Signaling in Colletotrichum trifolii during pathogenic development: Involvement of small G proteins.

摘要

Colletotrichum trifolii is a fungal pathogen of alfalfa that causes anthracnose disease. The pathogenicity of this fungus depends on cellular differences, which are regulated by various extracellular stimuli including signals from the host plant. The goals of this research were to investigate signaling related to pathogenesis in C. trifolii. Previously, it was reported that dominant active Ct-Ras strain (Val2) displayed aberrant hyphal growth and morphology and defects in development on minimal media, suggesting that this member of small G protein family plays a regulatory role in these processes of C. trifolii in a nutrient dependant manner. In this thesis, it was shown that proline bypasses the aberrant phenotypes caused by dominant active Ct-Ras, suggesting that Ct-Ras signaling is regulated in response to nutrient signal such as proline. Generation of various mutants of Ct-Ras in which expression was negatively regulated demonstrated that Ct-Ras is also important for conidial germination and hyphal growth. Cdc42 is another small G protein family molecule. In searching of possible downstream effector molecule of Ct-Ras, the biological function of Ct-Cdc42 was examined. The fact that Cdc42 mutants show similar phenotypes to Ct-Ras mutants and the dominant active Ct-Cdc42 mutant is also sensitive to proline imply that Ct-Cdc42 is positioned in the same signaling pathway as Ct-Ras is involved. The pleiotropic phenotypes of Val2 strain were suppressed by overexpression of dominant negative Ct-Cdc42. Conversely, a significant decrease in conidial germination of dominant negative Ct-Ras was partially recovered by overexpression of dominant active Ct-Cdc42. These results suggest that Ct-Cdc42 function as a downstream of Ct-Ras signaling in C. trifolii. Further analysis of Ct-Cdc42 mutants suggested that Ct-Cdc42 functions as a negative regulator in appressorium development. Lastly, the role of a protein kinase in C. trifolii was investigated. Lipid Induced Protein Kinase (LIPK) is a unique serine/threonine kinase whose transcription is induced by plant cutin or monomeric constituents of cutin. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria, while lipk gene disruptants did not developed appressoria and, thus, failed to infect intact host plant tissue. These results suggest that LIPK plays central role in triggering appressorium development in response to host surface chemistry.