Staff Publications

Staff Publications

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

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

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

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

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    Virulence contribution and recognition of homologs of the Verticillium dahliae effector Ave1
    Boshoven, Jordi C. - \ 2017
    Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. 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 - verticillium dahliae - 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.

    Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
    Song, Yin - \ 2017
    Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
    disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - verticillium dahliae - verticillium - tomaten - solanum lycopersicum - receptoren - genen - tabak - nicotiana glutinosa - aardappelen - solanum tuberosum - solanum torvum - humulus lupulus - katoen - gossypium hirsutum - transgene planten - arabidopsis thaliana

    Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

    Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
    Zeijl, Arjan van - \ 2017
    Wageningen University. Promotor(en): T. Bisseling, co-promotor(en): R. Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
    plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - rhizobium - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming

    Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.

    A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.

    Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.

    In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.

    Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.

    Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.

    Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.

    In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.

    The role of strigolactones and the fungal microbiome in rice during drought adaptation
    Andreo Jimenez, Beatriz - \ 2017
    Wageningen University. Promotor(en): H.J. Bouwmeester, co-promotor(en): C. Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789463437028 - 205
    drought resistance - drought - abiotic injuries - rice - oryza sativa - plant-microbe interactions - nutrient uptake - defence mechanisms - hormones - fungi - genes - droogteresistentie - droogte - abiotische beschadigingen - rijst - oryza sativa - plant-microbe interacties - voedingsstoffenopname (planten) - verdedigingsmechanismen - hormonen - schimmels - genen

    Rice is the most important food crop in the world, feeding over half the world’s population. However, rice water use efficiency, defined by units of yield produced per unit of water used, is the lowest of all crops. The aim of this thesis was to study the effect of plant hormones and the root microbiome on drought tolerance in rice. The new plant hormone, strigolactone, was shown to be upregulated under drought and to regulate drought tolerance in interaction with the drought-hormone abscisic acid. Using a large collection of rice genotypes grown in the field, we showed that the composition of the root associated fungal microbiome is determined by the rice genotype and can contribute to drought tolerance.

    Evasion of chitin-triggered immunity by fungal plant pathogens
    Rövenich, Hanna J. - \ 2017
    Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789463436137 - 133
    plant-microbe interactions - immunity - receptors - verticillium dahliae - cladosporium - plant pathogens - chitin - arabidopsis thaliana - fungi - plant-microbe interacties - immuniteit - receptoren - verticillium dahliae - cladosporium - plantenziekteverwekkers - chitine - arabidopsis thaliana - schimmels

    Plants establish intricate relationships with microorganisms that range from mutualistic to pathogenic. In order to prevent colonization by potentially harmful microbes, plant hosts employ surface-localized receptor molecules that perceive ligands, which are either microbe-derived or result from microbe-mediated plant manipulation. This recognition ultimately leads to the activation of host immunity. In order to circumvent recognition or suppress immune responses, microbes secrete effector proteins that deregulate host physiological processes. While the number of identified putative effectors has rapidly increased in recent years, their functions and the mechanisms governing their recognition have largely remained unexplored. To enhance our understanding of the molecular interplay between host and microbe, the work presented here was designed to identify further components involved in the recognition of the two fungal pathogens Verticillium dahliae and Cladosporium fulvum, as well as to characterize the functions of effector proteins produced by these pathogens during tomato infection.

    Plant growth promotion by Pseudomonas fluorescens : mechanisms, genes and regulation
    Cheng, X. - \ 2016
    Wageningen University. Promotor(en): Francine Govers; J.M. Raaijmakers, co-promotor(en): M. van der Voort. - Wageningen : Wageningen University - ISBN 9789462578753 - 192
    soil bacteria - pseudomonas fluorescens - plants - growth stimulators - soil suppressiveness - plant diseases - induced resistance - biochemistry - biosynthesis - plant-microbe interactions - transcriptomics - bodembacteriën - pseudomonas fluorescens - planten - groeistimulatoren - bodemweerbaarheid - plantenziekten - geïnduceerde resistentie - biochemie - biosynthese - plant-microbe interacties - transcriptomica

    Pseudomonas fluorescens is a Gram-negative rod shaped bacterium that has a versatile metabolism and is widely spread in soil and water. P. fluorescens strain SBW25 (Pf.SBW25) is a well-known model strain to study bacterial evolution, plant colonization and biocontrol of plant diseases. It produces the biosurfactant viscosin, a lipopeptide that plays a key role in motility, biofilm formation and activity against zoospores of Phytophthora infestans and other oomycete pathogens. In addition to viscosin, Pf.SBW25 produces other metabolites with activity against plant pathogens. The production of these yet unknown metabolites appeared to be regulated by the GacS/GacA two-component regulatory system (the Gac-system). The second P. fluorescens strain SS101 (Pf.SS101) studied in this thesis is known for its plant growth-promoting activities but the underlying mechanisms and genes are largely unknown. Therefore, in this study, we aimed to identify novel metabolites and biosynthetic genes in Pf.SBW25 and Pf.SS101, and to investigate their role in plant growth promotion and biocontrol. To this end, a multidisciplinary approach involving bioinformatic analysis of the genome sequences of strains Pf.SBW25 and Pf.SS101, microarray-based expression profiling, screening of genomic libraries, bioactivity assays, mass spectrometric image analysis (MALDI-IMS) and GC/MSMS analysis was adopted. In conclusion, we showed that the GacS/GacA two-component system as a global regulator of the expression of genes play important roles in antagonism of Pseudomonas fluorescens toward plant pathogenic microbes as well as in plant growth promotion and ISR. Growth promotion by P. fluorescens is associated with alterations in auxin biosynthesis and transport, steroid biosynthesis, carbohydrate metabolism and sulfur assimilation. Moreover, advanced chemical profiling allowed us to compare the metabolite profiles of free-living P. fluorescens and P. fluorescens living in association with plant roots. A better understanding of yet unknown mechanisms exploited by the various Pseudomonas fluorescens strains will lead to new opportunities for the discovery and application of natural bioactive compounds for both industrial and agricultural purposes.

    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 - passalora fulva - plant pathogenic fungi - virulence - proteinases - proteinase inhibitors - plant-microbe interactions - genomics - solanum lycopersicum - 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.

    Hoe levert onderzoek naar modder nieuwe medicijnen op?
    Vet, L.E.M. - \ 2015
    Universiteit van Nederland
    bodemkunde - bodembiologie - bodembiodiversiteit - lesmaterialen - boven- en ondergrondse interacties - plant-microbe interacties - bodemmicrobiologie - bodemweerbaarheid - biodiversiteit - soil science - soil biology - soil biodiversity - teaching materials - aboveground belowground interactions - plant-microbe interactions - soil microbiology - soil suppressiveness - biodiversity
    Onze bodem krioelt van micro-organismen. Aan zulke micro-organismen hebben we de ontdekking van onder meer penicilline te danken. Wat kunnen we nog meer aantreffen in die bio-schatkist onder onze voeten? Prof. dr. Louise Vet van Wageningen UR vertelt erover in haar college.
    Rhizobacterial modification of plant defenses against insect herbivores: from molecular mechanisms to tritrophic interactions
    Pangesti, N.P.D. - \ 2015
    Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462572836 - 224
    planten - rizosfeerbacteriën - insecten - multitrofe interacties - verdedigingsmechanismen - pseudomonas fluorescens - mamestra brassicae - pieris brassicae - plant-microbe interacties - insect-plant relaties - plant-herbivoor relaties - plants - rhizosphere bacteria - insects - multitrophic interactions - defence mechanisms - pseudomonas fluorescens - mamestra brassicae - pieris brassicae - plant-microbe interactions - insect plant relations - plant-herbivore interactions

    SUMMARY

    Plants as primary producers in terrestrial ecosystems are under constant threat from a multitude of attackers, which include insect herbivores. In addition to interactions with detrimental organisms, plants host a diversity of beneficial organisms, which include microbes in the rhizosphere. Furthermore, the interactions between plants and several groups of root-associated microbes such as mycorrhizae, plant growth promoting rhizobacteria (PGPR) and plant growth promoting fungi (PGPF) can affect plant interactions with foliar insect herbivores. The beneficial root-associated microbes are able to modify plant physiology by promoting plant growth and induced systemic resistance (ISR), in which the balance between both effects will determine the final impact on the insect herbivores. Using Arabidopsis thaliana Col-0, this thesis explores the molecular mechanisms on how plants integrate responses when simultaneously interacting with the rhizobacterium Pseudomonas fluorescens and the generalist and the specialist leaf-chewing insects Mamestra brassicae and Pieris brassicae respectively.

    A literature review on the state-of-the-art in the field of microbe-plant-insect interactions (Chapter 2) explores how root-associated microbes and insect folivores can influence each other via a shared host plant. For more than a decade, both ecological and mechanistic studies mostly focused on exploring these belowground and aboveground interactions using single microbe and single herbivore species. The importance of increasing the complexity of the study system in order to understand the interactions in more natural situations is being emphasized. Furthermore, this review discusses the role of plant hormones in regulating plant growth and defense against folivores, while simultaneously being involved in associations with root-associated microbes.

    Experimental evidence has shown patterns on the effects of mycorrhizal colonization on plant interactions with insect herbivores, and raises the question whether plant colonization by different groups of root-associated microbes has similar effects on particular categories of insect herbivores. In Chapter 3, plant-mediated effects of a non-pathogenic rhizobacterium on the performance of leaf-chewing insects, and the underlying mechanisms modulating the interactions, have been examined. Colonization of A. thaliana Col-0 roots by the bacterium P. fluorescens strain WCS417r resulted in decreased larval weight of the generalist leaf-chewing M. brassicae, and had no effect on larval weight of the specialist leaf-chewing P. brassicae. The crucial role of jasmonic acid (JA) in regulating rhizobacteria-mediated induced systemic resistance (ISR) against M. brassicae is confirmed by including plant mutants in the study. Interestingly, I also observed that rhizobacteria can induce systemic susceptibility to M. brassicae caterpillars. Comparison of M. brassicae performance and gene transcription in A. thaliana plants, grown in potting soil or a mixture of potting soil and sand in a 1:1 ratio, shows that in a mixture of potting soil and sand, rhizobacterial treatment had a consistently negative effect on M. brassicae, whereas the effect is more variable in potting soil. Rhizobacterial treatment primed plants grown in potting soil and sand for stronger expression of JA- and ethylene-regulated genes PDF1.2 and HEL, supporting stronger resistance to M. brassicae. Taken together, the results show that soil composition can be one of the factors modulating the outcome of microbe-plant-insect interactions.

    Chapter 4 further addresses the mechanisms underlying rhizobacteria-mediated ISR against the generalist leaf-chewing M. brassicae by integrating plant gene transcription, chemistry and performance of M. brassicae in wild type A. thaliana Col-0 plants and mutants defective in the JA-pathway, i.e. dde2-2 and myc2, in the ET pathway, i.e. ein2-1, and in the JA-/ET-pathway, i.e. ora59. Results of this study show that rhizobacterial colonization alone or in combination with herbivore infestation induced the expression of the defense-associated genes ORA59 and PDF1.2 at higher levels than activation by herbivore feeding alone, and the expression of both genes is suppressed in the knock-out mutant ora59. Interestingly, the colonization of plant roots by rhizobacteria alters the levels of plant defense compounds, i.e. camalexin and glucosinolates (GLS), by enhancing the synthesis of constitutive and induced levels of camalexin and aliphatic GLS while suppressing the induced levels of indole GLS. The changes are associated with modulation of the JA-/ET-signaling pathways as shown by investigating mutants. Furthermore, the herbivore performance results show that functional JA- and ET-signaling pathways are required for rhizobacteria-mediated ISR against leaf-chewing insects as observed in the knock-out mutants dde2-2 and ein2-1. The results indicate that colonization of plant roots by rhizobacteria modulates plant-insect interactions by prioritizing the ORA59-branch over the MYC2-branch, although the transcription factor ORA59 is not the only one responsible for the observed effects of rhizobacteria-mediated ISR against leaf-chewing insects.

    Taking a step further in increasing the complexity of the study system, Chapter 5 investigates how co-cultivation of P. fluorescens strains WCS417r and SS101 affects direct plant defense to the caterpillar M. brassicae. Inoculation of either P. fluorescens WCS417r or SS101 singly at root tips or simultaneously at two different positions along the roots resulted in a similar level of rhizobacterial colonization by each strain, whereas co-cultivation of both strains at either the root tips or at two different positions along the roots resulted in a higher colonization level of strain WCS417r compared to colonization by SS101. The results suggest that the site of inoculation influences the direct interactions between the two strains in the rhizosphere, as also confirmed by in vitro antagonism assays in the absence of plants. Both upon single inoculation and co-cultivation of both strains at the same or different sites along the roots, the two rhizobacterial strains induced the same strength of ISR against the caterpillar M. brassicae and the same degree of plant growth promotion. In the roots, colonization by the two strains as single or mixed culture resulted in a similar gene expression pattern of up-regulation of MYC2, down-regulation of WRKY70 and no effect on NPR1 expression, genes representing JA-signaling, SA-signaling and the node of crosstalk between the two pathways, respectively. We hypothesize that both rhizobacterial strains use negative crosstalk between JA- and SA-pathways as mechanism to suppress plant immunity and establish colonization. This study shows that competitive interactions between rhizobacterial strains known to induce plant defense in systemic tissue via different signaling pathways, may interfere with synergistic effects on ISR and plant growth promotion.

    While the effect of root-associated microbes on direct plant defense against insect herbivores has been studied previously, the effect of these microbes on indirect plant defense to herbivores is much less known. Chapter 6 explores how colonization by the rhizobacterium P. fluorescens strain WCS417r affects indirect plant defense against the generalist herbivore M. brassicae by combining behavioral, chemical and gene transcriptional approaches. The results show that rhizobacterial colonization of A. thaliana roots results in an increased attraction of the parasitoid Microplitis mediator to caterpillar-infested plants. Volatile analysis revealed that rhizobacterial colonization suppressed emission of the terpene (E)-α-bergamotene, and the aromatics methyl salicylate and lilial in response to caterpillar feeding. Rhizobacterial colonization decreased the caterpillar-induced transcription of the terpene synthase genes TPS03 and TPS04. Rhizobacteria enhanced both growth and indirect defense of plants under caterpillar attack. This study shows that rhizobacteria have a high potential to enhance the biocontrol of leaf-chewing herbivores based on enhanced attraction of parasitoids.

    Taken together, the research presented in this thesis has shown how single or combined applications of rhizobacteria affect interactions of plants with leaf-chewing insects in terms of direct and indirect resistance. Furthermore, results presented in this thesis have revealed some of the molecular mechanisms underlying plant-mediated interactions between rhizobacteria and leaf-chewing insects that can be used in developing practical approaches by applying beneficial root-associated microbes for improving plant resistance.

    Understanding the role of L-type lectin receptor kinases in Phytophthora resistance
    Wang, Y. - \ 2014
    Wageningen University. Promotor(en): Francine Govers, co-promotor(en): W. Shan; Klaas Bouwmeester. - Wageningen : Wageningen University - ISBN 9789462571327 - 214
    phytophthora - phytophthora capsici - oömycota - plantenziekteverwekkende schimmels - plant-microbe interacties - arabidopsis - transgene planten - genexpressie - receptoren - kinasen - genen - ziekteresistentie - immuniteit - phytophthora - phytophthora capsici - oomycota - plant pathogenic fungi - plant-microbe interactions - arabidopsis - transgenic plants - gene expression - receptors - kinases - genes - disease resistance - immunity

    Abstract

    Phytophthora pathogens are notorious for causing severe damage to many agriculturally and ornamentally important plants. Effective plant resistance depends largely on the capacity to perceive pathogens and to activate rapid defence. Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity upon recognition of matching effectors secreted by Phytophthora. However, Phytophthora pathogens are notoriously difficult to control due to their rapid adaptation to evade R protein-mediated recognition. Hence, exploring novel resistance components is instrumental for developing durable resistance. Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous and endogenous stimuli to initiate plant defence. One RLK that was previously identified as a novel Phytophthora resistance component is the Arabidopsis L-type lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of 45 members in Arabidopsis but whether or not the other members function in Phytophthora resistance was thus far unknown. The research described in this thesis was aimed at unravelling the role of LecRKs in plant immunity, in particular to Phytophthora pathogens.

    Chapter I describes various Phytophthora diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to Phytophthora pathogens.

    In Chapter II, we describe the development of a new Arabidopsis-Phytophthora pathosystem. We demonstrated that Phytophthora capsici is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and indole glucosinolates biosynthesis pathways are required for P. capsici resistance (Chapter IIa). The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem can be used as an additional tool to pinpoint essential components of Phytophthora resistance.

    We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of LecRKs in Phytophthora resistance. In Chapter III we describe a systematic phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other LecRKs function in Phytophthora resistance. We have also analysed whether the LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to bacterial or fungal pathogens, but none of the lines showed visible developmental changes under normal conditions or upon abiotic stress treatment. Combining these phenotypic data with LecRK expression profiles obtained from publicly available datasets revealed that LecRKs that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function display diverse expression patterns.

    Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora resistance was further investigated. In Chapter IV we describe that overexpression of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to Phytophthora, but also induced plant cell death. A mutation in the kinase domain abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce Phytophthora resistance as did deletion of the lectin domain. Cell death induction however, only required the kinase, not the lectin domain. Since transient expression of both LecRKs in Nicotiana benthamiana also resulted in increased Phytophthora resistance and induction of cell death, we used N. benthamiana to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for Phytophthora resistance. Collectively, these results illustrate that the Phytophthora resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death phenotype. By co-immunoprecipitation we identified putative interacting proteins, one of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis, the ABC transporter ABCG40, was found to interact in planta with both LecRK-IX.1 and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants showed compromised Phytophthora resistance, indicating that ABCG40 has a function in Phytophthora resistance.

    In Chapter V, we describe the generation of stable transgenic N. benthamiana plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of LecRK-IX.1 induced plant cell death. Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed increased resistance towards P. capsici or Phytophthora infestans. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora resistance upon interfamily transfer.

    Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in Phytophthora resistance. In Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both N. benthamiana and tomato lack LecRKs homologous to Arabidopsis LecRKs of clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional analysis of various Solanaceous LecRKs using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance. These results indicate that the role of clade IX LecRKs in Phytophthora resistance is conserved across plant species.

    In Chapter VII, the experimental data presented in this thesis are summarized and discussed in a broader context. We present an overview of the current understanding of LecRKs in plant immunity and discuss how LecRKs can be exploited to improve plant resistance.

    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 - phytophthora infestans - oomycota - plant pathogenic fungi - virulence - genes - plant-microbe interactions - disease resistance - defence mechanisms - susceptibility - gene silencing

    Summary

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

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

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

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

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

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

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

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

    The role of phosphatidylinositol-specific phospholipase-C in plant defense signaling
    Abd-El-Haliem, A.M. - \ 2014
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten. - Wageningen : Wageningen University - ISBN 9789462571181 - 186
    plantenziekten - ziekteresistentie - verdedigingsmechanismen - solanum lycopersicum - tomaten - signaaltransductie - fosfolipase c - plant-microbe interacties - passalora fulva - genetische analyse - nucleotidenvolgordes - plant diseases - disease resistance - defence mechanisms - solanum lycopersicum - tomatoes - signal transduction - phospholipase c - plant-microbe interactions - passalora fulva - genetic analysis - nucleotide sequences

    Plant innate immunity requires immune receptors that sense the presence of microbes and activate defense reactions. Phosphatidylinositol-phospholipase C (PI-PLC) activity was previously shown to be important for several types of plant defenses although its signaling mechanism is not fully understood. It is also not clear why plants possess several PI-PLC isoforms and how triggering immune receptors activates them. PI-PLC activation induces a transient release of free cytosolic Ca2+ and the turnover of specific low abundant signaling phospholipids in the plasma membrane. Both events are important signals in animals and plants. Here, the first genetic evidence linking PI-PLC signaling to plant defense against pathogens is provided. The structure of the tomato PI-PLC family was determined, the corresponding genes were cloned and the function of individual isoforms during defense was studied. We found that PLC4 and PLC6 encode active enzymes and that they have distinct roles in defense. Optimum activity requirements and substrate preferences were determined for three PI-PLC enzymes and their enzyme activity was found to be important for immune receptor-activated responses. The available information was used to draw a model explaining the role of PI-PLC signaling in immune receptor-mediated defense and resistance in plants.

    Bacterial canker resistance in tomato
    Sen, Y. - \ 2014
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Sjaak van Heusden. - Wageningen : Wageningen University - ISBN 9789462570764 - 140
    solanum lycopersicum - tomaten - plantenziekteverwekkende bacteriën - clavibacter michiganensis subsp. michiganensis - ziekteresistentie - wilde verwanten - solanum pimpinellifolium - inteeltlijnen - terugkruisingen - genetische analyse - detectie - plant-microbe interacties - plantenveredelingsmethoden - solanum lycopersicum - tomatoes - plant pathogenic bacteria - clavibacter michiganensis subsp. michiganensis - disease resistance - wild relatives - solanum pimpinellifolium - inbred lines - backcrosses - genetic analysis - detection - plant-microbe interactions - plant breeding methods

    Clavibacter michiganensis subsp. michiganensis (Cmm) is the pathogen causing bacterial canker in tomato. The disease was described for the first time in 1910 in Michigan, USA. Cmmis considered the most harmful bacteria threatening tomato. Disease transmission occurs via seed and symptoms become visible at least 20 days after infection. Due to its complex strategy and transmission, Cmm is under quarantine regulation in EU and other countries. There is no method to stop disease progress in plants after infection. Thus, disease management consists usually of chemical treatments as protection and by careful clean cultural practices. However, the use of resistant varieties is the most effective and environmentally friendly method. Unfortunately, there is no cultivar harboring effective resistance on the market although efforts to get resistant varieties already started in the 60s. Our aim in this thesis was to develop valuable genetic material for breeders in order to enable them to release resistant cultivars and provide comprehensive scientific knowledge for further detailed research about Cmm.

    Our scientific activity in this thesis started with the identification of new Cmm resistance sources and confirmation of existent ones. In Chapter 3 we have screened a collection of wild tomatoes for resistance to Cmm. We made use of Real Time TaqMan PCR for intensive phenotyping. Using wilting and bacterial concentration as parameters for evaluation of the sources, we have identified new sources and confirmed existent ones. We have decided to continue further with one new source, S. pimpinellifolium, and one existent source, S . arcanum.

    We continued our research in Chapter 4 with a genetic analysis of the new source coming from S. pimpinellifolium. A recombinant inbred line population between the resistant parent, S. pimpinellifolium, and the susceptible parent S. lycopersicum was evaluated in three different environments. Wilting, bacterial concentration, and stem discoloration were the scored parameters. Responses of resistance in different environments were determined and genomic regions responsible for different responses were mapped.

    In Chapter 5, we have continued our research by fine mapping of previously identified genomic regions and developing nearly isogenic lines containing those genomic regions. By doing fine mapping, we made use of old stock DNA and recently developed different types of SNP marker technology. Previously identified Quantitative Trait Loci(QTLs)could be more precisely delimited. During isogenic line development, embryo rescue was used in order to break the genetic barrier between our S. arcanum source and tomato. Marker assisted backcrossing was applied to obtain lines with a minimum of donor parent in a faster way. By using this method we gained two generations of backcrossing.

    In order to obtain comprehensive information about different Cmm isolates inTurkey, we have performed multi locus sequence analysis (MLST) analysis on a Cmm collection, which was collected in 20 years of time in different part of Turkey. In Chapter 6 a statistical analysis of this collection revealed that measurement of clonality of this collection was possible as well as it was possible to predict the virulence level of strainsusing a subset of housekeeping genes.

    All knowledge gained by our experiments and knowledge coming from literature about Cmm have led a Review paper, Chapter 2, by which comprehensive information about Cmm resistance sources, genetic analysis of these sources, detection methods of Cmm, infection strategies of Cmm and interaction with its host was discussed.

    In conclusion, two good Cmm resistance sources and several tools and methods are available for breeders. Genomic regions of these sources associated with resistance were determined. Wider knowledge about Cmm detection, Cmm infection and Cmm interaction with its host are available for further research.

    An integrated approach involving metabolomics and transcriptomics for a system-wide understanding of the interaction between tomato and Cladosporium fulvum
    Etalo, D.W. - \ 2014
    Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Matthieu Joosten; Ric de Vos. - Wageningen : Wageningen University - ISBN 9789461738219 - 270
    solanum lycopersicum - plantenziekteverwekkende schimmels - passalora fulva - plant-microbe interacties - metabolomica - genexpressie - genomica - ziekteresistentie - virulentie - solanum lycopersicum - plant pathogenic fungi - passalora fulva - plant-microbe interactions - metabolomics - gene expression - genomics - disease resistance - virulence
    Identification and characterization of novel effectors of Cladosporium fulvum
    Ökmen, B. - \ 2013
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare. - S.l. : s.n. - ISBN 9789461736383 - 190
    solanum lycopersicum - tomaten - plantenziekteverwekkende schimmels - passalora fulva - plant-microbe interacties - verdedigingsmechanismen - genomica - dna-sequencing - pathogeniteit - solanum lycopersicum - tomatoes - plant pathogenic fungi - passalora fulva - plant-microbe interactions - defence mechanisms - genomics - dna sequencing - pathogenicity

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

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

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

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

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

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

    Functional analysis of tomato immune receptor Ve1 and recognition of Verticillium effector Ave1
    Zhang, Z. - \ 2013
    Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit, co-promotor(en): C.M. Liu. - S.l. : s.n. - ISBN 9789461735461 - 191
    solanum lycopersicum - tomaten - celwanden - receptoren - immuunsysteem - liganden - plantenziekteverwekkende schimmels - verticillium - infectiviteit - modellen - plant-microbe interacties - solanum lycopersicum - tomatoes - cell walls - receptors - immune system - ligands - plant pathogenic fungi - verticillium - infectivity - models - plant-microbe interactions

    Similar to the animal innate immune system, plants employ extracellular leucine rich repeat (eLRR)-containing cell surface receptors to recognize conserved molecular structures that are derived from microbial pathogens. A number of these immune receptors, as well as the corresponding pathogen ligands, have been characterized. The interaction between the tomato Ve1 immune receptor and the Ave1 effector from the pathogenic fungus Verticillium serves as a model system for the study of plant innate immunity. The research described in this thesis was aimed at a further understanding of how the eLRR-containing cell surface receptor Ve1 confers recognition of the Ave1 ligand and how it activates downstream immune signaling.

    It has been shown that eLRR-containing cell surface receptors play important roles in development and innate immunity in various plant species.Chapter 1 gives an overview on the current status of research on eLRR-containing cell surface receptors, their co-receptors and corresponding ligands, with emphasis on structural aspects. The functions of distinct eLRR receptor domains, their role in structural conformation, ligand perception, signal transduction and receptor complex formation are extensively discussed.

    To facilitate studies on the Ve1-Ave1 model system, we describe the establishment of protocols to investigate Ve1-mediated recognition of Ave1 and immune signaling in tobacco in Chapter 2. We optimized an Agrobacterium tumefaciens transient expression assay (agroinfiltration) by testing various over-expression vectors, and found that co-expression of Ve1 and Ave1 leads to hypersensitive response (HR) only in particular tobacco species. We further report on virus-induced gene silencing (VIGS) in Nicotiana tabacum cv. Samsun that allows investigating signaling components involved in Ve1-mediated resistance. Collectively, we established N. tabacum as a model plant to study Ve1-mediated immunity.

    In Chapter 3, we further investigated whether co-expression of Ve1 and Ave1 in Arabidopsis leads to an HR, which may potentially be used as a straightforward screening method upon a random mutagenesis. However, although Ave1 is able to trigger an HR in resistant tomato and tobacco plants, co-expression of Ve1 and Ave1 did not activate an HR in Arabidopsis. These results suggest that the HR occurs as a consequence of Ve1-mediated resistance signaling, and it is not absolutely required for Verticillium resistance.

    In Chapter 4 we investigated the contribution of particular regions of Ve1 to the activation of immune signaling through domain swaps between Ve1 with its non-functional homolog Ve2. Agroinfiltration, as well as stable Arabidopsis transformation, revealed that chimeras in which the first thirty eLRRs of Ve1 were replaced with those of Ve2 remain able to induce HR and activate Verticillium resistance. However, a truncated Ve1 protein that lacks the first 30 eLRRs is no longer functional. We speculate that the non-functional Ve2 receptor may still interact with the Ave1 effector in the eLRR domain, but fails to activate immune signaling due to a non-functional C-terminus.

    In Chapter 5, site-directed mutagenesis was employed to further investigate the eLRR domain of Ve1. We designed alanine scanning mutants in the solvent-exposed residues across the convex surface of the eLRR domain. In each mutant, two of the five solvent-exposed residues in β-sheet of a single eLRR were substituted into alanines. Functionality of the mutants through agroinfiltration and stable transformation of Arabidopsis revealed three eLRR regions that are potentially required for ligand specificity and for co-receptor interaction. In addition, alanine substitution was employed to evaluate role of putative protein-protein interaction and endocytosis motifs in the transmembrane domain and the cytoplasmic tail of the Ve1 protein. However, no requirement of these domains for Ve1 functionality could be demonstrated.

    It has been demonstrated that eLRR-containing cell-surface immune receptors often recognize short peptide sequence stretches as epitopes of their ligands. In Chapter 6, we aimed to identify the surface epitope of the Verticilliumeffector Ave1 that is recognized by Ve1. Firstly, we assessed whether various Ave1 homologs are recognized by Ve1. Since we found that C-terminal fusion of a GFP tag to Ave1 compromised its recognition, we hypothesized that accessibility of the Ave1 C-terminus is essential for Ve1-mediated recognition. Ave1 truncations and domain swaps with Ave1 homologs that are not recognized by Ve1 showed that a nine amino acid sequence derived from the C-terminus of Ave1 is essential for recognition by Ve1. This nine amino acid epitope is sufficient to activate Ve1-mediated immunity.

    In Chapter 7 the highlights of the thesis are discussed and placed in a broader perspective. The current understanding of eLRR-containing cell surface receptors is discussed, taking the findings of this thesis into account, with specific emphasis on ligand perception and receptor complex formation. In addition, future perspectives on the future are sketched, and novel research questions are posed aimed to obtain further insights into how Ve1 may form complexes with various co-receptors and how Ave1 contributes to Verticillium pathogenicity.

    Verticillium wilt resistance in Arabidopsis and tomato: identification and functional characterization
    Yadeta, K.A. - \ 2012
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - S.l. : s.n. - ISBN 9789461733849 - 147
    solanum lycopersicum - arabidopsis thaliana - modellen - plant-microbe interacties - ziekteresistentie - verdedigingsmechanismen - verticillium - verwelkingsziekten - moleculaire plantenziektekunde - solanum lycopersicum - arabidopsis thaliana - models - plant-microbe interactions - disease resistance - defence mechanisms - verticillium - wilts - molecular plant pathology

    Vascular wilt pathogens, which comprise bacteria, fungi and oomycetes, are among the most destructive plant pathogens that affect annual crops as well as woody perennials, thus not only impacting world food and feed production but also natural ecosystems. Vascular wilt pathogens colonize the xylem vessels of their host plants and interfere with the normal transportation of water and nutrients from the roots to upper parts of the plant, thus causing wilting symptoms. The structure and composition of xylem vessels has a significant impact on the colonization of host plants by these pathogens. Presently, genetic resistance is the most preferred control strategy against this group of plant pathogens.

    Verticillium wilt disease, which is caused by the vascular fungal pathogen Verticillium spp., is among the major diseases in various horticultural crops in tropical, subtropical, and temperate agro-ecological regions. The genus Verticilllium comprises of three major plant pathogenic species; V. dahliae, V. albo-atrum,and V. longisporum. While V. dahliae and V. albo-atrum arecharacterized with the ability to infect broad host range, V. longisporum has relatively limited host range infecting mainly crucifers family. V. dahliae and V. albo-atrum isolates are categorized into race 1 and race 2 based on their ability to infect tomato plants containing a Ve1 resistance gene. On tomato, while race 1 isolates are contained by Ve1 resistance gene, race 2 isolates overcome Ve1-mediated resistance.

    Chapter 1is the introduction to the thesis that describes xylem defence responses that are directed against vascular wilt pathogens. Plants recognize xylem-invading vascular wilt pathogens by using extracellular or intracellular receptors. Pathogen recognition activates innate immune responses that include physical and chemical defense responses in the xylem vessels and the surrounding parenchyma cells. While physical defense responses often halt pathogen movement between vessels, chemical defense responses can eliminate the pathogen or inhibit its growth, thereby leading to resistance.

    In order to identify novel sources of Verticillium wilt resistance, a collection of activation-tagged Arabidopsis mutants was screened for plants that displayed enhanced Verticillium wilt resistance. Chapter 2 describes four mutants (A1 to A4) that showed enhanced resistance to not only V. dahliae, but also to V. albo-atrum, and the Brassicaceae pathogen V. longisporum. Further characterization of resistance in these mutants against other vascular wilt pathogens, Ralstonia solanacearum and Fusarium oxysporum f. sp. Raphani, and the foliar pathogens such Botrytis cinerea, Plectosphaerella cucumerina, Alternaria brassicicola, and Pseudomonas syringae pv. tomato, is presented in this chapter. Except for mutant A2, that showed enhanced resistance to R. solanacearum, and mutants A1 and A3, that showed enhanced susceptibility to P. syringae, all the mutants responded similar as wild-type plants to these pathogens. In chapter 2, we furthermore describe the cloning and functional characterization of the gene encoding the AT-hook DNA-binding protein AHL19 that is responsible for the enhanced resistance of the A1 mutant to Verticillium wilt disease. The Arabidopsis genome contains 29 AHL proteins (Fujimoto et al., 2004)some of which have been implicated in various biological processes including plant development (Lim et al., 2007; Xiao et al., 2009)and defense (Kim et al., 2007Kim et al., 2007; Lu et al., 2010). AHL19 provides Verticillium wilt resistance upon over-expression, whereas knock-out enhances susceptibility, indicating that AHL19 positively regulates Verticillium wilt resistance. AHL19 not only regulates Verticillium wilt resistance, but also affects plant development, as AHL19 over-expressing plants showed larger leaf size, delayed maturity, and low seed production (Yadeta et al., 2011).

    Chapter 3describes the cloning and functional characterization of EVR1 (for Enhanced Verticillium Resistance 1), the gene that is responsible for the enhanced Verticillium wilt resistance in mutant A2. Mutant A2 furthermore confers resistance to the bacterial vascular wilt pathogen R. solanacearum (Yadeta et al., 2011). While EVR1 over-expression enhances Arabidopsis resistance to three vascular wilt pathogens: V. dahliae, R. solanacearum, and F. oxysporum, knock-out enhances susceptibility to V. dahliae and R. solanacearum. Furthermore, EVR1 appears to regulate drought stress resistance. EVR1 is a single copy gene that encodes a protein of unknown function, and EVR1 homologs are only found in Brassicaceae species thus far. Interestingly, over-expression of the B. oleraceae EVR1 homolog in Arabidopsis conferred Verticillium wilt resistance. Moreover, over-expression of Arabidopsis and B. oleraceae EVR1 and BoEVR1 in the Solanaceous species N. benthamiana enhanced Verticillium wilt resistance. This suggests that the Brassicaceae-specific EVR1 gene can be used to engineer Verticillium wilt resistance in other plant families.

    Whereas chapters 2 and 3 focus on the identification of novel sources of Verticillium wilt resistance by screening a collection of Arabidopsis gain-of-function mutants, Chapter 4 describes the identification of novel Verticillium wilt resistance in wild tomato accessions. Six wild accessions were identified that displayed enhanced resistance to race 2 isolates. Surprisingly, however, these accessions did not show enhanced resistance to race 1 isolates. Using virus-induced gene silencing, the resistance signalling leading to race 2 resistance in the wild accessions was investigated, showing that the resistance signalling in the wild accessions is distinct from the signalling pathway employed by the resistance protein Ve1.

    Finally in chapter 5, the highlights of this thesis are discussed and placed in a broader perspective.

    Genetic analysis of symbiosome formation
    Ovchinnikova, E. - \ 2012
    Wageningen University. Promotor(en): Ton Bisseling; I.A. Tikhonovich, co-promotor(en): Erik Limpens. - S.l. : s.n. - ISBN 9789461733610 - 147
    rhizobium - fabaceae - endosymbiose - wortelknolletjes - genregulatie - moleculaire genetica - moleculaire biologie - plant-microbe interacties - rhizobium - fabaceae - endosymbiosis - root nodules - gene regulation - molecular genetics - molecular biology - plant-microbe interactions

    Endosymbiotic interactions form a fundament of life as we know it and are characterized by the formation of new specialized membrane compartments, in which the microbes are hosted inside living plant cells. A striking example is the symbiosis between legumes and nitrogen-fixing Rhizobium bacteria (rhizobia), which represents the most important source of biologically fixed nitrogen. The accommodation of rhizobia as novel nitrogen-fixing organelles, called symbiosomes, inside the cells of a novel organ, the root nodule, forms the heart of this ecologically and agriculturally important symbiosis. Understanding how these organelles are made will be keystone to exploit this symbiosis for sustainable agriculture in the future. In this thesis, we undertook a genetic approach to identity key components that control symbiosome formation especially in the genetically well-characterized garden pea (Pisum sativum) system. At the start of this thesis, the most extensive and morphologically best-characterized collection of mutants impaired in symbiosome formation was, and currently still is, available in pea. However, the cloning of the corresponding genes is severely hampered by its large genome size and recalcitrance to genetic transformation. Therefore, we used the model legume Medicago truncatula (Medicago) as reference genome to clone pea symbiosome mutants via a synteny-based cloning approach. We focused especially on three mutants in pea, named sym33, sym41 and sym31, which are affected most early in symbiosome formation: namely blocked in the release of bacteria from cell wall bound infection threads inside root nodule cells (sym33 and sym41) or induction of the subsequent differentiation of the symbiosomes (sym31).
    In Chapter 1, a general introduction is given on the process of symbiosome formation in legume root nodules. In this introduction, we focus on mechanisms by which these new nitrogen-fixing organelles are formed and address some of the recent insights, most of which were obtained after the start of this thesis, into plant components that control this process, which have been obtained from genetic studies in pea and the model legumes Medicago and Lotus japonicus (Lotus).
    Pea is part of the Papillionoid legume subfamily and closely related to the model legume Medicago. It has been shown that there is extensive synteny between the pea and Medicago genomes, which offers an efficient strategy to clone pea gene using Medicago as intergenomic cloning vehicle. In Chapter 2, we outline this synteny-based cloning approach and the molecular tools that we created to clone the pea genes required for symbiosome formation. In addition, we describe an efficient method to obtain transgenic roots via Agrobacterium rhizogenes mediated root transformation in pea that facilitates the functional analysis of pea genes in root endosymbioses.
    In Chapter 3, we report the cloning of the pea Sym33 and Medicago SYM1 genes those mutants are most strongly impaired in their ability to form symbiosomes, i.e. the release of rhizobia from the cell wall bound infection threads. Both pea Sym33 and Medicago SYM1 encode the interacting protein of DMI3, IPD3. IPD3 was shown to interact with DMI3/CCaMK, a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signalling pathway for both rhizobial symbiosis and arbuscular mycorrhiza. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. Further, we show that MtIPD3 is required for the expression of a nodule-specific remorin MtSYMREM1, which is required for proper infection thread growth and essential for symbiosome formation.
    In Chapter 4, we report the synteny-based cloning of the pea sym41 mutant that is also impaired in the release of the bacteria from the infection threads. We show that Sym41 represents a weak allele of the common symbiotic signalling gene PsSym19/MtDMI2, a leucine-rich repeat domain containing receptor kinase that is essential for both rhizobial and mycorrhizal endosymbioses. Sym41 contains a splice-site mutation in intron 9, by which the formation of a functional transcript is reduced by ~90%. The implication of Sym19/DMI2 together with the identified role of Sym33/IPD3 in symbiosome formation (Chapter 3) strongly indicate that rhizobia have co-opted the signalling pathway from the ancient arbuscular mycorrhiza to be hosted as new organelles inside root nodule cells.
    In Chapter 5, we describe the synteny-based mapping of pea sym31, a mutant impaired in symbiosome differentiation. By making use of the synteny with Medicago, we fine mapped the Sym31 gene to a region of ~2.5 cM, which corresponds to a <450 kb region in Medicago. In this syntenic region, one gene MtN3.1, a putative sugar transporter stands out as prime candidate to control symbiosome differentiation. We describe and discuss our efforts to determine the role of this gene in symbiosome differentiation in pea and Medicago.
    In Chapter 6, we summarize and discuss our current insight into symbiosome formation and its relation to the arbuscular mycorrhiza and we give a perspective on the future of cloning the pea genes required for endosymbioses.

    Plant Fungal Pathogens: Methods and Protocols
    Bolton, M.D. ; Thomma, B.P.H.J. - \ 2012
    New York, USA : Humana Press (Methods in Molecular Biology 835) - ISBN 9781617795008 - 648
    plantenziekteverwekkende schimmels - plant-microbe interacties - genomica - moleculaire plantenziektekunde - schimmelziekten - methodologie - protocollen - plant pathogenic fungi - plant-microbe interactions - genomics - molecular plant pathology - fungal diseases - methodology - protocols
    Over the course of evolution, fungi have adapted to occupy specific niches, from symbiotically inhabiting the flora of the intestinal tract of mammals to saprophytic growth on leaf litter resting on the forest floor. In Plant Fungal Pathogens: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study fungal plant pathogens. These include methods and techniques for model systems such as Arabidopsis thaliana as well as crop plants, aspects of fungal biology, genome annotation, next-generation sequencing, and fungal transformation and molecular tools for disease and/or pathogen quantification that are critical for revealing the role for a fungal gene of interest in disease development. Chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
    Multiplex SSR analysis of Phytophthora infestans in different countries and the importance for potato breeding
    Li, Y. - \ 2012
    Wageningen University. Promotor(en): Evert Jacobsen, co-promotor(en): Theo van der Lee; D.E.L. Cooke. - S.l. : s.n. - ISBN 9789461732798 - 206
    solanum tuberosum - aardappelen - plantenveredeling - plantenziekteverwekkende schimmels - phytophthora infestans - microsatellieten - populaties - ziekteresistentie - genetische merkers - moleculaire merkers - bio-informatica - genomica - plant-microbe interacties - solanum tuberosum - potatoes - plant breeding - plant pathogenic fungi - phytophthora infestans - microsatellites - populations - disease resistance - genetic markers - molecular markers - bioinformatics - genomics - plant-microbe interactions

    Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-19th century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland and other parts of Europe. Late blight remains the most important pathogen to potato and causes a yearly multi-billion US dollar loss globally. In Europe and North America, late blight control heavily relies on the use of chemicals, which is hardly affordable to farmers in developing countries and also raises considerable environmental concerns in the developed countries.
    The structure of P. infestans populations can change quickly by migration, sexual recombination and sub-clonal variation. Migration and the reconvening of the two mating types considerably raised the level of genetic diversity in the global P. infestans population, leading to a more variable population with a presumed higher level of adaptability as compared to the previously, purely asexually, reproducing population. How can the P. infestans population efficiently be monitored with such diverse genotypes? A high-throughput, high-resolution and easy-handled set of markers would be favorable for this purpose. Few genetic markers, if any, have found such widespread use as SSRs. Sequencing allows the identification of large numbers of microsatellites by bioinformatics. So far, however, only a limited number of informative microsatellite loci had been described for P. infestans and none have been mapped. This thesis first describes the development and mapping of SSR markers in P. infestans and integration with other SSRs to generate a multiplex SSR set and its application in the population analysis of P. infestans from four countries are described with the developed multiplex SSRs. Finally, the use of this knowledge in resistance breeding of potato is shortly indicated and discussed.
    Chapter 1 describes the historic population changes of P. infestans at the global level and the current population trends. It summarizes microsatellite as favorite molecular markers for studying pathogen population diversity and assesses monitoring of population dynamics in more detail for resistance breeding in potato.
    The selection and identification of new SSR markers is presented in Chapter 2. From EST and genomic sequences from P. infestans we identified 300 non-redundant SSR loci by a bioinformatic screening pipeline. Based on the robustness, level of polymorphism and map position eight SSR markers were selected, which were assembled in two multiplex PCR sets and labeled with two different fluorescent dyes to allow scoring after single capillary electrophoresis.
    This successful multiplex SSR approach encouraged the development of fast, accurate and high-throughput genotyping, in an one-step multiplex PCR method to facilitate worldwide screening of P. infestans populations. Published SSRs and the 8 new SSRs were integrated. All these SSR markers were re-evaluated and the 12 most informative SSRs were selected to set up a standard set for global application (Chapter 3). The 12-plex SSRs are distributed over different chromosomes, significantly increasing the resolution of genotyping compared to the previous set of 8 SSRs. The 12-plex SSRs were integrated to one-step fluorescence-based multiplex reaction, which plays a key role to facilitate highly paralleled genotyping and efficient dissection of the more complex P. infestans populations. This multiplex PCR for P. infestans populations is (i) simple, as only one PCR is needed to perform multi-locus typing with twelve markers; (ii) rapid, as the genotyping results can be available in 1 day; and (iii) reproducible and adapted to different laboratories. The genotyping data from different geographic populations were submitted to the Euroblight database. With the same SSR set and the bin set, a comparable global database can easily be achieved.
    As indicated earlier, more recent analyses of P. infestans populations highlight the appearance of many new genotypes via migration and/or sexual recombination. To practice the newly developed 12-plex SSR set and dissect the current population structure, several P. infestans populations from 4 different continents were selected for analysis. These include Chinese (Chapter 4), Dutch (Chapter 5), Ecuadorian (Chapter 6) and Tunisian (Chapter 7) populations.
    China has become the largest potato producing country not only for potato cultivation area but also in Megaton potato production. Interprovincial trade of consumption and seed potatoes is very important and frequent in China. Although both, the A1 and A2 mating types are found in China, to this date, no evidence of an active sexual cycle based on changes in allele frequency was found. With the ten SSRs, a large genotypic survey of in nation-wide collection of 228 P. infestans isolates was performed (Chapter 4). One of the three dominant clonal lineages CN-04 (A2) in this Chinese population was genetically similar to a major clonal lineage identified in Europe, called “Blue_13” with A2 mating type. It was not possible to critically assess the origin of this clonal lineage. This study is the first report of “Blue_13” outside Europe. The virulence spectrum of selected Chinese P. infestans isolates showed seven different virulence spectra varying from 3 to 10 differentials. The CN04 genotypes were identified as more aggressive and more virulent genotypes, one of whom had the full virulence pattern after using the potato differential set. Within the Chinese P. infestans population, the genotypes strongly clustered according to their six sampling provinces, which seems not to be influenced by the frequent interprovincial trading activities of seed potatoes. The mating type ratio and the SSR allele frequencies indicate that in China the contribution of the sexual cycle to P. infestans on population dynamics is minimal. It was concluded that the migration through asexual propagules and the generation of sub-clonal variation are the dominant driving factors behind the Chinese P. infestans population structure.
    The Netherlands has a long history of population studies on local P. infestans isolates and a substantial amount of commercial potato varieties growing in the field. One decade (2000-2009) of isolate sampling in 5 different regions provided the basis for a good understanding of the population dynamics in the Netherlands (Chapter 5). The surveyed population revealed the presence of several clonal lineages and a group of sexual progenies. The major clonal lineage with A2 mating type is known as “Blue_13”, but also two distinct clonal lineages with A1 mating type in this study have been identified. This survey witnesses that the Dutch population was undergoing dramatic changes in the ten years under study. The most notable change was the emergence and spread of A2 mating type strain “Blue_13”. The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation- and dispersal mechanism for successful genotypes. In addition to the neutral SSR markers a molecular marker for the virulence of isolates on potato lines that contain the Rpi-blb1 R-gene has been developed. Using this Avr-blb1 marker and the corresponding virulence assay we report, for the first time, the presence of Rpi-blb1 breaker isolates in the Netherlands even before a Rpi-blb1 containing resistant variety was introduced. The 12 breaker isolates only occurred in sexual progeny. So far the asexual spread of such virulent isolates has been limited because of the absence of Rpi-blb1 containing varieties in the field.
    Remarkably, on the other end of the world in the Andes, the region of potato origin, the situation is far less complex as far as P. infestans is concerned. There are more than 400 potato landraces in Ecuador and the planting habit by local farmers by traditional cultivation at small scale in the highlands is different from potato cultivation in other potato countries in North America or Europe (Chapter 6). Phytophthora isolates in Ecuador belong to two closely related species, P. infestans (on potato and tomato) and P. andina (on non-tuber bearing host), but SSR analysis of 66 isolates indicated that the two species are separated in two clearly distinguished genetic groups. Two ancient clonal lineages of P. infestans appeared to be dominant in Ecuador one is found only on tomato the other one only on potato. Within the potato isolates, but not in the tomato isolates, there is a large sub-clonal variation caused by (partial) polyploidization and loss of alleles.
    In Tunisia, potato is cultivated in three to four partly overlapping seasons while tomato is grown either in greenhouses or as aerial crop in most potato producing regions. Chapter 7 revealed, among 165 isolates of five regions, the presence of a major clonal lineage (NA-01, A1 mating type, Ia mtDNA haplotype) that seems to consist of races that are relatively simple. Another highly genetic diverse group of isolates was found containing more complex races and isolates with both mating types. Season clustering indicated that at least some of the new genotypes generated by sexual reproduction overlapped between seasons and such a sexual progeny may play an important role in the next season epidemics. On tomato, mostly asexual progeny was identified with two mtDNA haplotypes but less nuclear genotypes, compared to potato. This study shows that the P. infestans population is currently changing, and the old clonal lineage is being replaced by a more complex, genetically diverse and sexually propagating population in two sub-regions in Tunisia. Despite the massive import of potato seeds from Europe, the P. infestans population in Tunisia is still clearly distinct from the European population.
    Chapter 8 discusses the application of microsatellites in monitoring genetic diversity of late blight and the potential use in resistance breeding. Monitoring of the local P. infestans population for new virulent genotypes with the differential potato set in combination with screening for effector variation, allows early detection of adaptation of certain genotypes within the P. infestans population to particular resistance genes in a specific region. This provides the possibilities to determine which broad spectrum R-genes are still useful in order to adapt the control strategy by resistance breeding to the new situation. One way of doing that is to replace the existing varieties by other varieties with stacked non-broken R-genes obtained by marker assisted selection or to add additional R-genes to existing (R-gene containing) varieties by transformation. In a transgenic or cisgenic approach, additional broad spectrum R-genes could be added by re-transformation. As we have shown, the right R-gene management strategy in potato breeding, but also in potato production, should include the direct monitoring of local pathogen populations by using the differential set and the 12-plex SSR set.

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