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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Increased virulence of Globodera pallida during repeated rearing on different resistant potato cultivars explained by a simple model
Beniers, J.E. ; Nöllen, Y. ; Eck, H.J. van; Schouten, H.J. - \ 2019
Plant Pathology 68 (2019)3. - ISSN 0032-0862 - p. 445 - 453.
Globodera pallida - model - potato cyst nematodes - resistance - selection - virulence

Selection for virulence of Globodera pallida on potato cultivars was studied for four generations under controlled conditions. The reproduction rate (Pf/Pi) of a mixed Pa2/3 population increased by a factor of 61 during rearing on the partially resistant potato cv. Darwina compared to rearing on the susceptible cv. Irene. This was a result of selection for virulence on cv. Darwina, and achieving the Hardy–Weinberg equilibrium on cv. Irene. Increased virulence also significantly raised the reproduction rate on several other Solanum genotypes. These changes could be explained reasonably well by the monogenic inheritance of a virulence factor breaking the Grp1 locus. The virulence changes were probably mainly evoked by this gene only, inherited from S. vernei 1-3 or S. vernei 24/20. The Grp1 locus has probably provided the differential S. vernei hybrid (VTn)2 62-33-3 with its resistance to the Pa2 group and not to the Pa3 group. Alternation of cultivars did not halt selection if the cultivars highly differentiated between the Pa2 and Pa3 populations. Only when alternation was with cultivars that harboured a different resistance gene against Pa3 was selection for virulence delayed. Differences in virulence levels (i.e. reproduction rates) within the nematode population determined the rate of selection, not the resistance level itself. Selection of a Pa3 population for three generations on cv. Karakter not only increased the reproduction rate on cv. Karakter itself by a factor 4.2, but also raised the reproduction on other potato genotypes. A simple monogenic model could explain these changes in virulence.

Aphid populations showing differential levels of virulence on Capsicum accessions
Sun, Mengjing ; Voorrips, Roeland E. ; Vosman, Ben - \ 2018
Insect Science (2018). - ISSN 1672-9609
callose deposition - EPG - plant immunity - plant–aphid interaction - ROS accumulation - virulence

The green peach aphid, Myzus persicae, is one of the most threatening pests in pepper cultivation and growers would benefit from resistant varieties. Previously, we identified two Capsicum accessions as susceptible and three as resistant to M. persicae using an aphid population originating from the Netherlands (NL). Later on we identified an aphid population originating from a different geographical region (Switserland, SW) that was virulent on all tested Capsicum accessions. The objective of the current work is to describe in detail different aspects of the interaction between two aphid populations and two selected Capsicum accessions (one that was susceptible [PB2013046] and one that was resistant [PB2013071] to population NL), including biochemical processes involved. Electrical penetration graph (EPG) recordings showed similar feeding activities for both aphid populations on PB2013046. On accession PB2013071 the aphid population SW was able to devote significantly more time to phloem ingestion than population NL. We also studied plant defense response and found that plants of accession PB2013046 could not induce an accumulation of reactive oxygen species and callose formation after infestation with either aphid population. However, plants of PB2013071 induced a stronger defense response after infestation by population NL than after infestation by population SW. Based on these results, population SW of M. persicae seems to have overcome the resistance of PB2013071 that prevented feeding of aphids from NL population. The potential mechanism by which SW population overcomes the resistance is discussed.

The VraSR regulatory system contributes to virulence in Streptococcus suis via resistance to innate immune defenses
Velikova, Nadya - \ 2018
Virulence 9 (2018)1. - ISSN 2150-5594 - p. 681 - 682.
antibacterial drug targets - Streptococcus suis - swine industry - two-component systems - virulence - zoonotic pathogens
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.

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp
Haryadi, D. ; Verreth, J.A.J. ; Verdegem, M.C.J. ; Vlak, J.M. - \ 2015
Journal of Fish Diseases 38 (2015). - ISSN 0140-7775 - p. 419 - 428.
litopenaeus-vannamei - viral accommodation - baculovirus wsbv - scylla-serrata - mud crab - monodon - pathogenicity - host - infection - virulence
Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. ); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen-free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV-infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid-body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve-SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.
Paramyxovirus-based producton of Rift Valley fever virus replicon particles
Wichgers Schreur, P.J. ; Oreshkova, N. ; Harders, F. ; Bossers, A. ; Moormann, R.J.M. ; Kortekaas, J.A. - \ 2014
Journal of General Virology 95 (2014)12. - ISSN 0022-1317 - p. 2638 - 2648.
newcastle-disease virus - hemagglutinin-neuraminidase protein - fusion protein - vaccine vector - nsm protein - hn protein - virulence - transcription - bunyaviridae - expression
Replicon-particle-based vaccines combine the efficacy of live-attenuated vaccines with the safety of inactivated or subunit vaccines. Recently, we developed Rift Valley fever virus (RVFV) replicon particles, also known as nonspreading RVFV (NSR), and demonstrated that a single vaccination with these particles can confer sterile immunity in target animals. NSR particles can be produced by transfection of replicon cells, which stably maintain replicating RVFV S and L genome segments, with an expression plasmid encoding the RVFV glycoproteins, Gn and Gc, normally encoded by the M-genome segment. Here, we explored the possibility to produce NSR with the use of a helper virus. We show that replicon cells infected with a Newcastle disease virus expressing Gn and Gc (NDV-GnGc) were able to produce high levels of NSR particles. In addition, using reverse genetics and site-directed mutagenesis, we were able to create an NDV-GnGc variant that lacks the NDV fusion protein and contains two amino acid substitutions in, respectively, Gn and HN. The resulting virus uses a unique entry pathway that facilitates the efficient production of NSR in a one-component system. The novel system provides a promising alternative for transfection-based NSR production.
Lung pathogenicity of European genotype 3 strain porcine reproductive and respiratory syndrome virus (PRRSV) differs from that of subtype 1 strains.
Weesendorp, E. ; Rebel, J.M.J. ; Popma-de Graaf, D.J. ; Fijten, H.P.D. ; Stockhofe, N. - \ 2014
Veterinary Microbiology 174 (2014)1-2. - ISSN 0378-1135 - p. 127 - 138.
experimentally infected-pigs - lelystad virus - immunological responses - immune-responses - swine - cells - disease - expression - cytokines - virulence
Porcine reproductive and respiratory syndrome (PRRS) is difficult to control due to a high mutation rate of the PRRS virus (PRRSV) and the emergence of virulent strains. The objective of this study was to analyse early and late pathological responses in the respiratory tract after infection with the European PRRSV subtype 3 strain Lena in comparison to two European PRRSV subtype 1 strains: Belgium A and Lelystad-Ter Huurne (LV). For each virus strain, groups of twelve pigs were inoculated, and four pigs per group were euthanized at days 3, 7 and 35 post-infection (p.i.) for consecutive examination. Infection with strain Lena resulted in a more severe disease than with the subtype 1 strains, an inflammatory response within the first week of infection with expression of IL-1a in the lung and lymph node, and an influx of neutrophils and monocytes in bronchoalveolar lavage fluid (BALF). Infection with strain Belgium A or LV resulted in mild or no pathology within the first week of infection, but inflammatory cell influx in the lung interstititium was increased at the end of the experiment at day 35 p.i. At five weeks p.i., all strains induced a higher percentage of cytotoxic T cells and higher levels of IFN-¿ producing cells in BALF. This might have contributed to clearance of virus. In general, subtype 3 strain Lena induced a stronger early inflammatory response which led to more severe clinical disease and pathology. On the other hand, this may have supported an enhanced or faster clearance of virus in tissues, compared to subtype 1 strains.
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


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
Profiling the secretome and extracellular proteome of the potato late blight pathogen Phytophthora infestans
Meijer, H.J.G. ; Mancuso, F.M. ; Espadas, G. ; Seidl, M.F. ; Chiva, C. ; Govers, F. ; Sabido, E. - \ 2014
Molecular and Cellular Proteomics 13 (2014)8. - ISSN 1535-9476 - p. 2101 - 2113.
phosphate kinase domain - amino-acid-sequence - cell-wall proteins - coupled receptors - genome sequence - identification - family - infection - virulence - effectors
Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment.
Filamentous pathogen effector functions: of pathogens, hosts and microbiomes
Rövenich, H. ; Boshoven, J.C. ; Thomma, B. - \ 2014
Current Opinion in Plant Biology 20 (2014). - ISSN 1369-5266 - p. 96 - 103.
chitin-triggered immunity - secreted fungal effector - potato famine pathogen - cladosporium-fulvum - protease inhibitor - magnaporthe-oryzae - plant-pathogens - genome evolution - tomato - virulence
Microorganisms play essential roles in almost every environment on earth. For instance, microbes decompose organic material, or establish symbiotic relationships that range from pathogenic to mutualistic. Symbiotic relationships have been particularly well studied for microbial plant pathogens and have emphasized the role of effectors; secreted molecules that support host colonization. Most effectors characterized thus far play roles in deregulation of host immunity. Arguably, however, pathogens not only deal with immune responses during host colonization, but also encounter other microbes including competitors, (myco)parasites and even potential co-operators. Thus, part of the effector catalog may target microbiome co-inhabitants rather than host physiology.
Mangotoxin production of Pseudomonas syringae pv. syringae is regulated by MgoA
Carrion, V.J. ; Voort, M. van der; Arrebola, E. ; Gutiérrez-Barranquero, J.A. ; Vicente, A. de; Raaijmakers, J.M. ; Cazorla, F.M. - \ 2014
BMC Microbiology 14 (2014). - ISSN 1471-2180 - 13 p.
signal-transduction pathway - artemis comparison tool - 2-component system - response regulator - escherichia-coli - fluorescens pf-5 - rna recognition - tomato dc3000 - virulence - gaca
Background The antimetabolite mangotoxin is a key factor in virulence of Pseudomonas syringae pv. syringae strains which cause apical necrosis of mango trees. Previous studies showed that mangotoxin biosynthesis is governed by the mbo operon. Random mutagenesis led to the identification of two other gene clusters that affect mangotoxin biosynthesis. These are the gacS/gacA genes and mgo operon which harbors the four genes mgoBCAD. Results The current study shows that disruption of the nonribosomal peptide synthetase (NRPS) gene mgoA resulted in loss of mangotoxin production and reduced virulence on tomato leaves. Transcriptional analyses by qPCR and promoter reporter fusions revealed that mbo expression is regulated by both gacS/gacA and mgo genes. Also, expression of the mgo operon was shown to be regulated by gacS/gacA. Heterologous expression under the native promoter of the mbo operon resulted in mangotoxin production in non-producing P. syringae strains, but not in other Pseudomonas species. Also introduction of the mbo and mgo operons in nonproducing P. protegens Pf-5 did not confer mangotoxin production but did enhance transcription of the mbo promoter. Conclusions From the data obtained in this study, we conclude that both mbo and mgo operons are under the control of the gacS/gacA two-component system and that the MgoA product acts as a positive regulator of mangotoxin biosynthesis.
Increased difficulties to control late blight in Tunisia are caused by a genetically diverse Phytophthora infestans population next to the clonal lineage NA-01
Harbaoui, K. ; Hamada, W. ; Li, Y. ; Vleeshouwers, V.G.A.A. ; Lee, T.A.J. van der - \ 2014
Plant Disease 98 (2014)7. - ISSN 0191-2917 - p. 898 - 908.
genotypic diversity - durable resistance - potato - tomato - plant - netherlands - virulence
In Tunisia, late blight caused by Phytophthora infestans is a serious threat to potato and tomato. The Mediterranean weather conditions can be conducive to infection in all seasons and the host crops, tomato and potato, are grown year round. Potato is planted and harvested in two to four overlapping intervals from August to June and tomato is grown both in open fields and in greenhouses. The consequences of these agricultural practices and the massive import of seed potato on the genetic variation of P. infestans are largely unknown. We conducted a survey in which 165 P. infestans isolates, collected from five subregions in Tunisia between 2006 and 2008, on which we studied genotypic diversity through nuclear (simple-sequence repeat [SSR]) markers and combined this with a previous study on their mitochondrial haplotypes (mtDNA). The phylogenetic analysis revealed the presence of a major clonal lineage (NA-01, A1 mating type, mitochondrial haplotype Ia). Isolates belonging to this clonal lineage were found in all regions and showed a relatively simple virulence pattern on a potato differential set carrying different Solanum demissum resistance genes. Apart from isolates belonging to this NA-01 clonal lineage, a group of isolates was found that showed a high genetic diversity, comprising both mating types and a more complex race structure that was found in the regions where late blight on potato was more difficult to control. The population on potato and tomato seems to be under different selection pressures. Isolates collected from tomato showed a low genetic diversity even though potato isolates collected simultaneously from the same location showed a high genetic diversity. Based on the SSR profile comparison, we could demonstrate that the four major clonal lineages found in the Netherlands and also in other European countries could not be found in Tunisia. Despite the massive import of potato seed from Europe, the P. infestans population in Tunisia was found to be clearly distinct
The Ccpa regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs.
Willenborg, J. ; Greeff, A. de; Jarek, M. ; Valentin-Weigand, P. ; Goethe, R. - \ 2014
Molecular Microbiology 92 (2014)1. - ISSN 0950-382X - p. 61 - 83.
sugar phosphotransferase system - bacillus-subtilis - catabolite repression - virulence - bacteria - phosphoenolpyruvate - expression - pathogen - hpr - phosphocarrier
Streptococcus suis (S.¿suis) is a neglected zoonotic streptococcus causing fatal diseases in humans and in pigs. The transcriptional regulator CcpA (catabolite control protein A) is involved in the metabolic adaptation to different carbohydrate sources and virulence of S.¿suis and other pathogenic streptococci. In this study, we determined the DNA binding characteristics of CcpA and identified the CcpA regulon during growth of S.¿suis. Electrophoretic mobility shift analyses showed promiscuous DNA binding of CcpA to cognate cre sites in vitro. In contrast, sequencing of immunoprecipitated chromatin revealed two specific consensus motifs, a pseudo-palindromic cre motif (WWGAAARCGYTTTCWW) and a novel cre2 motif (TTTTYHWDHHWWTTTY), within the regulatory elements of the genes directly controlled by CcpA. Via these elements CcpA regulates expression of genes involved in carbohydrate uptake and conversion, and in addition in important metabolic pathways of the central carbon metabolism, like glycolysis, mixed-acid fermentation, and the fragmentary TCA cycle. Furthermore, our analyses provide evidence that CcpA regulates the genes of the central carbon metabolism by binding either the pseudo-palindromic cre motif or the cre2 motif in a HPr(Ser)~P independent conformation.
Latest developments on Streptococcus suis: an emerging zoonotic pathogen: part 2
Segura, M. ; Zheng, H. ; Greeff, A. de; Gao, G.F. ; Gremier, D. ; Jiang, Y. ; Chengping, L. ; Maskell, D. ; Oishi, K. ; Okura, M. ; Osawa, R. ; Schultsz, C. ; Schwerk, C. ; Sekizaki, T. ; Smith, H. ; Srimanote, P. ; Takamatsu, D. ; Tang, J. ; Tenenbaum, T. ; Tharavichitkul, P. ; Hoa, N.T. ; Valentin-Weigand, P. ; Wells, J.M. ; Wertheim, H. ; Zhu, B. ; Xu, J. ; Gottschalk, M. - \ 2014
Future Microbiology 9 (2014)5. - ISSN 1746-0913 - p. 587 - 591.
cerebrospinal fluid barrier - plexus epithelial-cells - in-vitro - bacterial interactions - swine pathogen - serotype-2 - infection - diversity - virulence - release
This second and final chapter of the report on the First International Workshop on Streptococcus suis follows on from Part 1, published in the April 2014, volume 9, issue 4 of Future Microbiology. S. suis is a swine pathogen and a zoonotic agent afflicting people in close contact with infected pigs or pork meat. Although sporadic cases of human infections had been reported worldwide, deadly S. suis outbreaks emerged in Asia. The severity of the disease underscores the lack of knowledge on the virulence and zoonotic evolution of this human-infecting agent. The pathogenesis of the infection, interactions with host cells and new avenues for treatments were among the topics discussed during the First International Workshop on S. suis (China 2013).
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