Cladosporium fulvum effector proteins and their role in pathogen virulence

Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould of tomato (Solanum esculentum). Chapter 1 is a “pathogen profile” describing the biology of the pathogen. During growth in the leaf apoplast, the intercellular space surrounding the mesophyll cells, the fungus secretes effector proteins that are thought to play a role in disease establishment. Eight of these effectors have been characterized in detail. For most of these effectors, cognate C. fulvum (Cf) resistance loci have been identified in tomato that mediate an immune response upon recognition of (the activity of) the cognate effector. In chapter 2, a targeted proteomics approach to investigate the role of these effector proteins and to identify possible in planta targets is described. C. fulvum proteins were expressed as recombinant fusion proteins carrying various affinity–tags at either their C– or N–terminus. Although these fusion proteins were correctly expressed and secreted into the leaf apoplast, detection of affinity–tagged C. fulvum proteins failed and affinity–purification did not result in the recovery of these proteins. However, when using C. fulvum effector protein–specific antibodies, specific signals were obtained for the different proteins. It was therefore concluded that the stability of the in planta expressed recombinant fusion proteins is insufficient, which resulted in removal of the affinity–tag from the fusion proteins, irrespective of C– or N–terminal fusion or the nature of the affinity–tag. Similar observations were made when the fusion proteins were expressed in other Solanaceous species, but not when expressed in Arabidopsis thaliana. Previous studies have demonstrated that Avr4 binds to chitin present in fungal cell walls, and that this binding by Avr4 can protect these cell walls against hydrolysis by plant chitinases. In chapter 3 it is described that Avr4–expression in Arabidopsis results in increased virulence of several fungal pathogens with exposed chitin in their cell walls, whereas the virulence of a bacterium and an oomycete remained unaltered. Heterologous expression of Avr4 in tomato increased the virulence of Fusarium oxysporum f. sp. lycopersici. Tomato GeneChip analysis was used to demonstrate that Avr4–expression in tomato results in the induced expression of only a handful of genes. Finally, silencing of the Avr4 gene in C. fulvum decreased fungal virulence on tomato. In conclusion, chapter 3 is the first report on the intrinsic function of a fungal avirulence protein that displays self-defense activity which is required for full pathogen virulence. In chapter 4, a study on the intrinsic biological function of Avr2 is presented. The Avr2 effector interacts with the apoplastic tomato cysteine protease Rcr3, which is required for Cf–2–mediated immunity. In this chapter it is demonstrated that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis resulted in enhanced susceptibility towards a number of extracellular fungal pathogens that include Botrytis cinerea and Verticillium dahliae, and microarray analysis of unchallenged Arabidopsis plants showed that Avr2 expression triggered a global transcription profile that is reminiscent of pathogen challenge. Cysteine protease activity profiling revealed that Avr2 inhibits multiple extracellular Arabidopsis cysteine proteases. In tomato, Avr2 expression resulted in enhanced susceptibility not only towards natural Avr2–defective C. fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. Cysteine protease activity profiling in tomato revealed that Avr2 inhibits multiple extracellular cysteine proteases including Rcr3 and its close relative PIP1. Finally, silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases required for plant basal defense in tomato. Chapter 5 describes the discovery and characterization of a novel effector protein of C. fulvum, Ecp6. To discover novel C. fulvum effectors that might play a role in virulence, two–dimensional polyacrylamide gel electrophoresis (2D–PAGE) was used to visualize proteins secreted during C. fulvum–tomato interactions. Three novel C. fulvum proteins were identified; CfPhiA, Ecp6, and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides, while Ecp6 contains lysine motifs (LysM domains), which are recognized as carbohydrate–binding modules. Finally, Ecp7 encodes a small, cysteine–rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNAi–mediated gene silencing it was demonstrated that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologs in other organisms, conserved Ecp6 orthologs were identified in various fungal species. Homology based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding. Chapter 6 presents global transcriptional profiling study to compare transcriptional changes in tomato during compatible and incompatible interactions with the foliar pathogenic fungus Cladosporium fulvum and the soil–borne vascular pathogenic fungus Verticillium dahliae. Although both pathogens colonize different host tissues, they display significant commonalities in their infection strategies as they both penetrate natural openings and grow strictly extracellular without the formation of haustoria. Furthermore, in incompatible interactions with both pathogens resistance is conveyed by extracellular transmembrane receptors that belong to the class of receptor–like proteins. For each of the two pathogens, the transcriptomes of the compatible and incompatible interaction largely overlapped. However, the C. fulvum–induced transcriptomes showed little overlap with the V. dahliae–induced transcriptomes, as most genes were uniquely regulated by one of the two pathogens. This also applied to both incompatible interactions, despite defense activation by the same type of resistance protein. Remarkably, of the relatively small subset of genes that was regulated by both pathogens a large portion showed an inverse regulation; induced by one pathogen and repressed by the other. With pathway reconstruction, interacting networks of tomato genes implicated in photorespiration, hypoxia and glycoxylate metabolism were identified that were repressed upon infection with C. fulvum and induced by V. dahliae. Similarly, auxin signaling was differentially affected by the two pathogens. In chapter 7, the general discussion, the implications are of the data that are presented in this thesis are discussed for the use of C. fulvum as a model, and for fungal pathogens in general. Furthermore, the use of heterologous expression systems to study fungal effectors is briefly discussed. In several of the chapters presented in this thesis, the use of microarrays has been instrumental to investigate the biology of C. fulvum and the role of specific effectors secreted by the pathogen. Therefore, an overview of the currently available in silico tools for reconstruction of cellular pathways based on plant gene expression datasets is presented.