B.P.H.J. Thomma
Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands

Since their discovery by Flor in the early forties of last century, gene-for-gene systems have intrigued many plant pathologists and disease resistance breeders. More than a decade ago the first Cladosporium fulvum avirulence gene (Avr9) and the matching tomato resistance gene (Cf-9) were cloned. So far, we have cloned three additional Avr genes and four Ecp genes that all encode small cysteine-rich peptides that are secreted by C. fulvum in the host apoplast after penetration of tomato leaves. Recognition of the peptide elicitors encoded by the Avr and Ecp genes is mediated by Cf resistance genes and eventually leads to a hypersensitive response (HR). C. fulvum can avoid recognition and subsequent induction of HR by various mechanisms: the Avr gene (i) is absent, (ii) contains point mutations in the ORF leading to protease-sensitive elicitors or frame shift mutations leading to truncated non-active elicitor proteins, (iii) contains point mutations in the ORF leading to production of stable non-active elicitors or (iv) contains transposon insertions in the Avr gene leading to a lack of Avr protein production. The biochemical basis of the gene-for-gene system implies that the Avr gene product directly interacts with the matching Cf gene product leading to HR. However, so far, no physical interaction between Avr and Cf proteins could be shown and the mechanism of Avr perception by Cf proteins is still unclear.
In contrast to Avr genes, the Ecp genes are present in all strains of C. fulvum and all encode active elicitors that can be recognized by some accessions of the wild tomato species Lycopersicon pimpinellifolium. The Cf genes occurring in those accessions have not yet been introduced in commercially grown tomato cultivars, and as a result the Ecp genes have not yet been under natural selection. Probably, single Avrs and Ecps play only a minor role in virulence. Presently, we try to simultaneously knock down several Avr and Ecp genes in C. fulvum by RNAi to study the effects on pathogen virulence. For two Avr proteins we have some indications for their biological function. The Avr4 elicitor appears to be a chitin-binding protein that can protect fungi against basic plant chitinases. Avr4 proteins encoded by virulent alleles in strains of C. fulvum are no longer recognised by Cf-4 plants, but still bind to chitin, suggesting that chitin-binding by Avr4 could represent a defensive virulence function. The Avr2 elicitor appears to be a cysteine protease inhibitor. For recognition of the Avr2 elicitor, in addition to Cf-2 protein, the tomato Rcr3 cysteine protease is required. Rcr3 can also be inhibited by Avr2, but whether inhibition of Rcr3 by Avr2 itself or the modulation of Rcr3 by Avr2 is required for Cf-2-mediated HR is not known yet. Although we envisage that, like for Avr proteins of other pathogens, Avr and Ecp proteins of C. fulvum most probably interact with virulence targets in tomato plants that are guarded by the Cf proteins, it is not clear whether Rcr3 represents such a virulence target.
Presently we are also studying downstream responses induced by C. fulvum elicitors both in susceptible (to identify virulence targets) and resistant (to identify defence-related genes) tomato plants, and in Arabidopsis. Plans for future work on gene-for-gene systems will be discussed.