The role of G protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea

To identify signal transduction pathways of the gray mold fungus Botrytis c inerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (PCR)-based approach to isolate two genes (bcg1 and bcg2) encoding cc subunits of heterotrimeric GTP-binding proteins. Both genes have homologues in other fungi: bcg1 is a member of the G alpha (i) class, whereas bcg2 has similarities to the magC gene of Magnaporthe grisea and the gna-2 gene of Neurospora crassa. Reverse-transcription (RT)-PCR ex periments showed clearly that both genes are expressed at very early stages in infected bean leaves. Gene replacement experiments were performed for b oth genes. bcg1 null mutants differ in colony morphology from the wild-type strain, do not secrete extracellular proteases, and show clearly reduced p athogenicity on bean and tomato. Conidia germination and penetration of pla nt tissue is not disturbed in bcg1 mutants, but the infection process stops after formation of primary lesions. In contrast, bcg2 mutants show wild-ty pe colony morphology in axenic culture and are only slightly reduced in pat hogenicity. Complementation of bcg1 mutants with the wild-type gene copy le d to the full recovery of colony morphology, protease secretion, and pathog enicity on both host plants. Application of exogenous cyclic AMP restored t he wild-type growth pattern of bcg1 mutants, but not the protease secretion , implicating an essential role of BCG1 in different signaling pathways.