Calcium Signal Transduction Pathway Involved In Magnaporthe Grisea Growth And Pathogenesis

The blast fungus, Magnaporthe grisea, causes a serious disease on a wide variety of grasses including rice, wheat, and barley. Rice blast is the most serious disease of cultivated rice and therefore poses a threat to the world's most important food security crop. Therefore, it is important to acquire a detailed molecular understanding of the mechanisms of this fungal phytopathogenicity to effectively control this pest. During infection and the disease process, fungi, including M. grisea are subjected to recognizing susceptible hosts, overcoming physical and chemical barriers to invade plant tissues, withstanding host defense mechanisms, extracting nutrients for proliferation, and eventually disseminating to a new host. Morphological transitions during the infection process are an integral aspect of fungal phytopathogenesis. In this regard, the virulence arsenal of a fungal phytopathogen may include the ability to exhibit dimorphism or morphological, the production of infection structures such as appressoria, and the ability to produce spores as agents of dispersal. These morphological aspects of virulence are tightly regulated by environmental signals and signaling pathways that operated throughout infection.Fungal cells employ sophisticated signal transduction programs to sense and respond to specific environmental cues that drives changes in cell morphology and physiology. Signaling mechanisms that regulate fungal proliferation and differentiation include the highly conserved MAP (mitogen-activated protein, MAP) kinase and cAMP signaling cascades and Ca2+ signaling cascades that are best characterized in mammalian cells and the model yeasts Saccharomyces cerevisiae. In M. grisea, many studies demonstrated MAP kinase and cAMP signaling cascades regulate hyphal growth polarity and morphogenesis, conidiation, and spore germination. Comparatively, only a few experiments suggested the Ca2+ signaling pathway was involved in the infection process of M grisea and the details of relationshp between Ca2+ signaling pathway and pathogenesis of M. grisea is unclear. In this study, inhibitors experiments elucidated the calcium signaling pathway is correlated with conidium germination and appressorium formation of M. grisea induced by hydrophobic surface; M. grisea cam (calmodulin) gene, the main protein of Ca2+ signaling system, was cloned and the role of cam gene in pathogenicity was analyzed by targetedgene replacement, antisense inhibition and over-expression. The results are showed as follows:(I )To ellustrate weather the calcium signaling pathway is correlated with conidium germination and appressorium formation of M. grisea induced by hydrophobic surface, conidia were treated by four kinds of inhibitors. Microscopic examination and statistical data showed: ( i )EGTA, a chelating agent selective for Ca2+, Verapamil, which could block Ca2+ channels, U-73122, an inhibitor of phospolipase C, KN93, a selective inhibitor of CaMK could inhibit (50%) germination and appressorium formation. (ii )Under the same concentrations of inhibitors, appressorium formation was inhibited more strongly than conidium germination. These inhibitors, except EGTA, caused the formation of small and abnormal appressorium. (iii)These inhibitors inhibited strongly conidium germination and appressorium formation in early state (l-4h). (iv)There were some roundish vesicle in the conidium inhibited completely. These results strongly suggested that Ca2+ signaling pathway was involved in conidium germination and appressorium formation induced by hydrophobic surface.(II )The calmodulin gene of Magnaporthe grisea was cloned by reverse transcriptase and polymerase chain reaction (RT-PCR). The sequences of DNA and predicted amino acid of calmodulin gene from M. grisea were 98% identical to those of the gene reported in GenBank (AF.103729) and 80% identical to those of the genes from other species of fungi. Southern blot analysis indicated it was single copy in genome of M. grisea. Relative quantitative RT-PCR demonstrated that CAM expression is highe...