detection, prediction and disease management
    Goud, J.C. - \ 2003
    Wageningen University. Promotor(en): Ariena van Bruggen, co-promotor(en): Aad Termorshuizen. - [S.I.] : S.n. - ISBN 9789058088727 - 98
    verticillium dahliae - verwelkingsziekten - houtachtige planten als sierplanten - plantenziekteverwekkende schimmels - virulentie - bodembiologie - epidemiologie - pathogeniteit - nederland - verticillium dahliae - wilts - ornamental woody plants - plant pathogenic fungi - virulence - soil biology - epidemiology - pathogenicity - netherlands

    Verticillium dahliae , the causal agent of verticillium wilt, is the cause of high losses in a number of crops, especially nursery trees. Existing methods for quantification of V. dahliae microsclerotia in the soil were compared. The distinction of V. dahliae and V. tricorpus was studied on two semi-selective media. The morphology was highly dependent on the medium. Discriminating morphological characteristics were successfully used to identify isolates. There were no virulence differences on trees between the two V. dahliae VCGs that occur in the Netherlands. Biological soil disinfestation reduced V. dahliae in the soil by 85% and Pratylenchus fallax nematodes by 99%, through the creation of anaerobic conditions. The relationships between soil inoculum densities and verticillium wilt in Acer platanoides and Catalpa bignonioides showed that up to 5% diseased plants occurred at 1-2 detected microsclerotia per g soil. Diseased plants often recovered, but had a higher chance of becoming diseased again.

    Variation in Phytophthora infestans: sources and implications
    Flier, W.G. - \ 2002
    Gewasbescherming 33 (2002)2. - ISSN 0166-6495 - p. 66 - 69.
    phytophthora infestans - plantenziekteverwekkende schimmels - aardappelen - tomaten - pathogeniteit - genetische variatie - ziekteresistentie - phytophthora infestans - plant pathogenic fungi - potatoes - tomatoes - pathogenicity - genetic variation - disease resistance
    Uitgebreide samenvatting van de dissertatie van Wilbert G. Flier over de oömyceet Phytophthora infestans (Monst.) de Bary, de veroorzaker van 'het kwaad' ofwel de aardappelziekte in de teelt van aardappelen en tomaten
    Meer resistente/virulente pathogenen?
    Kieboom, J. ; Abee, T. ; Hermans, A. ; Aarts, H. ; Berk, P. ; Jonge, R. de - \ 2002
    Voedingsmiddelentechnologie 35 (2002). - ISSN 0042-7934 - p. 29 - 29.
    pathogenen - voedselbesmetting - virulentie - pathogeniteit - voedselmicrobiologie - risicofactoren - conserveerkwaliteit - pathogens - food contamination - virulence - pathogenicity - food microbiology - risk factors - canning quality
    Toepassing van milde conserveringsmethoden kan leiden tot voedselpathogenen die meer virulent zijn. De risicobepaling van de blootstelling aan pathogene micro-organismen in levensmiddelen
    Variation in Phytophthora infestans : sources and implications
    Flier, W. - \ 2001
    Wageningen University. Promotor(en): R.F. Hoekstra; L.J. Turkensteen; W.E. Fry. - S.l. : S.n. - ISBN 9789058084200 - 206
    phytophthora infestans - plantenziekteverwekkende schimmels - solanum tuberosum - aardappelen - pathogeniteit - genetische variatie - ziekteresistentie - phytophthora infestans - plant pathogenic fungi - solanum tuberosum - potatoes - pathogenicity - genetic variation - disease resistance

    The oomycete pseudofungus Phytophthora infestans (Mont.) de Bary, the causal organism of late blight, is considered to be one of the most devastating pathogens affecting potatoes and tomatoes worldwide. In Europe, the pathogen caused severe epidemics on potatoes after its introduction in 1845. Late blight management became much more troublesome after the introduction of another P. infestans population of Mexican origin. The present population of P. infestans in the Netherlands and an increasing number of other European countries consists of both A1 and A2 mating type isolates and sexual reproduction has been reported. The goals of this thesis were to study late blight epidemiology and population biology of the present P. infestans population. Therefore, sources and patterns of variation for pathogenicity in sexual P. infestans populations were studied and the impact of increased levels of aggressiveness on the stability of partial resistance to late blight was investigated. Results showed that considerable levels of variation for aggressiveness are maintained in regional populations of P. infestans in the Netherlands and that oospores are readily produced in field crops and volunteer potato plants. Oospore production and viability proved to be highly dependent on combining abilities of parental strains and there are indications that oospores are able to survive in soils for three to four years. Increased aggressiveness and cultivar-by-isolate interactions between partial resistant potato cultivars and P. infestans strains negatively affect the stability of resistance. Population genetic studies in its centre of origin and diversity revealed differentiation within the Toluca Valley implying host specificity. From the results presented in this thesis, it is clear that the introduction of a sexually reproducing population of P. infestans in the Netherlands has had a major impact on late blight epidemics and population biology of the late blight pathogen. Sexual reproduction has therefore led to a genetically more diverse population of P. infestans in the Netherlands that is marked by an increased adaptability to host and environment.

    Specificity, pathogenicity and population dynamics of the endoparasitic nematode Heterodera arenaria in coastal foredune
    Stoel, C.D. van der - \ 2001
    Wageningen University. Promotor(en): L. Brussaard; J.W. Woldendorp; W.H. van der Putten. - S.l. : S.n. - ISBN 9789058084361 - 135
    plantenparasitaire nematoden - heterodera - ammophila arenaria - plantenziekteverwekkers - populatiedynamica - plantensuccessie - pathogeniteit - vastleggen van duinen - identificatie - biochemische technieken - plant parasitic nematodes - heterodera - ammophila arenaria - plant pathogens - population dynamics - plant succession - pathogenicity - sand dune stabilization - identification - biochemical techniques

    Key words : Heterodera , plant-parasitic nematodes, soil pathogens, Ammophila arenaria , occurrence, abundance, specificity, population dynamics, life history, pathogenicity, PCR-SSCP, molecular method, escape, sand burial, dispersal, migration, fitness, development time, survival, reproductive success, bottom-up, top-down.

    In natural ecosystems hardly any attention has been given to the population dynamics of plant-parasitic nematodes. In coastal foredunes, plant-parasitic nematodes are supposed to be involved in the degeneration and succession of the dominant sand-fixing grass Ammophila arenaria (Marram grass). The specificity, pathogenicity and population dynamics of the sedentary endoparasitic nematode Heterodera arenaria have been studied to determine if this species might be a key component of the soil pathogen complex of A. arenaria.

    H. arenaria was found to be specific to Elymus farctus and A. arenaria in the mobile area of the coastal foredunes. Colonisation of the newly deposited sand layer by H. arenaria corresponded well with the development of pathogenicity in a series of bioassays. However, direct addition of the nematode to A. arenaria did not result in growth reduction of the plant. So, H. arenaria behaves like a biotrophic parasite, which has a high specificity but is not aggressive. Therefore, H. arenaria did not seem to be directly involved in the degeneration of A. arenaria .

    Each year, the majority of the population of new H. arenaria cysts develops in the newly deposited sand layers. These layers are colonised by A. arenaria roots throughout the growing season. Migration to the new root layer may offer an individual nematode the benefit of early development and a larger potential offspring. The continuous release of juveniles in the field and their development in experiments indicate that release of juveniles from cysts is an ultimately determined process. Juveniles were found to emerge in November and many eggs or juveniles did not survive the winter period. The strategy of release, however, seems effective; the distance of migration could be too large to detect specific cues from the plant and the start of root formation in the field is highly variable. The emergence of juveniles late in the growing season could result in a second generation within the same year. The constant number of cysts per gram of roots suggests that the population density of H. arenaria is most likely a bottom-up directed process.

    Molecular genetic analysis of the pathogenicity of the potato cyst nematode Globodera rostochiensis
    Qin, L. - \ 2001
    Wageningen University. Promotor(en): J. Bakker; J. Helder, co-promotor(en): G. Smant. - S.l. : S.n. - ISBN 9789058084521
    plantenparasitaire nematoden - globodera rostochiensis - complementair dna - polymorfisme - genexpressie - pathogeniteit - moleculaire genetica - bio-informatica - plant parasitic nematodes - globodera rostochiensis - pathogenicity - complementary dna - polymorphism - gene expression - molecular genetics - bioinformatics

    A new strategy to identify pathogenicity factors from the potato cyst nematode Globodera rostochiensis is developed. cDNA-AFLP technology and in situ hybridization allowed us to efficiently select putative pathogenicity factors among thousands of expressed genes. As a result, an unprecedented number of putative pathogenicity factors from this obligatory plant parasite were cloned.

    A powerful bioinformatics tool named GenEST to link expression data generated from cDNA-AFLP directly to cDNA sequence data was developed. This computer program would be very useful for functional genomics studies in other systems as well.

    A RanBPM (Ran-Binding Protein in Microtubule organization)-like gene family was identified. The proteins encoded by these genes are probably secreted by the nematode into plant cells and might manipulate the host cell development by changing the dynamic instability of microtubules.

    A promoter region from the potato cyst nematode was shown to be functional in a distantly related nematode species Caenorhabditis elegans . This promoter could be a valuable tool to drive gene expression in transgenic plant-parasitic nematode species to assess the relative importance of putative pathogenicity factors.

    A paper based from one chapter of this thesis will be submitted to Science. In view of their press embargo policy, I will not discussed the detail of this particular finding here.

    Classic fowl plague of avian influenza
    Koch, G. van der; Groot, J.A. - \ 2000
    Tijdschrift voor Diergeneeskunde 125 (2000)10. - ISSN 0040-7453 - p. 322 - 326.
    vogels - aviaire influenzavirussen - influenza - virusziekten - pathogeniteit - diergeneeskunde - birds - avian influenza viruses - influenza - viral diseases - pathogenicity - veterinary science
    De uitbraak van aviaire influenza in Italie heeft duidelijk gemaakt dat laagpathogene stammen van het virus binnen korte tijd in het veld kunnen evolueren tot hoogpathogene stammen. In het artikel wordt de pathogenese van laag- en hoogpathogene virussen beschreven en toegelicht op welke genetische eigenschap van het virus het verschil in pathogenese berust
    Molecular tools to unravel the role of genes from Phytophthora infestans
    West, P. van - \ 2000
    Agricultural University. Promotor(en): P.J.G.M. de Wit; F.P.M. Govers. - S.l. : S.n. - ISBN 9789058081735 - 150
    aardappelen - solanum tuberosum - plantenziekteverwekkende schimmels - phytophthora infestans - genexpressie - moleculaire genetica - virulentie - pathogeniteit - potatoes - solanum tuberosum - plant pathogenic fungi - phytophthora infestans - gene expression - molecular genetics - virulence - pathogenicity

    The oomycete plant pathogen Phytophthora infestans is the causal agent of potato late blight. P. infestans is undoubtedly the best known and most studied Phytophthora species today. This is mainly because it is such a devastating pathogen that can cause complete destruction of a potato field in only a few days. In this thesis, we describe the characterisation of four P. infestans genes with presumed functions in pathogenicity and virulence, and the development of tools to study expression and function of two of these genes in P. infestans . The four genes are the in planta -induced genes ipiB and ipiO , the elicitin gene inf1 , and the stress-induced gene ric1 . We used theβ-glucuronidase reporter gene for expression analysis of the ipiO gene, and homology-dependent gene silencing for functional analysis of the inf1 gene. The latter resulted in the discovery of a new phenomenon, which we named internuclear gene silencing.

    In Chapter 2, we describe the characterisation of the in planta -induced genes ipiB and ipiO . IpiB constitutes a gene family with at least three members, ipiB1 , ipiB2 and ipiB3 , which are clustered in a head-to-tail arrangement. The ipiB genes are highly homologous throughout their promoter, coding and terminator sequences and encode three similar glycine-rich proteins of 301, 343 and 347 amino acids, respectively. The glycine-rich domains of the IPI-B proteins are predominantly composed of two repeats with the core sequences, A/V-G-A-G-L-Y-G-R and G-A-G-Y/V-G-G. The IPI-B proteins contain a putative signal peptide of 20 amino-acids, suggesting that the proteins are targeted to a specific organelle or to the outside of the cell. We speculate that the IPI-B proteins are structural proteins associated with the cell wall that are possibly involved in the development of infection structures. Also ipiO is a small gene family. Two members, ipiO1 and ipiO2 are closely linked and arranged in an inverted orientation. The ipiO genes encode almost identical 152 amino acid-proteins which do not have any significant homology with sequences present in data libraries. The IPI-O proteins contain a putative signal peptide which may target them to the extracellular matrix. A putative cell attachment sequence (RGD), functional in mammalian systems, was identified.

    Expression of the ipiO gene was analysed during several developmental stages of the life cycle of P. infestans (Chapter 3). IpiO mRNA was detected in zoospores, cysts, and germinating cysts, but not in sporangia or in mycelia grown in vitro . IpiO is also expressed during colonisation of potato leaves. In disease lesions, ipiO mRNA was detected in the water-soaked area and the surrounding healthy-looking plant tissue. IpiO mRNA could not be detected in necrotised tissue and sporulating areas of a lesion. Cytological assays were performed to determine more precisely the location and time of ipiO gene expression in planta . P. infestans transformants expressing a transcriptional fusion between the ipiO1 promoter and theβ-glucuronidase (GUS) reporter gene showed that GUS staining was specifically found in the subapical and vacuolated area of tips of invading hyphae. Therefore, we concluded that ipiO is expressed in hyphae during biotrophic stages of the infection process. We speculate that IPI-O has its function in the hyphal tips at the edge of the expanding lesion where the pathogen is invading healthy leaf cells. The IPI-O protein may well be localised at the interface between the invading hyphae and the plant cells, and could play a role in pathogenicity and/or virulence.

    To isolate more genes that might be involved in the interaction between P. infestans and potato, we used a small scale expressed sequence tag (EST) approach (Chapter 7). Twenty-two P. infestans cDNA clones were randomly selected from a potato- P. infestans interaction cDNA library. Five of these clones appeared to be derived from the same gene, ric1 . Two copies of the ric1 gene were identified and both genes share 98% homology at the nucleotide sequence level and 100% at the amino acid level. The open reading frame predicts a small protein sequence of 57 amino acids. The highly hydrophobic protein has two potential membrane spanning domains. The deduced amino acid sequence shows high homology with three putative plant proteins encoded by genes of which the expression is specifically induced during stress conditions. Expression of ric1 increased considerably during osmotic stress and at high pH. We hypothesise that RIC1 is a structural protein that is necessary to maintain membrane integrity, especially during unfavourable conditions.

    When culturing various Phytophthora species in liquid medium, all examined species secrete highly abundant 10 kDa proteins which have been shown to elicit a hypersensitive response when injected into tobacco leaves. It was hypothesised previously that these 10 kDa proteins, collectively called elicitins, function as plant species specific avirulence factors. In order to test this hypothesis, we set out to clone the elicitin gene inf1 , of P. infestans (Chapter 4) and subsequently generated mutants that no longer produced the INF1 protein (Chapter 5 & 6). An inf1 cDNA was isolated by heterologous hybridisation of a potato- P. infestans interaction cDNA library using parA1 , the gene encoding the major secreted elicitin of P. parasitica , as a probe. Inf1 encodes a 118 amino acid protein including a 20 amino acid signal peptide. Detailed expression studies show that inf1 is expressed in mycelium grown in various culture media, whereas expression was not detected in sporangia, zoospores, cysts and germinating cysts. The highest levels of expression of inf1 are observed in in vitro -grown mycelium and in planta during the late stages of infection when profuse sporulation and leaf necrosis occur. Expression is, however, down-regulated during early biotrophic stages of the interaction.

    To obtain P. infestans strains deficient in INF1 production, we explored a homology-dependent gene silencing strategy. Integrative transformation with antisense, sense and promoter-less constructs in P. infestans was performed to generate mutants. Inf1 mRNA and INF1 protein could not be detected in up to 20% of the transformants. The silenced state of the inf1 gene was shown to be mitotically stable under various conditions in vitro and in planta . In pathogenicity assays, the INF1-deficient strains remained pathogenic on the host plants potato and tomato. However, in contrast to the wild type and control transformant strains, INF1-deficient strains induced also disease lesions and extensive sporulation when inoculated on Nicotiana benthamiana . These results demonstrate that recognition of INF1 elicitin leads to active resistance in N. benthamiana to P. infestans and that INF1 functions as a plant species-specific avirulence factor.

    In Chapter 5, we describe experiments to identify the mechanism of silencing of the inf1 gene in P. infestans . Nuclear run-on assays showed that inf1 gene silencing is regulated at the transcriptional level. Interestingly, DNA methylation, a feature often associated with transcriptionally regulated gene silencing could not be detected in the inf1 gene sequences. Heterokaryons obtained by somatic fusion of an inf1 -silenced transgenic strain and a wild type strain displayed also stable gene silencing, demonstrating that inf1 silencing is dominant and acts in trans. The inf1 gene remained silenced in non-transgenic homokaryotic single zoospore isolates released from the silenced heterokaryons. Apparently, the presence of transgenes is not essential for maintaining the silenced status of the endogenous inf1 gene. Karyogamy was not demonstrated and hence, it is unlikely that the silenced state of the inf1 gene is transmitted from nucleus to nucleus by specific DNA-DNA interactions. Consequently, we propose a novel silencing phenomenon, called internuclear gene silencing, in which a diffusible silencing factor is involved in inducing stable gene silencing. We speculate that the proposed diffusible trans -acting silencing factor is either a protein, an aberrant RNA molecule or a complex consisting of RNA and protein. We envisage that such a molecule is transported from nucleus to nucleus where it may facilitate an inheritable change in inf1 -expression possibly initiated by changes in chromatin structure of the target gene or regions surrounding the target gene. Internuclear gene silencing is clearly a trans -inactivation phenomenon that is reminiscent of paramutation.

    In Chapter 8, we discuss how the results presented in this thesis will contribute to a better understanding of the biology and pathogenicity of P. infestans . On the long run, increased knowledge of this notorious pathogen will help in developing alternative methods to control potato late blight.

    The helper component-proteinase of cowpea aphid-borne mosaic virus
    Mlotshwa, S. - \ 2000
    Agricultural University. Promotor(en): A. van Kammen; J. Wellink; I. Sithole-Niang. - S.l. : S.n. - ISBN 9789058083401 - 111
    vigna unguiculata - vignabonen - potyvirus - kousenbandrolmozaïekvirus - pathogeniteit - weerstand - dna-sequencing - dna - genoomanalyse - genetische modificatie - genetische transformatie - ziekteresistentie - vigna unguiculata - cowpeas - potyvirus - blackeye cowpea mosaic virus - pathogenicity - resistance - dna sequencing - dna - genome analysis - genetic engineering - genetic transformation - disease resistance

    Cowpea aphid-borne mosaic potyvirus causes severe yield losses in cowpea, an important legume crop in semi-arid regions of Africa. We have elucidated the genomic sequence of the virus and subsequently focused our attention on the so-called helper component-proteinase (HC-Pro), a virus-encoded multifunctional protein with roles in different steps of the virus life cycle. Our study has shed more insight into some of the molecular properties of this protein. We have shown that HC-Pro is able to shut down host defense responses, and this puts HC-Pro at the core of the success of CABMV as a pathogen. The phenomenon also seems to benefit other viruses as they accumulate to higher levels and elicit enhanced symptoms in the presence of HC-Pro. On the other hand, we have found that the host does manifest an ability to counter the deleterious effects of HC-Pro. A full understanding of the molecular basis of this contest would enable the design of effective new strategies to protect plants from virus infections.

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