Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by self-inhibitors and requires surface attachment

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recentl y, such self-inhibitors were found to be present on the conidia of Magnapor the grisea, and plant surface waxes were found to relieve this self-inhibit ion. To determine whether the self-inhibitors suppress the expression of ea rly genes involved in the germination and differentiation of conidia, the c almodulin gene was chosen as a representative early gene, because it was fo und to be expressed early in Colletotrichum gloeosporioides and Colletotric hum trifolii differentiation. After calmodulin cDNA and genomic DNA from M. grisea were cloned, the promoter of the calmodulin gene was fused to a rep orter gene, that for green fluorescent protein (GFP), and transformed into the M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of M. grisea a ppressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this ge ne was restored when self inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also co nfirmed by RNA blotting. Concanavalin A inhibited surface attachment of con idia, GFP expression, and appressorium formation without affecting germinat ion. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formati on require surface attachment.