Functional analysis of the tomato Ve resistance locus against Verticillium wilt

Verticillium dahliae, V. albo-atrum and V. longisporum are soil-borne plant pathogens that are responsible for Verticillium wilt diseases in temperate and subtropical regions. Collectively they can infect over 200 hosts, including many economically important crops. Chapter 1 is a “pathogen profile” which describes the most important aspects of the biology of the Verticillium wilt pathogens. They colonize the xylem vessels of their host plants and cause symptoms such as wilting, chlorosis, stunting, necrosis and vein clearing. Verticillium species are notoriously difficult to control as there are no fungicides available to cure plants once they are infected. Therefore, genetic resistance is the preferred method for disease control. Chapter 2 describes the functional characterization of the tomato (Solanum lycopersicum) Ve locus. This locus is responsible for resistance against race 1 strains of V. dahliae and V. albo-atrum and comprises two closely linked inversely oriented genes, Ve1 and Ve2. Both genes encode cell surface receptor proteins of the extracellular leucine-rich repeat (eLRR) receptor-like protein (RLP) class of disease resistance proteins. In chapter 2, it is demonstrated that Ve1, but not Ve2, provides resistance in tomato against race 1 but not against race 2 strains of V. dahliae and V. albo-atrum. Using virus-induced gene silencing in tomato, the signaling cascade downstream of Ve1 was shown to require both EDS1 (enhanced disease susceptibility1) and NDR1 (non-race-specific disease resistance1). In addition, also NRC1 (NB-LRR protein required for hypersensitive response-associated cell death1), ACIF (Avr9/Cf-9–induced F-box1), MEK2 (MAP/ERK kinase2), and SERK3/BAK1 (somatic embryogenesis receptor kinase 3/brassinosteroid-associated kinase 1) act as positive regulators of Ve1 in tomato. In conclusion, Ve1-mediated resistance signaling only partially overlaps with signaling mediated by Cf proteins, type members of the eLRR-RLP-class of resistance proteins. In chapter 3 an attempt to introduce Nicotiana benthamiana as a model to facilitate the study of Ve1-mediated resistance is described. Challenge of wild type plants with several race 1 and race 2 strains of V. dahliae and V. albo-atrum demonstrated that N. benthamiana is susceptible to both Verticillium species. To obtain Verticillium wilt resistant plants, N. benthamiana was engineered to express the tomato Ve1 coding sequence. However, out of thirteen transgenic lines, six showed clear phenotypic aberrancies that included severe stunting and malformed leaves when compared to wild type plants. The seven Ve1-transgenic lines that did not show any phenotypic alterations were challenged with race 1 and race 2 strains. Although the pathogenicity assays indicated that in few lines Ve1 expression temporarily reduced disease development, most lines were as susceptible as wild type parental line. In conclusion, in chapter 3 it is demonstrated that the Ve1-transgenic N. benthamiana lines could not be used to study Ve1-mediated resistance signaling. In chapter 4, the use of Arabidopsis (Arabidopsis thaliana) as model to facilitate the study of Ve1-mediated resistance is presented. To this end, tomato Ve1 was expressed in susceptible Arabidopsis plants. Upon challenge with race 1 strains of V. dahliae or V. albo-atrum, Ve1-expressing plants were found to be resistant. In contrast, Ve1-expressing plants were susceptible to race 2 strains of both V. dahliae and V. albo-atrum. Furthermore, expression of Ve1 in Arabidopsis plants did not prevent colonization by V. longisporum strains. Through Ve1-expression in Arabidopsis defense signaling mutants, it was demonstrated that signaling downstream of Ve1 is highly conserved between tomato and Arabidopsis. In previous chapters it was shown that the receptor kinase SERK3/BAK1 is required for Ve1-mediated resistance in tomato as well as in Arabidopsis. In Arabidopsis, SERK3/BAK1 belongs to a gene family consisting of five members. In chapter 5, the requirement of the different SERK family members in Ve1-mediated resistance in Arabidopsis is investigated, revealing the requirement of SERK1 and, although to a lesser extent, SERK4 for resistance. Using virus-induced gene silencing, it was subsequently shown that SERK1 is also required for Ve1-mediated resistance in tomato. In conclusion, the results of chapter 5 demonstrate that Arabidopsis can be used as model to unravel the molecular mechanisms of Ve1-mediated resistance. In chapter 6, the recognition specificity of Ve1 was further investigated by performing domain-swaps with Ve2 and expressing the chimeric Ve proteins in Arabidopsis. Various domain swaps in which eLRRs from Ve1 were replaced by those of Ve2 suggest that the region between eLRR22 and eLRR35 is required for full Ve1-mediated resistance. However, plants expressing a Ve chimera in which eLRR1 to eLRR30 of Ve1 was replaced with those of Ve2 were resistant against Verticillium. Overall, these results suggest that Ve2 may still bind the elicitor in the eLRR domain, but its C-terminal domain is not able to activate a successful defense response. Finally in Chapter 7, highlights of this thesis are discussed and placed in a broader perspective. </p

Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum

Introduction: Verticillium spp. are soil-borne plant pathogens responsible for Verticillium wilt diseases in temperate and subtropical regions; collectively they affect over 200 hosts, including many economically important crops. There are currently no fungicides available to cure plants once they are infected. Taxonomy: Kingdom: Fungi, phylum: Ascomycota, subphylum, Pezizomycotina, class: Sordariomycetes, order: Phyllachorales, genus: Verticillium. Host range and disease symptoms: Over 200 mainly dicotyledonous species including herbaceous annuals, perennials and woody species are host to Verticillium diseases. As Verticillium symptoms can vary between hosts, there are no unique symptoms that belong to all plants infected by this fungus. Disease symptoms may comprise wilting, chlorosis, stunting, necrosis and vein clearing. Brown vascular discoloration may be observed in stem tissue cross-sections. Pathogenicity: Verticillium spp. have been reported to produce cell-wall-degrading enzymes and phytotoxins that all have been implicated in symptom development. Nevertheless, evidence for a crucial role of toxins in pathogenicity is inconsistent and therefore not generally accepted. Microsclerotia and melanized mycelium play an important role in the disease cycle as they are a major inoculum source and are the primary long-term survival structures. Resistance: Different defence responses in the prevascular and the vascular stage of Verticillium wilt diseases determine resistance. Although resistance physiology is well established, the molecular processes underlying this physiology remain largely unknown. Resistance against Verticillium largely depends on the isolation of the fungus in contained parts of the xylem tissues followed by subsequent elimination of the fungus. Although genetic resistance has been described in several plant species, only one resistance locus against Verticillium has been cloned to date

GWA Mapping of Anthocyanin Accumulation Reveals Balancing Selection of MYB90 in Arabidopsis thaliana

Induction of anthocyanin accumulation by osmotic stress was assessed in 360 accessions of Arabidopsis thaliana. A wide range of natural variation, with phenotypes ranging from green to completely red/purple rosettes, was observed. A genome wide association (GWA) mapping approach revealed that sequence diversity in a small 15 kb region on chromosome 1 explained 40% of the variation observed. Sequence and expression analyses of alleles ofthe candidate gene MYB90 identified a causal polymorphism at amino acid (AA) position 210 of this transcription factor of the anthocyanin biosynthesis pathway. This amino acid discriminates the two most frequent alleles of MYB90. Both alleles are present in a substantial part of the population, suggesting balancing selection between these two alleles. Analysis of the geographical origin of the studied accessions suggests that the macro climate is not the driving force behind positive or negative selection for anthocyanin accumulation. An important role for local climatic conditions is, therefore, suggested. This study emphasizes that GWA mapping is a powerful approach to identify alleles that are under balancing selection pressure in nature

Tomato Transcriptional Responses to a Foliar and a Vascular Fungal Pathogen Are Distinct

Plant activation of host defense against pathogenic microbes requires significant host transcriptional reprogramming. In this study, we compared transcriptional changes in tomato during compatible and incompatible interactions with the foliar fungal pathogen Cladosporium fulvum and the vascular fungal pathogen Verticillium dahliae. Although both pathogens colonize different host tissues, they display distinct commonalities in their infection strategy; both pathogens penetrate natural openings and grow strictly extracellular. Furthermore, resistance against both pathogens is conveyed by the same class of resistance proteins, the receptor-like proteins. For each individual pathogen, the expression profile of the compatible and incompatible interaction largely overlaps. However, when comparing between the two pathogens, the C. fulvum-induced transcriptional changes show little overlap with those induced by V. dahliae. Moreover, within the subset of genes that are regulated by both pathogens, many genes show inverse regulation. With pathway reconstruction, networks of tomato genes implicated in photorespiration, hypoxia, and glycoxylate metabolism were identified that are repressed upon infection with C. fulvum and induced by V. dahliae. Similarly, auxin signaling is differentially affected by the two pathogens. Thus, differentially regulated pathways were identified with novel strategies that allowed the use of state-of-the-art tools, even though tomato is not a genetic model organis

Ve1-mediated resistance against Verticillium does not involve a hypersensitive response in Arabidopsis

The recognition of pathogen effectors by plant immune receptors leads to the activation of immune responses that often include a hypersensitive response (HR): rapid and localized host cell death surrounding the site of attempted pathogen ingress. We have demonstrated previously that the recognition of the Verticillium dahliae effector protein Ave1 by the tomato immune receptor Ve1 triggers an HR in tomato and tobacco. Furthermore, we have demonstrated that tomato Ve1 provides Verticillium resistance in Arabidopsis upon Ave1 recognition. In this study, we investigated whether the co-expression of Ve1 and Ave1 in Arabidopsis results in an HR, which could facilitate a forward genetics screen. Surprisingly, we found that the co-expression of Ve1 and Ave1 does not induce an HR in Arabidopsis. These results suggest that an HR may occur as a consequence of Ve1/Ave1-induced immune signalling in tomato and tobacco, but is not absolutely required for Verticillium resistance

Genome-Wide Association Mapping of Fertility Reduction upon Heat Stress Reveals Developmental Stage-Specific QTLs in Arabidopsis thaliana

For crops that are grown for their fruits or seeds, elevated temperatures that occur during flowering and seed or fruit set have a stronger effect on yield than high temperatures during the vegetative stage. Even short-term exposure to heat can have a large impact on yield. In this study, we used Arabidopsis thaliana to study the effect of short-term heat exposure on flower and seed development. The impact of a single hot day (35°C) was determined in more than 250 natural accessions by measuring the lengths of the siliques along the main inflorescence. Two sensitive developmental stages were identified, one before anthesis, during male and female meiosis, and one after anthesis, during fertilization and early embryo development. In addition, we observed a correlation between flowering time and heat tolerance. Genome-wide association mapping revealed four quantitative trait loci (QTLs) strongly associated with the heat response. These QTLs were developmental stage specific, as different QTLs were detected before and after anthesis. For a number of QTLs, T-DNA insertion knockout lines could validate assigned candidate genes. Our findings show that the regulation of complex traits can be highly dependent on the developmental timing