Studies of calcium-calmodulin signaling and genes induced by hard-surface contact of Colletotrichum gloeosporioides Conidia

The germinating conidia of many phytopathogenic fungi on plant hosts must differentiate into an infection structure called the appressoria in order to penetrate their hosts. Induction of this differentiation in the avocado pathogen Colletotrichum gloeosporioides by chemical signals such as the host's surface wax or the fruit ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 hr. Our efforts to elucidate the molecular events in the early phase of the hard surface contact found that EGTA (at 5 mM) and U73122 (at 12 nM), an inhibitor of phospholipase C, inhibited (50%) appressorium formation. Measurement of calmodulin (CaM) transcripts with a CaM cDNA we cloned from C. gloeosporioides showed that CaM was induced by hard surface contact maximally at 2 h and then declined; ethephon enhanced this induction. The CaM antagonist, compound 48/80, completely inhibited the conidial germination and appressorium formation at a concentration of 30 muM implication involvement of CaM in this process. A selective inhibitor of CaMK, KN93 (at 20 muM), inhibited (50%) appressoria formation, blocked melanization and caused formation of abnormal appressoria. These results strongly suggest that hard surface contact induces Ca 2+-calmodulin signaling that primes the conidia to respond to host signals by differentiation into appressoria.; To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode C olletotrichum hard-surface induced proteins are designated CHIP genes. In this study, we report characterization of CHIP2 and CHIP3 genes that would encode proteins of molecular mass of 65- and 64 kDa, respectively. RNA blots showed that CHIP2 and CHIP3 are induced by a 2 h hard-surface contact. However, disruption of these genes did not affect the appressorium, forming ability and did not show significant decrease in virulence on avocado fruits suggesting that C. gloeosporioides might have genes functionally redundant to CHIP2 and CHIP3 or that these genes induced by hard surface contact control processes not directly involved in pathogenesis.