Identification and characterization of novel effectors of Cladosporium fulvum

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.

Bibliographic Details

Main Author:	Ökmen, B.
Other Authors:	de Wit, Pierre
Format:	Doctoral thesis biblioteca
Language:	English
Subjects:	defence mechanisms, dna sequencing, genomics, passalora fulva, pathogenicity, plant pathogenic fungi, plant-microbe interactions, solanum lycopersicum, tomatoes, dna-sequencing, genomica, pathogeniteit, plant-microbe interacties, plantenziekteverwekkende schimmels, tomaten, verdedigingsmechanismen,
Online Access:	https://research.wur.nl/en/publications/identification-and-characterization-of-novel-effectors-of-cladosp
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