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.

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Virulence contribution and recognition of homologs of the Verticillium dahliae effector Ave1
Boshoven, Jordi C. - \ 2017
Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463436441 - 183
verticillium dahliae - plant pathogenic fungi - plant pathogens - disease resistance - virulence factors - virulence - immunity - host parasite relationships - plant-microbe interactions - symbiosis - mutagenesis - resistance breeding - plantenziekteverwekkende schimmels - plantenziekteverwekkers - ziekteresistentie - virulente factoren - virulentie - immuniteit - gastheer parasiet relaties - plant-microbe interacties - symbiose - mutagenese - resistentieveredeling

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.

Characterization of Coxiella burnetii outbreak strains
Kuley, Runa - \ 2017
Wageningen University. Promotor(en): Mari Smits; Jerry Wells, co-promotor(en): Alex Bossers. - Wageningen : Wageningen University - ISBN 9789463431514 - 226
coxiella burnetii - q fever - outbreaks - strains - characterization - pathogenesis - zoonoses - virulence - dna sequencing - polymerase chain reaction - livestock farming - netherlands - q-koorts - uitbraken (ziekten) - stammen (biologisch) - karakterisering - pathogenese - zoönosen - virulentie - dna-sequencing - polymerase-kettingreactie - veehouderij - nederland

Q fever is a worldwide zoonotic infectious disease caused by the bacterium Coxiella burnetii. During 2007-2010, the largest Q fever outbreak was reported in The Netherlands, where more than 4000 human cases were registered showing a serious burden of the disease. During this outbreak, goats harboring predominantly the CbNL01 genotype strain were identified as the major source of disease in humans and drastic measures such as mass culling of infected goats were implemented to reduce the spread of the pathogen and control the disease. In order to minimize such complications in the future, it is crucial to have a thorough understanding of the disease causing pathogen and to develop effective Q fever vaccines. The causes of the large Dutch outbreak are not well-understood and one of the main reasons speculated were the hyper-virulent behavior of the circulating C. burnetii isolates. The research described in this thesis focuses on the characterization of C. burnetii outbreak strains isolated from infected goats, cattle, sheep and human clinical materials. Our studies were initiated to better understand the bacterial pathogenesis, virulence, evolution, adaptations in various environments, host immune responses and to identify pathogen related factors that have modulated the disease outbreak. We specifically aimed to identify the virulence factors and mechanisms that contributed to the increased zoonotic potential of the strain associated with the Dutch Q fever outbreak.

The studies presented in this thesis majorly applied Pathogenomic approaches at the genome and transcriptome level to decipher host-pathogen interactions and to develop new tools to study C. burnetii infections. A transcriptome analysis of the outbreak C. burnetii strain of the CbNL01 genotype grown under in vivo and in vitro conditions resulted in the identification of distinct metabolic adaptations and virulence mechanisms of the bacterium. Detailed comparative analysis of complete genome sequences of C. burnetii strains showed a high similarity between strains of the same genotype. Genome sequences of the Dutch outbreak CbNL01 genotype strains were more divergent than the genome sequences of the less prevalent CbNL12 genotype strains and the NM reference strain. The analysis also showed that the high virulence of the outbreak strains was not associated with acquiring novel virulence-related genes arguing against the idea that the Dutch outbreak was due to emergence of hyper-virulent strains though horizontal gene transfer. Among the prominent genetic differences in the CbNL01 outbreak strains compared to CbNL12 and NM, were the presence of several point mutations and increased transposon mediated genome plasticity, which might have contributed to its epidemic potential. Point mutations, especially in a large number of membrane proteins, could also have contributed to the increased zoonotic potential of CbNL01 strains allowing this clone to escape the host immune responses in goats and humans. In addition, mutations in critical genes involved in virulence and evasion of the host immune system could be potentially involved in the increased virulence of the CbNL01 outbreak strains. On the contrary, studies on host immune responses in an in vivo (experimental infections in mice) and an in vitro (human PBMC’s stimulation) model did not show any difference associated with the strain genotype. However, differences in immune responses were found to be associated with the host-origin of the C. burnetii strains. Among different host-origin strains, strains derived from goats and humans generated significantly lower innate and adaptive immune responses than strains derived from cattle, whereas no differences in immune responses were observed when strains were grouped based upon their genotype. These observations support immune evasions as a major virulence strategy of goat and human strains in hosts and further suggest that bacteria originating from goats have a greater potential to cause outbreaks in humans. This indicates that for Q fever prevention purposes goats should be efficiently monitored for the presence of C. burnetii. Taken together, the results described in this thesis suggest that the virulence potential of C. burnetii strains is not only based on genetic differences, but also on other host-adaptation mechanisms such as transposition of genomic elements and/or differential regulation of gene expression. Finally, the results from this thesis provide a framework for future studies in the development of vaccines and diagnostic tools for Q fever.

Unravelling aspects of spatial and temporal distribution of Verticillium dahliae in olive, maple and ash trees and improvement of detection methods
Keykhasaber, Mojtaba - \ 2017
Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit, co-promotor(en): Jelle Hiemstra. - Wageningen : Wageningen University - ISBN 9789463430142 - 163
olea europaea - olives - acer - fraxinus - plant pathogenic fungi - verticillium dahliae - distribution - virulence - detection - olijven - plantenziekteverwekkende schimmels - distributie - virulentie - detectie

Vascular wilts caused by xylem-colonizing pathogens are among the most devastating plant diseases that affect a wide range of plant species worldwide. Information on the distribution of V. dahliae in infected trees helps to design an appropriate and efficient sampling method for reliable detection of the pathogen in diseased trees. In Chapter 3, the distribution of V. dahliae in young twigs and leaves of infected olive trees is studied by real-time quantification of V. dahliae DNA. Analysis of twig and leaf samples collected from different sides of the crown of infected olive trees showed a non-uniform distribution of the fungus within infected parts of diseased olive trees. It was demonstrated that testing of combined samples comprising subsamples from at least 5 twigs from different sides of the tree, or 5-10 random leaves, can reliably detect the pathogen. V. dahliae isolates that infect olive trees can be classified as defoliating (D) isolates that are highly virulent, or non-defoliating (ND) isolates that are generally less aggressive. Discrimination of these pathotypes is important in order to predict the severity of disease, and decide on appropriate disease management strategies. This is particularly important due to the alarming spread of highly virulent isolates of the D pathotype worldwide. In Chapter 4, a novel method is designed for accurate discrimination and sensitive detection of D and ND isolates of V. dahliae. Through comparative genomics of multiple D and ND isolates of V. dahliae a region was identified that is present in all sequenced ND isolates, while absent from all D isolates. Based on this presence-absence polymorphism, a set of primers was designed spanning this region that was able to generate differentially sized amplicons for isolates that belong to the different pathotypes. Additionally, a nested-PCR assay was designed to increase the sensitivity and improve detection of D and ND isolates in planta. In Chapter 5, the relation of the dynamics in pathogen distribution in infected plants to the differences in extent and severity of disease caused by D and ND isolates in resistant and susceptible olive genotypes is studied. To this end, the distribution of a D (V117) and a ND (V4) isolate of V. dahliae in root-inoculated young plants of a susceptible (Picual) and a partially resistant cultivar (Frantoio) of olive and its relationship to the disease progression was investigated using real-time PCR. The amount of pathogen DNA detected in the two cultivars correlated with their susceptibility to Verticillium wilt, with lower quantities of V4 and V117 DNA detected in ‘Frantoio’ than in ‘Picual’. Also quantities of pathogen DNA in V117-inoculated plants were higher than quantities of pathogen DNA in V4-inoculated plants. The distribution patterns of D and ND isolates in the lower, middle and top parts of tested olive cultivars showed that differences in symptom severity were related to amounts of the pathogen in lower and middle parts of the trees, since colonization of the pathogen in top parts of the stem of inoculated plants was minor and was not significantly different between treatments. Moreover, microscopic analysis of infection and colonization processes of V. dahliae in olive plants inoculated with GFP-labelled isolates revealed that colonization of the above ground tissues of infected olive plants is by means of conidia transported upward with the xylem sap stream. In Chapter 6 we investigated the spatial and temporal distribution of V. dahliae in relation to disease progression and recovery in stem-inoculated maple and ash trees. These species differ strongly in vascular anatomy with maple having a diffuse porous xylem anatomy whereas ash has a ring porous xylem anatomy. Results showed that that differences in the xylem anatomy of ash and maple did not significantly affect the speed and extent of the upward spread of the pathogen in stem-inoculated trees. Nevertheless, the xylem of ash trees is much less supportive for growth and survival of V. dahliae than that of maple trees, as in the year after inoculation disease incidence and also quantities of V. dahliae detected in maple trees were significantly higher than in ash trees. Moreover, V. dahliae could not be reisolated at all from ash trees that had recovered from disease. However, it could be detected by PCR in some cases in the xylem formed in the year of inoculation, never in the xylem formed in the year after inoculation. Nevertheless, V. dahliae easily could be detected in the wood of diseased ash and maple trees in the year after inoculation. Notably, despite the presence of a layer of terminal parenchyma cells between growth rings, in ash trees showing disease symptoms in the year after inoculation V. dahliae was present in the xylem of the new growth ring. It was also observed that in stem-inoculated trees V. dahliae can move downward from the point of inoculation into the root collar, which may provide an avenue for infection of new growth rings in ash trees.

A multidisciplinary approach to study virulence of the entomopathogenic fungus Beauveria bassiana towards malaria mosquitoes
Valero Jimenez, C.A. - \ 2016
Wageningen University. Promotor(en): Bas Zwaan; Willem Takken, co-promotor(en): Sander Koenraadt; Jan van Kan. - Wageningen : Wageningen University - ISBN 9789462578548 - 131 p.
beauveria bassiana - entomogenous fungi - virulence - vector control - mosquito-borne diseases - malaria - anopheles - culicidae - entomopathogene schimmels - virulentie - vectorbestrijding - ziekten overgebracht door muskieten

Although globally malaria mortality rates have fallen by 48% between 2000 and 2015, malaria is still killing an estimated 438,000 people each year. An effective way to alleviate the burden of malaria is to control its vector (malaria mosquitoes) using insecticides. This can be achieved either with insecticide-treated bed nets (ITNs) or through indoor residual spraying of insecticides (IRS). However, because of rapidly expanding insecticide resistance, there is a need to find alternatives to control the mosquitoes. Entomopathogenic fungi (EPF) could constitute an effective biological control tool, as is able to reduce malaria transmission under laboratory and field conditions. However, fundamental knowledge on the mechanisms and regulation of the infection process of the fungus, as well as insights into the defensive responses of the host insect to EPF, is limited. Therefore, the main goal of this thesis was to study virulence of the entomopathogenic fungus B. bassiana towards malaria mosquitoes using a multidisciplinary approach.

Chapter 2 provides an overview of existing knowledge of genes influencing virulence in EPF, with a special focus on B. bassiana. The infection cycle and virulence mechanisms are discussed, and put in a framework of novel strategies and experimental methods that are needed to better understand virulence and improve the usage of EPF as a biocontrol agent.

The study of natural variation in fungal virulence is a first step towards understanding the genetic mechanisms involved, because it reveals the extent of variation in the different components of virulence and their overall role. Chapter 3 describes the natural variation in virulence for 29 B. bassiana isolates that were tested on malaria mosquitoes. Furthermore, the phenotypic characteristics of the fungal isolates such as sporulation, spore size and growth were evaluated and their relationship with virulence analysed.

Based on the ample natural variation observed in fungal virulence, in Chapter 4, a comparative genomics analysis was performed on five selected isolates of contrasting virulence. In order to understand mechanisms underlying contrasting virulence, a comparison on gene gain/loss, single nucleotide polymorphisms (SNPs), secreted proteins, and secondary metabolites was performed. Insight is provided to the magnitude of the complexity of a trait such as virulence and suggests candidate genes that can be further studied using a functional analysis approach.

Chapter 5 focuses on an experimental evolution approach in which B. bassiana was solely using insects as a nutritional source for ten consecutive passages through malaria mosquitoes. Two isolates of B. bassiana that differed in virulence were compared to their respective ancestors, and they were assayed in virulence, fungal outgrowth, mycelial growth rate (MGR), and sporulation. Passage of the entomopathogenic fungi B. bassiana through the insect host resulted in an altered capacity to grow on different substrates while maintaining the ability to kill insects.

Chapter 6 presents a discussion on the main findings of this thesis and describes future perspectives to study virulence of the entomopathogenic fungi Beauveria bassiana in the context of biological control of malaria mosquitoes.

Discovery, characterization and applications of natural DNA transformation in Streptococcus suis
Zaccaria, E. - \ 2015
Wageningen University. Promotor(en): Jerry Wells, co-promotor(en): Peter van Baarlen. - Wageningen : Wageningen University - ISBN 9789462576056 - 171
streptococcus suis - virulence - pathogenesis - gene expression - direct dna uptake - virulence factors - infection - modeling - virulentie - pathogenese - genexpressie - directe dna-opname - virulente factoren - infectie - modelleren

Streptococcus suis is Gram-positive bacterium and its natural habitat is the upper respiratory tract of pigs, and in particular the tonsils and nasal cavity. Although it is considered to be a normal member of the adult pig microbiome, it can cause serious diseases in pigs and humans. S. suis is in fact one of the most important swine pathogens world-wide, causing a wide variety of diseases in pigs including septicemia, arthritis, endocarditis, and meningitis that leads often to a rapid death within 1-2 days. Although most human infections are considered the consequence of occupational exposure, in the last years the number of human cases has increased and isolates with multi-resistance genes have been isolated. Human infection caused by S. suis are characterized by a similar symptomatology as in pigs. Despite the economic loss in the pork industry due to S. suis infection and its importance as emerging zoonotic agent, experimental studies of S. suis virulence and pathology have been hampered by the lack of efficient methods for genetic transformation and the lack of a simple, cost-effective model to investigate S. suis virulence.

In some streptococcal species, genetic transformation can be carried out very efficiently as these species can be experimentally induced to take-up and recombine homologous extracellular DNA. The discovery of natural competence in some streptococci and the potential of opening up new avenues for genetic analysis of S. suis, was the motivation for investigating natural competence in this important pathogen.

In Chapter 2 we showed that a peptide pheromone induces competence in S. suis. The induction was dependent on ComX, a sigma factor that controls the streptococcal late competence regulon; the SigX-inducing peptide (XIP); and ComR, a regulator of comX. XIP was identified as an N-terminally truncated variant of ComS. This has resulted in the development of a novel methodology that will enable diverse research groups to accelerate discovery of novel features of S. suis ecology and pathology, especially with respect to virulence.

In Chapter 3 we investigated the genetic regulation of competence in S. suis and we provided a hypothetical model of the S. suis transformasome. We verified the essential role of the S. suis major pilin, and CinA for efficient competence development, supporting the notion that our predicted multi-protein transformasome indeed appears to function as described for other streptococci. We have also characterised the differential metabolic states that enable competence, and the metabolic state associated with competence exit (Chapter 4).

In Chapter 5 we investigated for the first time the use a zebrafish larvae model to assess the relative virulence of S. suis strains in porcine infections. Because of its convenience and cost-effectiveness, this model may be used to assay virulence of environmental S. suis strains, in particularly those of clinical relevance to infection of pigs and humans. Furthermore, a large number of bacterial mutants and strains can be screened for their virulence and in vivo pathogenicity, opening up new avenues to investigate the so far undiscovered pathways mediating successful host infection by S. suis.

In Chapter 6 we applied these two innovative methods (the competence system and the zebrafish larval model) to characterize two different two-component systems (TCS) of S. suis. TCS are important players in the regulation of bacterial adaptation to changes in environmental conditions, including those encountered in the host during infection. In this study, we studied the role of the two TCS of S. suis 2 strain S10 in virulence and in the survival of the bacteria in the bloodstream and host tissue.

Chapter 7 summarizes and discusses the key results and the future prospective of the thesis research.

Identification and functional characterization of proteases and protease inhibitors involved in virulence of fungal tomato pathogens
Karimi Jashni, M. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462574571 - 183
passalora fulva - plantenziekteverwekkende schimmels - virulentie - proteïnasen - proteïnaseremmers - plant-microbe interacties - genomica - solanum lycopersicum - tomaten - eiwitexpressieanalyse - plant pathogenic fungi - virulence - proteinases - proteinase inhibitors - plant-microbe interactions - genomics - tomatoes - proteomics

Pathogens cause disease on both animal and plant hosts. For successful infection and establishment of disease, pathogens need proper weaponry to protect themselves against host defenses and to promote host colonization to facilitate uptake of nutrients for growth and reproduction. Indeed, plant pathogens secrete various types of effector molecules (proteins and secondary metabolites) to manipulate host responses for their own needs. Secreted proteases and protease inhibitors (PIs) are such effector molecules. Proteases can hydrolyze plant defense proteins and PIs can inhibit plant proteases that are part of the host surveillance system. Despite the importance of proteases and PIs secreted by fungal pathogens, little information about their role in virulence is available. The recent advances in genomics, bioinformatics, transcriptomics and proteomics have facilitated identification and functional analysis of proteases and PIs relevant to plant-fungus interactions.

Chapter 1 is an introduction to the thesis outlining the general concept of plant-microbe interactions. It briefly describes the current knowledge of pathogenicity mechanisms employed by fungal plant pathogens and defense mechanisms employed by their host plants. It further introduces proteases and PIs and their potential role in modifying pathogenesis-related (PR) proteins to facilitate fungal virulence. It completes with an outline of the PhD research project.

In chapter 2, we analyzed and compared the number of putatively secreted proteases present in the genomes of 30 fungi with different lifestyles. The analysis showed that fungi with a saprotrophic and hemibiotrophic lifestyle contain more secreted protease genes than biotrophs. Surprisingly, the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs and saprotrophs. We analyzed all C. fulvum protease genes both at the transcriptome and proteome level by means of RNA-Seq/RT-qrtPCR and mass spectrometry analyses, respectively. Results showed that many proteases of C. fulvum are not expressed during growth in planta, likely sustaining the biotrophic growth pattern of this fungus.

In chapter 3, using an alignment-based gene prediction tool, we identified pseudogenes containing disruptive mutations (DMs) that likely lead to the production of nonfunctional proteins, including a group of putatively secreted proteases from C. fulvum. Fewer DMs were observed in other fungi including Dothistroma septosporum, a hemibiotrophic pine needle pathogen and close relative of C. fulvum, and suggested that the difference in pseudogenization of proteases between these two pathogens might in part explain their different lifestyle.

In chapter 4, we analyzed the tomato genome and identified 30 candidate chitinases genes, of which six encoded chitin binding domain (CBD)-containing chitinases. Transcriptome and proteome data were collected after inoculation of tomato with several fungal pathogens and allowed the identification of two CBD-chitinases (SlChi2 and SlChi13) with a putative role in protecting tomato against C. fulvum and F. oxysporum f. sp. lycopersici (F. oxysporum), respectively. Purified CBD-chitinases SlChi1, SlChi2, SlChi4 and SlChi13 were incubated with secreted protein extracts (SPEs) from seven fungal tomato pathogens and we could show that SPEs from F. oxysporum, Verticillium dahliae, and Botrytis cinerea modified SlChi1 and SlChi13. LC-MS/MS analysis revealed that incubation with SPE from F. oxysporum removed the N-terminal 37 and 49 amino acids, comprising part and complete CBD domain from SlChi1 and SlChi13, respectively. Removal of the CBD of SlChi1 and SlChi13 by SPE of F. oxysporum reduced the antifungal activity of the two chitinases. We identified a fungal metalloprotease (FoMep1) and a subtilisin serine protease (FoSep1) that synergistically cleaved both SlChi1 and SlChi13. Transgenic F. oxysporum in which the genes encoding these two proteases were knocked out by homologous recombination lost the ability to cleave the two chitinases and were compromised in virulence on tomato compared to the parental wild type. These results suggest an important role of the two chitinases in defense of tomato against this pathogen.

In chapter 5, we searched for host target(s) of the apoplastic effector Avr9 secreted by C. fulvum during infection of tomato. Based on the structural homology of Avr9 with carboxy peptidase inhibitors, we hypothesized that the host target of Avr9 might be apoplastic proteases. To isolate and identify Avr9 targets in apoplastic fluids, we used synthetic biotinylated Avr9, and performed pull-down and far-western blotting assays with apoplastic fluids from tomato inoculated with a C. fulvum race lacking the Avr9 gene. However, we found no specific Avr9-interacting proteins from pull-down complexes analyzed by mass spectrometry or by far-western blotting. Then, we hypothesized that glycosylation of Avr9 might be required for its biological function. The results of mass spectrometry analysis revealed that Avr9 is N-glycosylated when secreted by C. fulvum, containing at least two GlcNac and six mannose residues. The necrosis-inducing activity of glycosylated and non-glycosylated Avr9 was assayed but appeared not significantly different; however, we could not produce sufficient amounts of (biotinylated)-glycosylated Avr9 to perform pull-down assays for identification of potential glycosylated Arv9-interacting proteins by mass spectrometry.

Previous studies as well as the results present in this PhD thesis showed that fungal pathogens secrete a plethora of effectors including proteases and PIs. Many of identified proteases and PIs mediate effector-triggered immunity in host plants. In chapter 6, we reviewed the recent advances on the various roles of proteases and PIs in compromising basal defense responses induced by microbe-associated molecular patterns.

Chapter 7 is a summarizing discussion of the PhD thesis. We showed determinative roles of proteases and PIs in shaping plant-pathogen interactions. The expression and pseudogenization studies on proteases of C. fulvum showed that the genome content does not necessarily reflect the lifestyle of this fungus. This is true for many classes of fungal genes, including proteases. Fungi contain many different types of proteases whose functions may partly overlap. This hampers the discovery of their biological functions. We could demonstrate that two different types of proteases (metalloprotease (FoMep1) and subtilisin serine protease (FoSep1)) of F. oxysporum act synergistically to modify and reduce antifungal activity of two plant CBD-chitinases. Identifying additional proteases is achievable by a targeted proteomics approach using known targets as we did in chapter 4. However, identification of biological functions of proteases is a technical challenge when targets are not known. Multi-gene targeting of protease and PI genes is required to reveal their function in plant-pathogen interactions, which can only be addressed by using advanced genetic tools in future research.

Phytophthora infestans RXLR effector AVR1 and its host target Sec5
Du, Y. - \ 2014
Wageningen University. Promotor(en): Francine Govers, co-promotor(en): Klaas Bouwmeester. - Wageningen : Wageningen University - ISBN 9789462571310 - 188
phytophthora infestans - oömycota - plantenziekteverwekkende schimmels - virulentie - genen - plant-microbe interacties - ziekteresistentie - verdedigingsmechanismen - vatbaarheid - uitschakelen van genexpressie - oomycota - plant pathogenic fungi - virulence - genes - plant-microbe interactions - disease resistance - defence mechanisms - susceptibility - gene silencing

Summary

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating potato diseases worldwide. To successfully colonize its host, P. infestans secretes a plethora of RXLR effectors that translocate into host cells to modulate plant defense. The RXLR effectors form the largest and most diverse effector family in oomycete plant pathogens, and include several that were demonstrated to trigger host resistance mediated by intracellular host immune receptors. Chapter 1 is a summary focussing on the molecular mechanisms underlying host–pathogen interactions. It introduces the multi-layered innate immune system of plants, as well as the strategies that pathogens exploit to circumvent and suppress host defense. Furthermore, it highlights the importance of vesicle-trafficking during plant defense.

The central subject of this thesis is AVR1, one of the race-specific avirulence (AVR) factors of P. infestans. AVR1 triggers plant resistance mediated by its corresponding potato Nucleotide-binding Leucine-rich repeat (NLR) resistance protein R1. P. infestans isolates that are avirulent on R1-containing potato cultivars always contain AVR1, while virulent isolates lack AVR1 but contain a related gene that we baptized as AVR1-like. AVR1 has all hallmarks of a typical RXLR effector; it contains a signal peptide, an RXLR domain and a C-terminal effector domain that contains two W motifs and one Y motif. In addition, it has, at the very end a stretch of 38 amino acids in length that we named the Tail (T)-region. AVR1-like, or in short A-L, shares high sequence similarity with AVR1. However, due to a premature stop codon the 38 amino acid T-region is missing.

Chapter 2 explores the conserved motifs and regions in the C-terminal effector domain of AVR1 that are required to trigger R1-mediated hypersensitive response (HR). Various truncated and chimeric constructs of AVR1 and A-L were generated and assayed for their ability to elicit R1-mediated HR. Results show that the T-region of AVR1 plays an important role in HR activation. Furthermore, we revealed that R1 recognizes two epitopes in AVR1, one located in the C-terminal region containing the conserved W and Y motifs, and one comprised by the T region.

In Chapter 3 the subcellular localization of AVR1 and R1 was investigated. Both were demonstrated to be nucleocytoplasmic proteins. We artificially modified the nucleocytoplasmic partitioning of AVR1 and R1 using nuclear localization and export signals (NLS/NES), and studied the effect on R1-AVR1 recognition. This revealed that nuclear localization of both AVR1 and R1 is important to induce R1-mediated immunity. In addition, we showed that AVR1-mediated suppression of CRN2-induced cell death is dependent on cytosolic localization of AVR1.

In Chapter 4, we investigated how AVR1 modulates host defense. In a yeast two-hybrid screening we identified the exocyst subunit Sec5 as a host target for AVR1. Interaction between AVR1 and Sec5 was confirmed in planta by co-immunoprecipitation and bimolecular fluorescent complementation. Although A-L shares high sequence similarity with AVR1, we found that it is not able to interact with Sec5. Sec5 was shown to be required for proper plant defense against P. infestans. The role of Sec5 in plant response upon pathogen attack was further supported by its role in callose deposition and in secretion of the pathogenesis-related protein PR-1, which indicates that Sec5 plays a crucial role in vesicle trafficking during host defense. AVR1 is able to suppress callose deposition while A-L is not, which suggests that P. infestans manipulates host vesicle trafficking by secretion of AVR1 to target Sec5. Overall, our findings unravelled a novel strategy that oomycete pathogens exploit in order to modulate host defense.

In Chapter 5 we further analysed the potential virulence activities of AVR1 and A-L. Both AVR1 and A-L were able to promote P. infestans colonization, indicating that both are genuine P. infestans virulence factors. Moreover, AVR1 was found to suppress not only callose deposition, but also Sec5-dependent cell death induced by the P. infestans elicitors INF1 and CRN2. In contrast, A-L was neither able to suppress Sec5-dependent nor Sec5-independent cell death. The conserved C-terminal motifs and regions required for virulence activity of AVR1 were investigated using AVR1 truncated constructs. In addition, the conserved C-terminal motifs and regions of AVR1 required for Sec5 interaction were studied by Y2H assays. Although the T-region of AVR1 was found to be sufficient to facilitate P. infestans colonization and suppression of CRN2-induced cell death, it could not fully accommodate the interaction of AVR1 with Sec5. Instead, both the Y motif and the T-region of AVR1 appear to be required for Sec5 targeting.

Next to Sec5, the role of other exocyst subunits in Phytophthora resistance was studied (Chapter 6). The evolutionary relationships of exocyst subunits from three Solanaceous plants, i.e. Nicotiana benthamiana, tomato and potato, were investigated in comparison to their Arabidopsis orthologs. Virus-induced gene silencing in N. benthamiana of the majority of the exocyst subunit genes (exo84s were not yet included) showed that, except for some Exo70 members, all other tested exocyst subunits are required for plant defense against P. infestans and callose deposition. In addition, all of the analysed exocyst subunit gene-silenced tomato plants showed gain of susceptibility to both P. infestans and Phytophthora capsici.

In Chapter 7, our findings obtained in this thesis on the mechanisms of AVR1-triggered host immunity and susceptibility are discussed in a broader perspective with emphasis on the current developments in the field of effector biology.

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 - genetic analysis - genetic variance - pathogenicity - virulence - genes - biological control - insect pests
Towards generating broad-spectrum resistance to pathogens in plants: studies on a down-stream signalling NB-LRR of tomato
Sueldo, D.J. - \ 2014
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten; Wladimir Tameling. - Wageningen : Wageningen University - ISBN 9789461738974 - 209
solanum lycopersicum - tomaten - ziekteresistentie - verdedigingsmechanismen - receptoren - pathogenesis-gerelateerde eiwitten - bindende eiwitten - virulentie - mutanten - genetische kartering - tomatoes - disease resistance - defence mechanisms - receptors - pathogenesis-related proteins - binding proteins - virulence - mutants - genetic mapping
Functional analysis of LysM effectors secreted by fungal plant pathogens
Kombrink, A. - \ 2014
Wageningen University. Promotor(en): Bart Thomma; Pierre 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.

An integrated approach involving metabolomics and transcriptomics for a system-wide understanding of the interaction between tomato and Cladosporium fulvum
Etalo, D.W. - \ 2014
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Matthieu Joosten; Ric de Vos. - Wageningen : Wageningen University - ISBN 9789461738219 - 270
solanum lycopersicum - plantenziekteverwekkende schimmels - passalora fulva - plant-microbe interacties - metabolomica - genexpressie - genomica - ziekteresistentie - virulentie - plant pathogenic fungi - plant-microbe interactions - metabolomics - gene expression - genomics - disease resistance - virulence
Analysis of Tomato spotted wilt virus effector-triggered immunity
Ronde, D. de - \ 2013
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - S.l. : s.n. - ISBN 9789461737212 - 190
tomatenbronsvlekkenvirus - plantenvirussen - ziekteresistentie - immuniteit - virulentie - genkartering - genetische merkers - genetische analyse - capsicum annuum - paprika's - tomato spotted wilt virus - plant viruses - disease resistance - immunity - virulence - gene mapping - genetic markers - genetic analysis - sweet peppers

ResistanceinCapsicumagainsttheTomatospottedwiltvirus(TSWV),typespeciesof the Tospovirusgenuswithinthe Bunyaviridaefamily,employsthe singledominant resistancegeneTsw.Thisresistance hasmeanwhilebeenbrokenbyresistance breaking (RB) TSWV isolates and is causing increasing problems in many different (Capsicumcultivating)countries.Theresearchdescribedhereaimedtoidentify andcharacterise theviralproteintriggeringTswresistanceandprovidefurther insightintothemechanismofTsw-mediatedresistance.Knowledgegainedfrom thegeneticandphenotypiccharacterisationofTsw-resistancebreakingisolateswas usedtodevelopdiagnosticmarkersfordetectionofTsw-breakingpathotypesin fieldcultivations.

TheNSsRNAsilencingsuppressor(RSS)proteinwasidentifiedastheavirulence determinant ofTsw-mediatedresistance(Chapter2).WhiletheNSsproteinfrom theTSWVresistanceinducer(RI)isolatewasactiveasRNAsilencingsuppressorand avirulencedeterminant,theNSsproteinfromtwodifferentTSWVRBisolateslacked bothfunctionsasevidencedfromtransientassays.Surprisingly,thecorresponding resistancebreakingvirusisolatesstillexhibitedRNAisuppressoractivity. Noneof the other viral proteins were able to aid in the transient recovery of RSS activity. Electrophoreticmobilityshift assays(EMSAs)usingplantextractscontaining transientlyexpressedNSsproteinsshowedashift ofsiRNAswithNSsRI,indicative forbinding,butnotwithNSsRB.InagreementwiththelocalleafRSSassaysusinga virusinfection,plantextractsofvirusinfectedleaveswereabletoshiftthesiRNAs, showing recovery of the RSS activityduring virus infection.

The linkage of RNAi suppression and avirulence in NSs was further investigated bymutationalanalysis(Chapter3).AlargesetofNSsmutantswasgeneratedusing alaninesubstitutions ofauthenticTSWVNSsaminoacidsandwastestedfortheir abilitytotriggerTsw-mediatedHRandabilitytosuppressRNAi.Theseassaysshowed thatthe N-terminaldomainofNSscarried mostimportantresiduesinvolvedwith bothactivities. However,singlemutationscouldbeintroducedthatdisruptedone function,whilemaintainingtheotheroneandviceversaindicatingthatRSSactivity andavirulencewerenotfunctionally linked.SwappingofdomainsbetweenNSsRI andNSsRB notonlyconfirmedtheimportanceoftheN-terminaldomainbutalso thespecificitywithintheTSWVspecies,sincedomainswapsbetweenNSsRIandNSs fromGRSV,arelatedbutdistinct Tospovirus,couldnottransfertheAvrphenotype toGRSV.MutationofaGW/WG-motifintheNterminalregionofNSsRI leadtoa lossofbothfunctionsandindicatedthatthismotif, knowntobeinvolvedinAGO1 interactionof other viral RSS, was of biological relevance for TSWV NSs.

Theputativeinteraction ofAGO1andNSswasinvestigatedbyusingdifferent approaches to co-immunoprecipitate (Co-IP) on transiently co-expressed tagged- AGO1and(His-)NSs(Chapter4).Initialindicationsforsuchinteraction were obtained,howeverfurthersupportforthisputativeinteraction willhavetocome fromcomplementaryexperiments,e.g. Yeast-2-hybrid (Y2H), FRET-FLIM or BiFC.

Severaladditional TSWVisolateswereanalysedthatbesidestheknownresistance inducing-and resistance breaking-phenotype showed a temperature-dependent phenotype(Chapter5).IsolatesclassifiedtothistypeexhibitedanRIphenotypeat standardgreenhouseconditions (~22°C)whileatelevatedtemperatures(≥28°C), butstillbelowtemperaturesthatinactivatedtheR-geneproduct(≥31°C),wereable tobreaktheresistance.Viruschallengingassaysatvariousconditionsindicatedthat inductionofTswresistanceatalower temperaturebythesesocalledtemperature dependentresistancebreakingisolates(TempRB)involveddenovosynthesisofthe avirulenceprotein,i.e.NSs,andthat proteinfoldingmight play arole. NSsproteins clonedandexpressedfromthisadditional newsetofTSWVresistanceinducing, resistancebreakingandtemperature dependentresistancebreakingisolates revealedvariableresultsregardless oftheircorrespondingvirusphenotype,when tested for their abilitytoinduceTsw-mediated HR andsuppress RNAi at normal greenhouseconditions(22°C).However,similarassaystoanalysetheiractivity attheelevatedtemperature(28°C)failedwhenusingAgrobacteriummediated transientassays.Sofar,themechanismoftemperature dependencyhasnotbeen clarified yetandneedsfurtherinvestigation.Usingtheinformationobtained,a diagnostictoolwasdevelopedtoscreenforthepotential presenceofresistance breakingisolatesofTSWVusingreversetranscription-polymerasechainreaction amplification(RT-PCR).Aprimersetwasdesignedtargetinganimportantcodon ataaposition79andshowedtobeabletodistinguishRB-isolatesfromRI-isolates. However,afewRB-isolatesstillescapedfromdetection indicatingthelimitedand conditionaluse of this tool.

In summary, NSs has been identified as Avr-determinant of Tsw-mediated resistance,butthisfunctionisnottightlylinkedtoitsRNAisuppressor-activity. Preliminarydataindicateaputativeinteraction betweenAGO1andNSs.Besides the typicalRIandRBphenotypes,athirdphenotypicclassofTSWVisolates has beenidentified thatexhibitsatemperaturedependencyontriggeringTsw- mediatedresistance andpossiblyinvolvesanalteredproteinfoldingofNSs.A diagnostic toolhasbeendevelopedtodetectresistancebreakingisolatesinthe fieldbasedonRT-PCR,butthistoolstillallowsforescapesofRBisolates.Theresults onNSsarediscussedinlightofitsroleaseffectorwithinthe‘Zig-zag-model’of planthostdefenceresponses.Finally,TSWVNSsisbriefly discussedandcompared totheanimal-infecting(NSs)paralogsoftheBunyaviridaefamily,alsoinlightof functional andstructuralhomologiesbetweenthesensorsofinnateimmunityin plant(R-genes)and animal (NLRs/TLRs) cell systems.

Interactions of Streptococcus suis with host mucosa
Ferrando, M.L. - \ 2012
Wageningen University. Promotor(en): Jerry Wells, co-promotor(en): Hilde Smith; Peter van Baarlen. - S.L. : s.n. - ISBN 9789461732262 - 198
streptococcus suis - slijmvlies - micro-organismen - bacteriën - gastheer-pathogeen interacties - genexpressie - virulentie - glucanen - enzymen - varkens - mucosa - microorganisms - bacteria - host pathogen interactions - gene expression - virulence - glucans - enzymes - pigs
Hoge resolutie typering van Coxiella burnetii
Janse, I. ; Bossers, A. ; Roest, H.I.J. ; Rotterdam, B. van - \ 2011
Bilthoven : RIVM (RIVM briefrapport / Rijksinstituut voor Volksgezondheid en Milieu 330302001/2011) - 38
q-koorts - coxiella burnetii - epidemiologie - genotypen - virulentie - geiten - schapen - q fever - epidemiology - genotypes - virulence - goats - sheep
Dit rapport beschrijft het onderzoek wat uitgevoerd is in het kader van het project ‘Hoge Resolutie Typering Coxiella burnetii’’. Het doel van dit project was om de genoomsequenties van een aantal Nederlandse isolaten van de bacterie Coxiella burnetii, de veroorzaker van Q-koorts, in kaart te brengen om hiermee de ontwikkeling van verbeterde typeringsmethodes mogelijk te maken. Op basis van genoomsequenties kan onderzocht worden of de Nederlandse uitbraken bijvoorbeeld samenhangen met veranderde virulentiekenmerken. Genoom informatie is ook essentieel om een verbeterde, op de Nederlandse situatie toegespitste typering mogelijk te maken.
Moleculaire risk assessment Escherichia coli O157 in Nederland
Franz, E. ; Wal, F.J. van der; Hoek, A. ; Heuvelink, A.E. - \ 2011
Bilthoven : RIVM (RIVM briefrapport / Rijksinstituut voor Volksgezondheid en Milieu 330411001/2011) - 15
escherichia coli - risicoschatting - virulentie - voedselveiligheid - genotypen - risk assessment - virulence - food safety - genotypes
Infecties door Shiga toxine-producerende Escherichia coli O157 (STEC O157) zijn een bedreiging voor de volksgezondheid gezien de ernstige klinische gevolgen bij voornamelijk jonge kinderen en de potentie voor voedselgerelateerde uitbraken. Verschillende STEC O157 isolaten kunnen erg verschillen in genetische samenstelling. In deze studie is de mate van genotypische gelijkenis tussen STEC O157 isolaten uit (gezonde) runderen en humane ziektegevallen onderzocht aan de hand van vier moleculaire typeringsessays.
The Cladosporium fulvum Avr2 protein behaves both as a virulence and an avirulence factor
Klooster, J.W. van t - \ 2010
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - [S.l. : S.n. - ISBN 9789085856672 - 176
passalora fulva - cladosporium - virulentie - genen - eiwitten - solanum lycopersicum - proteïnasen - ziekteresistentie - gastheer-pathogeen interacties - plant-microbe interacties - virulence - genes - proteins - proteinases - disease resistance - host pathogen interactions - plant-microbe interactions
Plants are not able to move or escape and have to confront environmental challenges like nutrient and water deprivation, low and high temperatures, and biotic stress imposed by pathogens like viruses, bacteria, fungi, nematodes and insects that all compete for plant nutrient sources. The outcome of a plant-pathogen interaction can vary from mild symptoms that are hardly harmful to the host to complete destruction of the host plant. Plants have evolved various mechanisms to counter-attack infections by pathogens. Mechanisms of evasion or suppression of basal host defense by pathogens on the one hand, and specific recognition of a pathogen by its host and activation of downstream defense signaling on the other hand, are complex and both organisms have to come up with sophisticated strategies to survive their encounters. In principle these encounters have two possible outcomes: (i) a pathogen successfully infects the host plant, which is also referred to as a compatible interaction (the pathogen is virulent and the host plant is susceptible), or (ii) the pathogen cannot successfully infect the host plant which stays healthy, also referred to as an incompatible interaction (the pathogen is avirulent and the host plant is resistant).
Nearly 70 years ago, Harold Flor (1942) studied the genetics of the interaction between the flax rust fungus Melampsora lini and flax, Linum usitatissimum. Based on these studies he postulated the so-called gene-for-gene hypothesis (Flor 1942) which states that for each dominant resistance (R) gene in the host there is a matching dominant avirulence (Avr) gene in the pathogen. Co-occurrence and expression of both genes leads to an incompatible interaction that is often associated with a hypersensitive response (HR).
The interaction between the fungus Cladosporium fulvum (syn. Passalora fulva) and the host tomato (Solanum lycopersicum) is an excellent model to study plant-pathogen interactions and obeys to the gene-for-gene hypothesis. C. fulvum is a biotrophic pathogen that causes leaf mold of tomato, avoids breaching the cell wall and exclusively colonizes the tomato leaf apoplast while establishing a long-term feeding relationship with the living cells of the host. During the infection process, the fungus secretes several effector molecules, relatively small, cysteine–rich proteins. They are likely to contribute to pathogen fitness and play a role in pathogen virulence. According to the 'Zig-Zag' model that explains the evolutionary development of plant-pathogen interactions, effectors are required for ETS (effector -triggered susceptibility). Tomato plants that carry cognate Cf resistance genes recognize the effector and elicit a defense response known as the hypersensitive response (HR), nowadays known as effector-triggered immunity (ETI). In this thesis I have focused on several molecular and biochemical aspects of the Avr2 and Cf-2 gene pair and on an additional gene, Rcr3 (required for Cladosporium resistance), that is required for Cf-2-mediated resistance with an emphasis on the role of Avr2 in ETS and ETI in the C. fulvum-tomato interaction.

The gene-for-gene hypothesis postulated by Harrold Flor has inspired many plant pathologists and initiated numerous plant-pathogen studies as discussed in chapter 1. This hypothesis has lead to the characterization of various host plant R genes and cognate pathogen Avr genes from fungi, bacteria and oomycetes. Plant resistance proteins are the basic molecules that mediate a defense reaction, triggered by cognate effectors directed against the pathogens, are found extracellularly as well as intracellularly and are divided in classes based on the composition of different subdomains that may have various functions. Particularly the LRR domain(s) are involved in recognition, regulating protein activation and signal transduction and are highly adjustable in diverse binding specificities to self and non-self molecules. The nucleotide binding (NB) domain acts a switch for activation of downstream host defenses, often resulting in HR. Inappropriate R protein folding and activation is controlled by intramolecular interactions between the various domains and by hetero-multimeric protein complexes.
Studies on interactions of plants, especially Arabidopsis thaliana, with prokaryotic pathogens have resulted in major scientific breakthroughs with respect to the gene-for-gene hypothesis. Research on the bacterial Type Three Secretion System and the delivery of the effectors has indentified sophisticated mechanisms for perception and recognition of pathogens and regulation of host resistance.
The functions of effectors of eukaryotic plant pathogens remain largely unknown so far. Oomycete pathogens such as Phytophthora infestans produce various types of effectors during infection of their hosts. One class of oomycete effectors localizes to, and operates in, the extracellular matrix while the other class acts inside the host plant cell. Recent studies on the interaction of the flax rust fungus Melampsora lini with flax (Linum usitatissimum) has revealed a number of direct Avr-R protein interactions in vitro. These interactions are expected to occur in the haustorial matrix which is produced by the fungus during host infection. Secreted Avr proteins of C. fulvum interact exclusively with the corresponding extracellular Cf proteins of tomato. The C. fulvum-tomato pathosystem is one of the most well-studied plant pathogen interactions and revealed important insights in perception and recognition of Avr proteins. For many years it was assumed that the interactions beween C. fulvum Avrs and tomato R proteins occurred in a direct manner, but proof for such interactions has never been obtained. Indirect interactions were more likely and obeyed to the guard hypothesis wherein the Avr protein interacts with a host target and this interaction is monitored, or guarded, by the Cf- protein.


Chapter 2 reports on the avirulence function of Avr2 in the Cf-2-mediated resistance that also requires the extracellular tomato cysteine protease Rcr3. The interaction between Avr2, Cf-2 and Rcr3 obeys to the guard hypothesis. Purified heterologously expressed and affinity-tagged Rcr3 and Avr2 were applied in co-immunoprecipitation assays and revealed a physical interaction between Avr2 and Rcr3 independent of additional plant and or fungal factors. It is shown that Avr2 binds and inhibits Rcr3, and blocking of the active site of Rcr3 by the irreversible cysteine protease inhibitor E-64 eliminates this interaction. The interaction with and the inhibition of Rcr3 by Avr2 occurs in a pH-dependent fashion and the pH optimum for Rcr3 activity and its inhibition by Avr2 coincides with the pH of the tomato apoplast. Cysteine protease activity profiling showed that, in addition to Rcr3, Avr2 inhibits several other apoplastic cysteine proteases in tomato, but this inhibition did not lead to Cf-2-mediated HR. Infiltration of purified active Rcr3, or E-64-inactivated Rcr3, in combination with Avr2 in Cf-2/rcr3 tomato leaves revealed that only the Avr2-Rcr3 inhibition complex triggers Cf-2-dependent HR. It is proposed that Avr2 modifies Rcr3 which is recognized by Cf-2 and initiates the HR. This study represents the first indirect fungus-plant gene-for-gene interaction that obeys to the guard hypothesis.

In chapter 3 the focus is on the virulence function of Avr2, and it is demonstrated that Avr2 has an indisputable intrinsic biological virulence function for C. fulvum during infection of tomato. Silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. Heterologous expression of Avr2 in tomato resulted in enhanced susceptibility towards natural Avr2-defective C.fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. In A. thaliana, Avr2 expression resulted in enhanced susceptibility to various extracellular fungal pathogens including Botrytis cinerea and Verticillium dahliae. Microarray analysis of unchallenged A. thaliana plants showed that Avr2 expression induced a global transcription profile that is comparable to the profile upon pathogen challenge. Cysteine protease activity profiling and LC-MS/MS analyses showed that Avr2 inhibits multiple extracellular A. thaliana cysteine proteases. Similar results were obtained for tomato, showing that Avr2 inhibits multiple cysteine proteases including Rcr3 and its close relative Pip1. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases that are required for basal host defense.






In chapter 4 the emphasis is on Avr2 protein features and the mode of inhibition of Rcr3. Like many other Avr genes, Avr2 lacks homology with sequences deposited in public databases. The mature Avr2 protein contains 8 cysteine residues and biochemical analyses revealed that all of these are involved in disulphide bridging, showing a unique disulphide bridge pattern. Based on a bioinformatics analysis, site-specific mutations were made in the Avr2 protein and affinity-tagged wild-type and mutant proteins were produced by heterologous expression in the yeast Pichia pastoris. After affinity purification, all proteins were infiltrated in tomato Cf-2 plants, and proteins with altered HR inducing activity were tested for their ability to inhibit Rcr3. From these assays it became evident that especially the C-terminal six amino acids that also include one disulphide bridge are essential for the interaction with and inhibition of Rcr3. All these data show that Avr2 is a novel type of cysteine protease inhibitor.

Chapter 5 is a general discussion about the role of plant cysteine proteases and cysteine protease inhibitors in plant-pathogen interactions. Microbial pathogens and host plants both employ cysteine proteases and cysteine protease inhibitors as weapons for attack and defence. This so-called arms race has led to multiple attacks and counter-attacks that have shaped co-evolution between pathogens and their host plants. Examples of some prokaryotic plant pathogens that employ cysteine proteases as effector proteins to suppress plant defense will be discussed, in addition to some eukaryotic pathogens that use cysteine protease inhibitors for the same purpose. Examples of plant cysteine proteases will be discussed that are involved in multiple processes including plant development, plant defense and processes in programmed cell death.





The interplay between a Phytophthora RXLR effector and an Arabidopsis lectin receptor kinase
Bouwmeester, K. - \ 2010
Wageningen University. Promotor(en): Francine Govers; Pierre de Wit. - [S.l. : S.n. - ISBN 9789085856474 - 223
phytophthora infestans - arabidopsis - solanum tuberosum - genen - genomen - virulentie - kinasen - lectinen - receptoren - genexpressie - plant-microbe interacties - genes - genomes - virulence - kinases - lectins - receptors - gene expression - plant-microbe interactions
Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management.
Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature.
LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora.
In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments.
This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object.
Inventarisatie van de virulentie binnen Nederlandse Globodera pallida populaties
Beers, T.G. van; Molendijk, L.P.G. - \ 2009
Lelystad : PPO AGV (PPO-AGV / Rapport ) - 31
globodera pallida - plantenparasitaire nematoden - virulentie - inventarisaties - nederland - plant parasitic nematodes - virulence - inventories - netherlands
De vaststelling van de Nederlandse standaard populaties G. pallida, gebruikt door de aardappelkwekers voor toetsingen tbv de rassenlijst, is ruim 10 jaar geleden. Mogelijk is deze selectie van populaties niet relevant meer omdat er ‘nieuwe’ populaties zijn die nog agressiever zijn dan de als zeer virulent aangemerkte ‘Rookmaker’ populatie. Binnen het project zullen, afhankelijk van financiering, nog een aantal populaties in een virulentietoetsing bekeken. Voor de toekomst zou het aan te bevelen zijn dat de bemonsterende instanties onverwacht extreem vermeerderende populaties centraal zouden aanmelden. Binnen dit project wordt nog een plan geschreven om tot een opzet van een monitoringsysteem voor virulentie in Nederland te komen.
Phytophthora: kampioen aanpassing
Kessel, G.J.T. ; Evenhuis, A. ; Bosch, G.B.M. van den; Förch, M.G. ; Topper, C.G. - \ 2009
phytophthora infestans - genotypen - fenotypen - virulentie - fungiciden - aardappelen - ziekteresistentie - genotypes - phenotypes - virulence - fungicides - potatoes - disease resistance
Poster met onderzoeksinformatie. Uit onbeschermd opgekweekte proefveldjes zijn Phytophthora infestans isolaten verzameld en gekarakteriseerd.
Inventarisatie virulentie van Nederlandse Globodera pallida populaties
Molendijk, L.P.G. ; Beers, T.G. van - \ 2009
gewasbescherming - ziekteresistentie - globodera pallida - aardappelen - virulentie - inventarisaties - nematodenbestrijding - nederland - akkerbouw - plant protection - disease resistance - potatoes - virulence - inventories - nematode control - netherlands - arable farming
Resistentie tegen het aardappelcysteaaltje Globodera pallida is het belangrijkste instrument in het beheersen van dit aaltje. Om zeker te zijn dat de huidige resistentietoetsing zinvolle resistente rassen tegen aardappelmoeheid oplevert voor de praktijk, is inzicht in de virulentie van Nederlandse veldpopulaties nodig
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