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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Antimicrobial peptides and the interplay between microbes and host : towards preventing porcine infections with Streptococcus suis
Gaiser, Rogier A. - \ 2016
University. Promotor(en): Jerry Wells, co-promotor(en): Peter van Baarlen. - Wageningen : Wageningen University - ISBN 9789462578913 - 239
antimicrobial peptides - streptococcus suis - infections - bacteria - microorganisms - host pathogen interactions - pigs - antimicrobiële peptiden - infecties - bacteriën - micro-organismen - gastheer-pathogeen interacties - varkens

The increasing prevalence of antibiotic resistance in pathogenic bacteria and the potential future implications for human and animal morbidity and mortality, health-care costs and economic losses pose an urgent worldwide problem. As a result, exploration of alternative strategies to combat antibiotic resistant bacteria have intensified over the last decades. The work described in this thesis focused on the study of naturally occurring antimicrobial peptides (AMPs) and other bioactive molecules produced by bacteria as potential alternatives to prevent or treat infections with pathogenic bacteria. A large part of the thesis aimed to increase knowledge about the role of the microbiota (the collection of microbes present at a certain location of the body) of the oral cavity or small intestine in the abundance of Streptococcus suis, a pathogenic bacteria that mostly causes disease in young pigs. We identified commensal bacteria that displayed strong and selective antagonism against this S. suis. Several bacteria that showed strong growth inhibition of S. suis in the lab through the production of AMPs were isolated and characterised. This thesis increased the understanding of the role of host- and microbiota-derived biologically active small molecules in microbe-microbe and microbe-host interplay. Such knowledge may contribute to the development of novel therapeutic solutions to treat antibiotic resistant bacteria, such as beneficial microbial communities (i.e. next-generation probiotics) or biotechnological applications of natural or modified AMPs.

Host-interaction effector molecules of Lactobacillus plantarum WCFS1
Lee, I.C. - \ 2016
University. Promotor(en): Michiel Kleerebezem, co-promotor(en): P.A. Bron. - Wageningen : Wageningen University - ISBN 9789462576858 - 183 p.
lactobacillus plantarum - molecules - probiotics - immunomodulatory properties - lipoproteins - interactions - molecular interactions - host pathogen interactions - moleculen - probiotica - immunomodulerende eigenschappen - lipoproteïnen - interacties - moleculaire interacties - gastheer-pathogeen interacties

Abstract

Lactobacillus plantarum is found in various environmental habitats, including fermentation products and the mammalian gastrointestinal tract, and specific strains are marketed as probiotics, which are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. Throughout the studies of the mechanisms underlying probiotic activity, it became apparent that the probiotic effects are often species and/or strain specific. This situation has led more researchers to focus on the molecular characteristics of probiotic strains intending to link specific molecular structures to specific probiotic functions, and thereby deduce the mechanisms of molecular communication of probiotics. This thesis focuses on potential cell envelope effector molecules involved in interaction with the mammalian host cells, including lipoteichoic acid (LTA), lipo- and glyco-proteins, and extracellular polysaccharides (EPS), of L. plantarum WCFS1, a model strain for probiotic lactobacilli with a well-annotated genome sequences and sophisticated genetic engineering tools. First, existing research regarding the potential roles in probiotic functionality of Lactobacillus surface molecules in terms of their biosynthesis pathways and structure variations as well as interaction with host Pattern Recognition Receptors (PRRs) and immunomodulatory properties of these molecules are summarized and compared to provide an overview of the state-of-the-art in probiotic effector molecule research. Subsequently, specific molecules that reside in the cell envelope of L. plantarum WCFS1 were study for their role in bacterial physiology, as well as their role as ligands in Toll-like receptor (TLR) 2 signaling and immunomodulatory properties using human-cell co-incubation models. Our results showed that the deficiency of LTA had a drastic impact on cell division, cell morphology and growth in L. plantarum WCFS1, while LTA-deficient cells also elicited more pro-inflammatory responses in PBMCs rather than the expected loss of pro-inflammatory capacity as was observed with similar mutants of Lactobacillus acidophilus NCFM. Further studies on the signaling capacity of the purified LTA from L. plantarum WCFS1 revealed that these molecules are poor TLR2 activators, which is in clear contrast to the highly potent TLR2 stimulatory capacity of LTA obtained from Bacillus subtilis, implying that structural differences of the LTA produced by different bacteria are prominent determinants of their TLR2 signaling capacity and immunomodulatory properties. Lipoproteins of L. plantarum WCFS1 were studied using a derivative strain that is deficient in prolipoprotein diacylglyceryltransferase (Lgt), which transfers acyl chain moieties onto lipoproteins. The lipid moiety was shown to be important for proper anchoring of lipoproteins and TLR1/2 signaling capacity, but did not affect TLR2/6 signaling, suggesting that lipoproteins of L. plantarum WCFS1 are predominantly (if not exclusively) triacylated. The Lgt deficient strain elicited more pro-inflammatory responses in PBMCs as compared to the wild type, indicating that the native lipoproteins could play a role in dampening inflammation upon host-probiotic interaction. In addition, we explored the protein glycosylation machinery in L. plantarum WCFS1, responsible for the glycosylation of the major autolysin (Acm2) of this bacterium, which was previously shown to be O-glycosylated with N-acetylhexosamine conjugates. Using sequence similarity searches in combination with a lectin-based glycan detection and mass spectrometry analysis, two glycosyl-transferases, GtfA and GtfB (formerly annotated as TagE5 and TagE6, respectively), were shown to be required for the glycosylation of Acm2 and other unidentified L. plantarum WCFS1 glycosylated proteins. These results provide the first example of a general protein-glycosylation machinery in a Lactobacillus species. Finally, extracellular polysaccharides (EPS) in L. plantarum were studied in two strains that produce large amounts of EPS: L. plantarum SF2A35B and Lp90, in comparison to the lowly producing model strain WCFS1. Based on genome sequence comparison, both of the high producer strains were found to possess strain-specific and unique polysaccharide gene clusters. These gene clusters were deleted and the mutants were shown to have lost the capacity to produce large amounts of EPS, and were studied in relation to their properties in host-bacteria interaction. The results illustrate strain-specific and variable impacts of the removal of the EPS in the background of individual L. plantarum strains, supporting the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction. Overall, this thesis showed that surface molecules not only play important roles in bacterial physiology, but also in the interaction with the host mucosa through pattern recognition receptors expressed by the host cells. With the growing amount of evidence of structural variations in surface molecules, which are influenced by genetic background, physiological status, environmental factors, and other biological processes, these molecules form a unique signature associated with each strain that as a consequence elicits a strain-specific response when interacting with host cells.

Resistentieveredeling - Verdedigingsmechanisme : Kennisclip Bogo-project e-learning
Hop, M.E.C.M. - \ 2016
Groen Kennisnet
resistance breeding - susceptibility - resistance - tolerance - host pathogen interactions - plant protection - teaching materials - disease resistance - resistentieveredeling - vatbaarheid - weerstand - tolerantie - gastheer-pathogeen interacties - gewasbescherming - lesmaterialen - ziekteresistentie
Deze kennisclip maakt onderdeel uit van de lesmodule Resistentie Veredeling van het CIV T&U.
Discovery of inhibitors of bacterial histidine kinases
Velikova, N.R. - \ 2014
University. Promotor(en): Jerry Wells, co-promotor(en): A. Marina. - Wageningen : Wageningen University - ISBN 9789462571112 - 225
gastheer-pathogeen interacties - bacteriën - kinasen - histidine - geneesmiddelresistentie - antibiotica - microbiologie - host pathogen interactions - bacteria - kinases - drug resistance - antibiotics - microbiology
Discovery of Inhibitors of Bacterial Histidine Kinases Summary

The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based drug discovery approach, we have identified small-molecule histidine-kinase inhibitors with antibacterial effect against multi-drug resistant strains, including clinical isolates of multi-drug resistant bacteria such as MRSA. Furthermore, we have shown broadening of the antibacterial spectrum and lowering the toxicity of the histidine-kinase inhibitors using nanoparticles. The results open up exciting possibilities for development of novel antibacterial(nano)medicines.

WUR - Weerbaarheid 1
Hofland-Zijlstra, J.D. - \ 2014
TuinbouwTV
glastuinbouw - plantenziekteverwekkende schimmels - meeldauw - bestrijdingsmethoden - tests - schimmelsporen - meting - gastheer-pathogeen interacties - greenhouse horticulture - plant pathogenic fungi - mildews - control methods - fungal spores - measurement - host pathogen interactions
Filmpje over weerbaarheid van planten en hoe dit goed en snel getoetst kan worden. In dit filmpje ook aandacht voor een eenvoudig techniek om sporen te vangen in de lucht.
Antimicrobial peptides with therapeutic potential from skin secretions of polyploid frogs of the Pipidae family
Mechkarska, M.P.M. - \ 2013
University. Promotor(en): Jerry Wells. - [S.l.] : s.n. - ISBN 9789461735508 - 224
kikkers - pipidae - secreties - antimicrobiële peptiden - gastheer-pathogeen interacties - frogs - secretions - antimicrobial peptides - host pathogen interactions

The emergence of pathogenic bacteria and fungi resistant to commonly used antibiotics poses a serious threat to public health and necessitates novel treatment approaches in order to control infections. Antimicrobial peptides (AMPs) are one of the central components of the system of innate immunity and due to their non-specific and highly destructive mechanism of killing, pathogens will develop resistance at lower rates than conventional antibiotics.Skin secretions of frogs from the family Pipidae are a rich source of AMPs which show potential for development into therapeutic agents.

Until recently, the only representatives of the Pipidae family frogs from which dermal AMPs had been identified were the diploid frog Silurana tropicalis, the tetraploid frog Xenopus laevis and the octoploid frog Xenopus amieti. Therefore, this program of research was undertaken with the aim to isolate, purify and characterize AMPs with therapeutic potential from skin secretions of other polyploid species of African clawed frogs of the Pipidae family. Emphasis is given to the application of the AMPs as markers to elucidate the taxonomic relationships and evolutionary history of the frogs. The study also investigates the effects which polyploidization and interspecies hybridization have had on the multiplicity of AMPs in frog skin secretions.

Chapter 2 and Chapter 3 present data from the peptidomic analysis of norepinephrine-stimulated skin secretions of two well-characterized and closely related tetraploid Xenopus species – X. borealis and
X. clivii. Multiple peptides with varying degrees of antimicrobial activity were isolated. Structural characterization demonstrated that they were orthologous to magainins, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragments (CPFs) and xenopsin-precursor fragments (XPFs), previously isolated from S. tropicalis, X. laevis and X. amieti. CPF-B1 and CPF-C1 were the most abundant AMPs in the skin secretions of X. borealis and X. clivii respectively. CPF-B1 (GLGSLLGKAFKIGLKTVGKMMGGAPREQ) was active against clinical isolates of the hospital-associated pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB) with MIC = 5 µM and showed low hemolytic activity against human erythrocytes (HC50 >200 µM). CPF-C1 (GFGSLLGKALRLGANVL.NH2) also showed potent activity against a range of Gram-negative bacteria. CPF peptides, therefore, represent promising candidates for development into therapeutic agents for use against these emerging antibiotic-resistant pathogens.

The genera Silurana and Xenopus are united in the subfamily Xenopodinae and have a complex evolutionary history. Chapter 4 includes data from the peptidomic analysis of skin secretions from an incompletely characterized tetraploid species termed “S. new tetraploid 1”with chromosome number 2n=40 and the octoploid species X. andrei (2n=72). The species represent model systems in which to study of the fate of duplicated AMP genes following putative allopolyploidization events. Multiple peptides belonging to the PGLa, XPF, and CPF familes were identified. The primary structures of the AMPs from X. andrei indicate aclose phylogenetic relationship between this species and the previously studied X. amieti. Three CPF peptides from “S. new tetraploid 1” showed potent, broad-spectrum antimicrobial activity and are present in high abundance. In contrast, only a single CPF peptide was isolated in low yield from the X. andrei secretions. There is no increase in the multiplicity of the AMPs in skin secretions of “S. new tetraploid 1”and the octoploid X. andrei when compared to the diploid
S. tropicalis (2n=20) and the tetraploid X. laevis (2n=36). It is concluded that nonfunctionalization (gene silencing) has been the most common fate of duplicated AMP genes following polyploidization in the Silurana and Xenopus lineages.

AMPs constitute a characteristic ‘‘fingerprint’’ of a particular frog species that may be used for an unequivocal taxonomic classification. Two populations of the tetraploid X. muelleri, occupying separate non-contiguous ranges in east and west Africa, are studied in Chapter 5. Their taxonomic relationship is unclear and it has been proposed that the western population represents a separate species referred to as
X. muelleri West while the eastern population retains the original name X. muelleri. A comparison of the primary sequences of AMPs in skin secretions reveals that no orthologous peptide from the two populations of X. muelleri has the same amino acid sequence. Additionally, the X. muelleri secretions, like those from X. clivii, did not contain a PGLa peptide whereas the X. muelleri West secretions contained two members of this family. The data indicate that X. muelleri West is more closely related to X. borealis than to X. muelleri and so provide strong support for the proposal that X. muelleri and
X. muelleri West should be described as separate species.

In contrast to species in the subfamily Xenopodinae, frogs from the subfamily Pipinae have not been investigated as a source of AMPs. The AMP profile in skin secretions from Hymenochirus boettgeri as a representative of genus Hymenochirus (subfamily Pipinae) is described in Chapter 6. A novel family of five structurally-related peptides, designated as hymenochirins, was identified. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAV AKMV.NH2) is C-terminally α-amidated whereas hymenochirins-2B - 5B have the general structure XKIPX2VKDTLKKVAKG X2SX2 AGAX3.COOH. The most abundant peptide in the secretions was hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ). Synthetic replicates of hymenochirin-1B - 4B possess broad-spectrum antimicrobial activity and relatively weak hemolytic activity and so represent potential candidates for development into therapeutically valuable agents against drug-resistant pathogens. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of S. tropicalis and X. laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae.

The F1 hybrid frogs X. laevis x X. muelleri represent a model of interspecies hybridization in the Pipidae family that does not result in an increase in ploidy. They arestudied in Chapter 7 and the data obtained provide an insight into the mode of inheritance of AMPs. A total of 18 different AMPs were isolated from skin secretions of the female hybrids. In addition to the complement of AMPs from the parent species, three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin 2 from X. laevis by a single amino acid substitution (Gly13 ®Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.

The thesis is completed by a general discussion in Chapter 8 of theresults and conclusions in Chapters 2-7. The potential of AMPs from skin secretions of frogs belonging to the Pipidae family is reviewed from three different aspects: promising candidates for development into therapeutic valuable anti-infective agents; reliable taxonomic and phylogenetic markers; and tools to study the fate of duplicated genes in Xenopus and Silurana. The interspecies Xenopus hybrids are proposed as a suitable model to perform future studies on the mode of inheritance of skin AMPs.

Molecular analysis of candidate probiotic effector molecules of Lactobacillus plantarum
Remus, D.M. - \ 2012
University. Promotor(en): Michiel Kleerebezem, co-promotor(en): P.A. Bron. - S.l. : s.n. - ISBN 9789461733733 - 187
lactobacillus plantarum - probiotica - genomica - gastheer-pathogeen interacties - probiotics - genomics - host pathogen interactions

Probiotics are health-promoting microorganisms that exert their beneficial effects in several ways. While it is known that probiotic bacteria interact with cells of the host gastrointestinal tractand modulate cell-signaling responses by which they might promote health, the underlying molecular mechanism and probiotic “effector” molecules that are responsible for these effects remain largely unexplored. In this thesis, effector molecules of the lactic acid bacterium Lactobacillus plantarum were discovered that interact with diverse host cell types and manipulate cell-associated signaling pathways. This work represents crucial steps to better understand the exact mode of probiotic action, which is a prerequisite for their controlled, safe, purpose-directed, and person-specific applications in the context of health improvement and disease prevention.

Interactions of Streptococcus suis with host mucosa
Ferrando, M.L. - \ 2012
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
Homologues of Cladosporium fulvum effector proteins are present in species of Dothideomycetes, are recognized by cognate Cf tomato resistance proteins, and can be exploited in molecular resistance breeding.
Stergiopoulos, I. ; Burg, H.A. van den; Ökmen, B. ; Beenen, H.G. ; Kema, G.H.J. ; Wit, P.J.G.M. de - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 149 - 149.
cladosporium - plantenziekteverwekkende schimmels - pathogenesis-gerelateerde eiwitten - passalora fulva - gastheer-pathogeen interacties - plant-microbe interacties - plant pathogenic fungi - pathogenesis-related proteins - host pathogen interactions - plant-microbe interactions
Tot nu toe werden effectoreiwitten van schimmels beschouwd als soortspecifiek en homologen van Cladosporium fulvum effectors werden nooit eerder aangetoond in andere schimmelsoorten. Nu is er bewijs gevonden voor het bestaan van homologe C. fulvum effectors in soorten van Dothideomycetes die pathogeen zijn op ver verwante monocotyle en dicotyle plantensoorten.
Phytophthora infestans, een dynamische ziekteverwekker
Govers, F. - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 128 - 132.
phytophthora infestans - aardappelen - ziektebestrijding - moleculaire genetica - genetische modificatie - phytophthora - genetisch bepaalde resistentie - oömycota - resistentieveredeling - gastheer-pathogeen interacties - potatoes - disease control - molecular genetics - genetic engineering - genetic resistance - oomycota - resistance breeding - host pathogen interactions
Samenvatting van de inaugurele rede van Francine Govers op 11 juni 2009. Dit artikel beschrijft de stand van zaken in het onderzoek aan oömyceten en in het bijzonder aan Phytophthora infestans, de veroorzaker van de aardappelziekte. Er wordt ingegaan op ziektebestrijding en resistentieveredeling, de diversiteit van Phytophthora en zijn gastheren en de genetische blauwdruk: het DNA, de genen, de effectoren en de resistentiegenen.
The Cladosporium fulvum Avr2 protein behaves both as a virulence and an avirulence factor
Klooster, J.W. van t - \ 2010
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.





Functional genomics of Phytophthora infestans effectors and Solanum resistance genes
Champouret, N. - \ 2010
University. Promotor(en): Richard Visser; Evert Jacobsen, co-promotor(en): Vivianne Vleeshouwers. - [S.l. : S.n. - ISBN 9789085856580 - 154
phytophthora infestans - solanum - wilde verwanten - ziekteresistentie - genen - resistentieveredeling - genexpressieanalyse - plant-microbe interacties - gastheer-pathogeen interacties - wild relatives - disease resistance - genes - resistance breeding - genomics - plant-microbe interactions - host pathogen interactions
Potato (Solanum tuberosum L.) is nowadays the most important non-cereal food crop in the world. It is prone to huge annual losses due to late blight, the disease caused by the oomycete pathogen Phytophthora infestans. Modern management of late blight necessitates the use of multiple resistance (R) genes, which requires efficient pipelines for identification, isolation and characterization of R genes. This thesis employs effectoromics, i.e. the use of effectors (pathogenic secreted protein) to probe corresponding R gene(s) in a host plant and sort out their functional redundancy and specificity. Using cytoplasmic RXLR effectors of P. infestans to probe resistant Solanum germplasm for late blight R genes, we were able to: (i) assess the biodiversity of Avr-blb1, characterize the genomic structure of virulent P. infestans isolates on Rpi-blb1 plants and thus provide a technical solution for long-term disease management; (ii) identify the centre of origin of R3a, characterize R3a gene homologues and a functional R gene (Rpi-sto2), and (iii) uncover the potential co-evolution at both R and Avr side for the R2/PiAvr2-PexRD11 interactions, providing more diversity and specificity of R2 homologues, which may be valuable for potato breeding
Multi-scale modeling of potato late blight epidemics
Skelsey, P. - \ 2008
University. Promotor(en): Bert Holtslag, co-promotor(en): Wopke van der Werf; Geert Jan Kessel; Walter Rossing. - S.l. : S.n. - ISBN 9789085049418 - 257
phytophthora infestans - solanum tuberosum - epidemieën - ziektemodellen - sporenverspreiding - virulentie - epidemiologie - geïntegreerde plagenbestrijding - gastheer-pathogeen interacties - epidemics - disease models - spore dispersal - virulence - epidemiology - integrated pest management - host pathogen interactions
Keywords: Solanum, invasion, Gaussian plume model, functional connectivity, landscape design

Proper landscape-scale deployment of disease resistant genotypes of agricultural crop species could
make those crops less vulnerable to invasion by resistance breaking genotypes. Here we develop a
multi-scale, spatio-temporal model of the potato late blight pathosystem (Phytophthora infestans -
Solanum tuberosum) to investigate spatial strategies for the deployment of host resistance. The
model comprises a landscape generator, a potato late blight model, and a suite of aerobiological
models, including an atmospheric dispersion model. Spatial phenomena are solved using Fast Fourier
transforms.
Increasing the number of host genotypes caused the greatest reduction in epidemic extent,
followed by reduction of the proportion of potato in the landscape, lowering the clustering of host
fields, and reducing the size of host fields. Simulation results showed that spatial spread through
short-distance “island hopping” is not a prerequisite for P. infestans invasions, and it appeared not
possible to generate host free zones at the landscape level that were large enough to provide
worthwhile levels of resilience against disease invasion from one host area to another. Deployment
of host resistance in genotype mixtures had a large effect on disease invasion. A new functional
connectivity parameter, characterizing the probability of successful infection following spore
dispersal, proved to be useful in interpreting these results.
Variation in simulation results revealed the importance of using an atmospheric dispersion
model for dispersal, with large weather data sets, and many random landscape iterations. The
specific coincidence in time and space between weather conditions and the geographic locations of
source and target sites defined true landscape connectivity and determined model results regarding
inoculum exchange between fields.
Given the apparent capacity of P. infestans for long distance transport of viable inoculum, it can
be concluded that spatial resistance deployment strategies that center on the creation of spatial
barriers to disease at scales up to several kilometers may not be effective in mitigating invasions of
virulent pathogen strains. Strategies that induce finer-grained spatial and genotypic heterogeneities
in host populations are more limiting to epidemic spread. Genotype mixing was an effective option
for generating agricultural landscapes that are comparatively resilient to disease invasion.
Characterization of genes coding for small hypervariable peptides in Globodera rostochiensis
Bers, N.E.M. van - \ 2008
University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - [S.l.] : S.n. - ISBN 9789085049579 - 229
globodera rostochiensis - plantenparasitaire nematoden - peptiden - genen - genexpressie - solanum tuberosum - arabidopsis - nicotiana - gensplitsing - gastheer-pathogeen interacties - moleculaire interacties - plant parasitic nematodes - peptides - genes - gene expression - gene splicing - host pathogen interactions - molecular interactions
Plant parasitic nematodes secrete a cocktail of effector molecules, which are involved
in several aspects of the interaction with the host, eg. in host defense suppression, in
migration and in feeding cell formation. In this thesis, we performed the first study on
10 novel peptide genes, believed to be important for parasitism of the potato cyst
nematode, Globodera rostochiensis. Nine of the peptide genes described here belong
to the SECPEP gene family. The SECPEP genes are all expressed in the dorsal
esophageal gland, which is one of the main sites for the production of effector
molecules. This, together with the predominant expression in preparasitic and early
parasitic juvenile nematodes, makes it very likely that the SECPEPs code for effector
peptides essential for succesful infection and feeding site formation.
In chapter 2, we show that diversifying selection is a likely driver of the molecular
evolution of the SECPEPs. The sequences of the mature peptides appear to be highly
diverse, while the non)coding 3’UTR and intronic regions as well as the region coding
for the signal peptide for secretion are relatively conserved. In fact, a pairwise
comparison of the SECPEPs reveals no significant sequence similarity between family
members at all. In chapter 5 we further speculate on a possible role for RNA)editing as
a mechanism to yield hypervariability in the SECPEPs, because the sequence diversity
at the transcript level significantly exceeds that of the genomic locus. Chapter 5 further
elaborates on the analysis of trans)splicing in SECPEP1 transcripts. We show that
SECPEP1 transcripts are trans)spliced to a surprising diversity of novel spliced)leader
sequences. The first approach to unravel the role of the members of the SECPEP family
in plant parasitism, is described in chapter 4. We generated transgenic potato and
Arabidopsis plants expressing SECPEP3 while using the CaMV 35S promotor. The
phenotype associated with SECPEP3 in both potato and Arabidopsis plants includes a
reduction of root growth and an alteration of the leaf morphology. The SECPEP3
peptide harbors several sequence motifs first found in the cyclin)dependent kinase
inhibitors ICK1/KRP1, SIM and Smr1. We, therefore, suggest a role for SECPEP3 in cell
cycle alteration in nematode feeding site formation. Although the SECPEP genes show
only a low level of primary sequence similarity, all code for positively charged,
hydrophilic peptides with a C)x)G γ)core motif (chapter 2). These are characteristics
typical for host defense peptides, and in chapter 6 we investigate whether these
characteristics are also found for other peptides involved in plant)parasite interactions.
We show that a considerable number of these effector peptides share a positive
charge, hydrophilicity and C)x)G γ)core motif with the SECPEPs, and we speculate on a
role for the positive charge in peptide)ligand interaction.
In chapter 3 we describe the NEMPEP peptide, secreted by G. rostochiensis. NEMPEP
is also a positively charged, hydrophilic peptide with a C)x)G γ)core motif, although it is
genetically unrelated to the SECPEP gene family. During the life cycle of G.
rostochiensis, the expression pattern of NEMPEP reveals a striking regulation. NEMPEP
is highly expressed in preparasitic juveniles and in the parasitic life stages after initial
feeding cell formation. However, NEMPEP expression was hardly detectable in the
juveniles just after entering the plant root. Several disease resistance genes condition
nematode resistance at the onset of parasitism. The downregulation of NEMPEP at
exactly this timepoint could be a strategy to avoid recognition by the host’s immune
system. In planta expression of NEMPEP, as a fusion to GFP, shows that NEMPEP
accumulates in the nucleolus of tobacco cells. Potato plants transformed with
35S::NEMPEP were slow at forming roots and the internodes between the leaflets were
shortened. This, together with a reduced transformation efficiency, led us to
hypothesize a role for NEMPEP in cytokinin signaling (Chapter 3).
Currently, there are two models regarding the functional role of the SECPEPs and
NEMPEP. The first one concerns a role as an antimicrobial peptide, which could protect
the host plant against secondary infections by opportunistic microbes. As a competing
hypothesis, the high hydrophilicity of the peptides may point to a role as peptide
hormone. As such, they may be involved in redirecting cell cycle or hormonal regulation
upon feeding cell formation.
Cladosporium fulvum effector proteins and their role in pathogen virulence
Esse, H.P. van - \ 2008
University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - [S.l. : S.n. - ISBN 9789085049272 - 214
solanum lycopersicum - tomaten - plantenziekteverwekkende schimmels - passalora fulva - genen - virulentie - genexpressie - genomen - gene silencing - gastheer-pathogeen interacties - plant-microbe interacties - tomatoes - plant pathogenic fungi - genes - virulence - gene expression - genomes - host pathogen interactions - plant-microbe interactions
Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould of tomato (Solanum esculentum). Chapter 1 is a “pathogen profile” describing the biology of the pathogen. During growth in the leaf apoplast, the intercellular space surrounding the mesophyll cells, the fungus secretes effector proteins that are thought to play a role in disease establishment. Eight of these effectors have been characterized in detail. For most of these effectors, cognate C. fulvum (Cf) resistance loci have been identified in tomato that mediate an immune response upon recognition of (the activity of) the cognate effector.
In chapter 2, a targeted proteomics approach to investigate the role of these effector proteins and to identify possible in planta targets is described. C. fulvum proteins were expressed as recombinant fusion proteins carrying various affinity–tags at either their C– or N–terminus. Although these fusion proteins were correctly expressed and secreted into the leaf apoplast, detection of affinity–tagged C. fulvum proteins failed and affinity–purification did not result in the recovery of these proteins. However, when using C. fulvum effector protein–specific antibodies, specific signals were obtained for the different proteins. It was therefore concluded that the stability of the in planta expressed recombinant fusion proteins is insufficient, which resulted in removal of the affinity–tag from the fusion proteins, irrespective of C– or N–terminal fusion or the nature of the affinity–tag. Similar observations were made when the fusion proteins were expressed in other Solanaceous species, but not when expressed in Arabidopsis thaliana.
Previous studies have demonstrated that Avr4 binds to chitin present in fungal cell walls, and that this binding by Avr4 can protect these cell walls against hydrolysis by plant chitinases. In chapter 3 it is described that Avr4–expression in Arabidopsis results in increased virulence of several fungal pathogens with exposed chitin in their cell walls, whereas the virulence of a bacterium and an oomycete remained unaltered. Heterologous expression of Avr4 in tomato increased the virulence of Fusarium oxysporum f. sp. lycopersici. Tomato GeneChip analysis was used to demonstrate that Avr4–expression in tomato results in the induced expression of only a handful of genes. Finally, silencing of the Avr4 gene in C. fulvum decreased fungal virulence on tomato. In conclusion, chapter 3 is the first report on the intrinsic function of a fungal avirulence protein that displays self-defense activity which is required for full pathogen virulence.
In chapter 4, a study on the intrinsic biological function of Avr2 is presented. The Avr2 effector interacts with the apoplastic tomato cysteine protease Rcr3, which is required for Cf–2–mediated immunity. In this chapter it is demonstrated that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis resulted in enhanced susceptibility towards a number of extracellular fungal pathogens that include Botrytis cinerea and Verticillium dahliae, and microarray analysis of unchallenged Arabidopsis plants showed that Avr2 expression triggered a global transcription profile that is reminiscent of pathogen challenge. Cysteine protease activity profiling revealed that Avr2 inhibits multiple extracellular Arabidopsis cysteine proteases. In tomato, Avr2 expression resulted in enhanced susceptibility not only towards natural Avr2–defective C. fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. Cysteine protease activity profiling in tomato revealed that Avr2 inhibits multiple extracellular cysteine proteases including Rcr3 and its close relative PIP1. Finally, silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases required for plant basal defense in tomato.
Chapter 5 describes the discovery and characterization of a novel effector protein of C. fulvum, Ecp6. To discover novel C. fulvum effectors that might play a role in virulence, two–dimensional polyacrylamide gel electrophoresis (2D–PAGE) was used to visualize proteins secreted during C. fulvum–tomato interactions. Three novel C. fulvum proteins were identified; CfPhiA, Ecp6, and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides, while Ecp6 contains lysine motifs (LysM domains), which are recognized as carbohydrate–binding modules. Finally, Ecp7 encodes a small, cysteine–rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNAi–mediated gene silencing it was demonstrated that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologs in other organisms, conserved Ecp6 orthologs were identified in various fungal species. Homology based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.
Chapter 6 presents global transcriptional profiling study to compare transcriptional changes in tomato during compatible and incompatible interactions with the foliar pathogenic fungus Cladosporium fulvum and the soil–borne vascular pathogenic fungus Verticillium dahliae. Although both pathogens colonize different host tissues, they display significant commonalities in their infection strategies as they both penetrate natural openings and grow strictly extracellular without the formation of haustoria. Furthermore, in incompatible interactions with both pathogens resistance is conveyed by extracellular transmembrane receptors that belong to the class of receptor–like proteins. For each of the two pathogens, the transcriptomes of the compatible and incompatible interaction largely overlapped. However, the C. fulvum–induced transcriptomes showed little overlap with the V. dahliae–induced transcriptomes, as most genes were uniquely regulated by one of the two pathogens. This also applied to both incompatible interactions, despite defense activation by the same type of resistance protein. Remarkably, of the relatively small subset of genes that was regulated by both pathogens a large portion showed an inverse regulation; induced by one pathogen and repressed by the other. With pathway reconstruction, interacting networks of tomato genes implicated in photorespiration, hypoxia and glycoxylate metabolism were identified that were repressed upon infection with C. fulvum and induced by V. dahliae. Similarly, auxin signaling was differentially affected by the two pathogens.
In chapter 7, the general discussion, the implications are of the data that are presented in this thesis are discussed for the use of C. fulvum as a model, and for fungal pathogens in general. Furthermore, the use of heterologous expression systems to study fungal effectors is briefly discussed. In several of the chapters presented in this thesis, the use of microarrays has been instrumental to investigate the biology of C. fulvum and the role of specific effectors secreted by the pathogen. Therefore, an overview of the currently available in silico tools for reconstruction of cellular pathways based on plant gene expression datasets is presented.

Phosphorylation and proteome dynamics in pathogen-resistant tomato plants
Stulemeijer, I.J.E. - \ 2008
University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten. - [S.l. : S.n. - ISBN 9789085048855 - 208
solanum lycopersicum - passalora fulva - plantenziektekunde - signaaltransductie - fosforylering - eiwitsynthese - kinasen - verdedigingsmechanismen - plant-microbe interacties - gastheer-pathogeen interacties - eiwitexpressieanalyse - resistentieveredeling - plant pathology - signal transduction - phosphorylation - protein synthesis - kinases - defence mechanisms - plant-microbe interactions - host pathogen interactions - proteomics - resistance breeding
Microbial plant pathogens impose a continuous threat on global food production. Similar to disease resistance in mammals, an innate immune system allows plants to recognise pathogens and swiftly activate defence. For the work described in this thesis, the interaction between tomato and the extracellular fungal pathogen Cladosporium fulvum serves as a model system to study host resistance and susceptibility in plant-pathogen interactions. Resistance to C. fulvum in tomato plants follows the gene-for-gene hypothesis, which requires the presence of a Cf resistance gene in tomato and presence of the cognate avirulence gene (Avr) in C. fulvum. Upon perception of the Avr by a tomato plant, a typical hypersensitive response (HR) is induced that renders the plant resistant to C. fulvum. In the years preceding this thesis work, most research was focussed on understanding which Avrs are produced by C. fulvum and how these Avrs are actually perceived by resistant plants (Chapter 1). The goal of the work described in this thesis is to reveal downstream signalling cascades triggered upon Avr perception. Therefore, the HR was studied by using a model system in which the Cf-4 protein of tomato and the Avr4 protein from C. fulvum were simultaneously expressed in tomato seedlings. Since the Cf-4/Avr-induced responses are inhibited at 33°C and high humidity, these Cf-4/Avr4 seedlings initiate a synchronized and reproducible HR after incubation at 33°C and a subsequent shift to 20°C, which allows studying downstream responses.
To prevent pathogen proliferation in the resistant plant, defence signalling cascades need to be activated extremely fast upon pathogen recognition. Therefore, many downstream signalling cascades depend on post-translational modifications (PTMs) that allow a rapid, reversible, controlled and highly specific transduction of perceived signals. An overview of the various types of PTMs and their role in the resistance response of plants to pathogens is provided in Chapter 2. In addition, examples are provided of successful pathogens that manipulate PTMs.
Protein phosphorylation seems to play an important role in the Cf-4/Avr4-triggered HR, since Avr4 perception leads to the specific activation of at least three mitogen-activated protein kinases, LeMPK1, -2 and -3, which requires phosphorylation by an upstream kinase (Chapter 3). Each of these three kinases seems to have a different role in downstream defence signalling, since the kinases were shown to have different phosphorylation specificities and therefore most likely have different downstream target substrates. Furthermore, these kinases appear to play a different role with regard to HR and full resistance to C. fulvum in tomato (Chapter 3).
Since protein phosphorylation was shown to play an important role in Cf-4/Avr4-induced defence signalling, the phosphoproteome of Cf-4/Avr4 and control seedlings after HR initiation was studied using a new approach (Chapter 4). This approach led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Quantification revealed 13 phosphoproteins with an altered abundance in the Cf-4/Avr4 seedlings as compared to the control, which implies HR-induced differential phosphorylation of these proteins. Phosphorylation-mediated regulation of the activity of these proteins pointed to a swift decrease in photosynthetic activity upon HR-initiation, which was confirmed by experiments in which the actual efficiency of the photosynthesis in the Cf-4/Avr4 seedlings was determined upon induction of the HR. Furthermore, a shift from aerobic to anaerobic respiration, which possibly results from oxygen depletion caused by a massive oxidative burst consuming large amounts of oxygen, seems to take place upon initiation of the HR. Finally, differential phosphorylation of the four cytoplasmic isoforms of the Hsp90 chaperone protein was observed, suggesting that they play distinct roles during defence signalling (Chapter 4).
In addition to the HR, other associated defence responses are initiated upon recognition of C. fulvum. One of these responses is the secretion of defence-related proteins into the apoplast, which is the environment where C. fulvum operates. Therefore, the dynamics of the apoplastic proteome of resistant, Cf-4-expressing plants and susceptible tomato plants lacking Cf-4, were studied after inoculation with a strain of C. fulvum that secretes Avr4 (Chapter 5). Analysis of the apoplastic proteome revealed a slow accumulation of defence proteins in the apoplast of susceptible plants, which is most likely the result of perception of general elicitors of C. fulvum by tomato. In resistant plants, the same set of proteins accumulates in the apoplast, but this occurs much faster and to higher levels. The accelerated response is caused by the Cf-4/Avr4-initiated HR that also leads to cell death. The HR, in combination with the accelerated protein secretion, renders the plants resistant to C. fulvum. In addition, in susceptible plants C. fulvum seems to specifically downregulate genes encoding cell wall proteins of which the accumulation possibly hampers nutrient and water uptake and thereby proliferation of the pathogen in the tomato apoplast. Possibly, an effector of C. fulvum targets a receptor for general elicitors, thereby suppressing transcription of these genes (Chapter 5).
Most data described in this thesis have been obtained from Cf-4/Avr4 seedlings in which the HR can be inhibited by incubating the plants at 33°C. The present data suggest that this temperature-sensitivity occurs at the site of signal perception. Possibly, cytoplasmic Hsp90 stabilizes R protein complexes localized at the plasma membrane. Upon high temperature stress, an increased demand for Hsp90 occurs in the cells to stabilize unfolding proteins that play a role in basal cellular processes, which could lead to the release and subsequent degradation of R protein complexes, rendering defence signalling temperature-sensitive (Chapter 6). The temperature-sensitivity of the Cf-4/Avr4-initiated HR provides a very clean and reproducible tool to study the HR, in the absence of the fungus that produces the Avr. Furthermore, the data described in this thesis provide evidence that the Cf-4/Avr4 seedlings recover from the temperature stress before the specific Cf-4/Avr4-triggered HR is initiated. The possibility to separate the events directly associated with the HR from the full resistance response of the plant to the invading fungus, provides new insight into the complexity of plant defence responses and their specific suppression upon successful colonization by C. fulvum (Chapter 6). Comparison of the defence response to other processes that occur in the cell underlines that resistance and HR execution cannot be seen as an independent and separate process in resistant plants that have recognized a pathogen. On the contrary, signalling cascades seem to depend on similar components and on cascades that possibly converge, eventually leading to a similar response (Chapter 6). Finally, an up to date model for the Cf-4/Avr4-triggered HR and resistance is proposed, based on data that have been published before and the results obtained with the research described in this thesis (Chapter 6).

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