Molecular events in Colletotrichum gloeosporioides triggered by hard-surface contact and early expression of calmodulin gene required for appressorium formation in Magnaporthe grisea is inhibited by self-inhibitors and requires surface attachment

The avocado pathogen Colletotrichum gloeosporioides, like many plant pathogenic fungi, must differentiate from the germ-tube into an infection structure called the appressorium in order to penetrate into its host. Either avocado surface wax or the host's ripening hormone ethylene can induce conidial germination and appressorium formation. Contact with a hard surface is necessary for ethylene and avocado wax to induce appressorium formation in C. gloeosporioides.; To study molecular events triggered by hard surface contact, fungal genes expressed during hard surface treatment by C. gloeosporioides conidia have been isolated, based on a differential display method. The genes are designated C. gloeosporioides hard-surface induced protein genes (chips). CHIP1 encodes a 16.2 kDa ubiquitin-conjugating enzyme, which shows 82% identity to Saccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. CHIP1 can complement the proteolysis deficiency of Saccharomyces cerevisiae ubc4ubc5 mutant, indicating that ubiquitin dependent protein degradation is involved in conidial germination and appressorial differentiation. Two other cloned full-length cDNAs (CHIP3, 6) showed no significant homology to any known proteins. CHIP3 is a putative seven helix transmembrane receptor.; Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of Magnaporthe grisea and plant surface waxes were found to relieve this self-inhibition. To determine whether the self inhibitors suppress the expression of early genes involved in conidia differentiation, expression of calmodulin was chosen as a marker for the early genes, because this gene was found to be expressed early in C. gloeosporioides and C. trifolii differentiation. After cloning calmodulin cDNA and genomic DNA from M. grisea, the promoter of the calmodulin gene was fused to a reporter gene, green fluorescent protein (GFP), and transformed into M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed that the transcription of calmodulin decreased significantly when self-inhibition of M. grisea appressorium formation occured because of high conidia density or addition of exogenous self-inhibitors, and was restored when self-inhibition was relieved by plant surface waxes. The fluorescence increase correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blots. Concanavalin A (Con A) inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.