clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum.

A screen for insertional mutants of Colletrichum lindemuthianum, the causative agent of common bean anthracnose, led to the identification of a non-pathogenic, lightly colored transformant. This mutant is unable to induce disease symptoms on intact or wounded primary leaves of seedlings and plantlets of Phaseolus vulgaris. In vitro, it exhibits normal vegetative growth, sporulation and conidial germination, but the cultures remain beige instead of becoming black. Microscopic examination revealed that this mutant forms fewer appressoria than the wild-type strain, and these are misshapen and poorly melanized. Molecular analyses indicated that the mutagenic plasmid had targeted clap1, a gene encoding a putative copper-transporting ATPase sharing 35% identity with the human Menkes and Wilson proteins and the product of the CCC2 gene of Saccharomyces cerevisiae. Complementation of the non-pathogenic beige mutant with a wild-type allele of clap1 restored both pathogenicity and pigmentation. Conversely, replacement of the wild-type allele with a disrupted clap1 gene gave rise to non-pathogenic beige transformants. Compared with the wild-type strain, extracts from clap1 mutants were found to have very low levels of phenol oxidase activity. These observations suggest that the clap1 gene product may be involved in the pathogenicity of C. lindemuthianum strains because of its role in delivering copper to secreted cuproenzymes, such as the phenol oxidases that mediate the polymerization of 1,8-dihydroxynaphthalene to melanin.

Specific HR-associated recognition of secreted proteins from Cladosporium fulvum occurs in both host and non-host plants.

The resistance of tomato (Lycopersicon esculentum) to the pathogenic fungus Cladosporium fulvum complies with the gene-for-gene concept. Host resistance is based on specific recognition of extracellular fungal proteins, resulting in a hypersensitive response (HR). Five proteins secreted by C. fulvum were purified and the encoding cDNA clone was obtained from two novel ones among them. Various tomato breeding lines and accessions of Lycopersicon pimpinellifolium were tested for their recognitional specificity by injection of the purified proteins or potato virus X-based expression of the cDNA. We found that HR-associated recognition of one or more of these proteins, in addition to recognition of the race-specific elicitors AVR4 and AVR9 of C. fulvum, occurs among Lycopersicon species. Studies on the inheritance of this recognition confirmed that single dominant genes are involved. Furthermore, one of the extracellular proteins of C. fulvum is specifically recognized by Nicotiana paniculata, which is not a host for C. fulvum. These results indicate that plants have a highly effective surveillance system for the presence of 'foreign' proteins, which, together with the high mutation rate of pathogens, can explain the complex gene-for-gene relationships frequently observed in pathosystems.

Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum.

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the gene-for-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.