Two novel Venturia inaequalis genes induced upon morphogenetic differentiation during infection and in vitro growth on cellophane.

Venturia inaequalis is a hemibiotrophic ascomycete that causes apple scab. Germ tubes, from conidia or ascospores, penetrate the leaf or fruit surface directly via appressoria-like swellings; subsequently the hyphae divide laterally to form a stroma between the cuticle and the outer wall of the epidermal cells. This morphological switch can be mimicked by growing the fungus in vitro on cellophane discs. The aim of this work was to identify genes upregulated in planta using growth on cellophane as a model. Four cDNA clones were found to be induced by growth on cellophane, and qRT-PCR showed two of these genes were up-regulated over a thousand fold in infected apple leaves compared to liquid culture. The predicted proteins for both genes possess putative signal peptides for secretion but have no similarity to sequences in publicly available databases. Both genes encode proteins with novel, imperfect repeat domain structures, the number of which vary in an isolate-specific fashion. Cin1 has seven or eight repeats of about 60 amino acids with four conserved cysteine residues per repeat, while Cin3 has four or five repeats of 32 amino acids with no cysteines. Both proteins appear to have evolved through internal duplication. Cin3, in particular, shows considerable between-strain variation in domain structure, indicating a high degree of recombination at this locus and revealing that the repeat structure has most likely arisen by unequal crossing-over. Results of this study support the hypothesis that cellophane-grown V. inaequalis mimics aspects of biotrophic infection and provide the first insights into novel fungal genes expressed during apple scab infection and their mechanisms of evolution.