Functional Analysis Of A Putative Rho-GEF Protein MorRgf1 In Magnaporthe Oryzae

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of fundamental cellular processes, including cell morphology, gene expression and vesicular trafficking. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation. Guanine nucleotide exchange factor （GEF） has a crucial role in activating Rho GTPases by exchange GDP for GTP. MoRgfl （MGG₀3064）, a putative RHO-GEF protein in Magnaporthe oryzae, has a putative RhoGEF domain, which is homologous to RhoGEF in Saccharomyces cerevisiae. Phylogenetic analysis showed that MoRgfl is highly homologous to N.cRgfl and S.cRom2 which are both identified as a GEF protein for Rhol in N. crassa and S. cerevisiae respectively. Based on the bioinformatics analysis, we infer that the MoRgfl may function as one GEF for Rhol protein. In this study, we generated MoRGF1 （MGG₀3064） deletion mutant by homologous recombination-based approach. The grow rate of the deletion mutant was much slower than that of wild-type strain （Ku80）, but the distance of two neighbor septa is nearly as normal, indicating that MoRGF1 gene maybe related with cell division cycle. In addition, the deletion mutants showed lower capacity to endure SDS than that of WT, as well as lost the ability to form conidia. Furthermore, the MoRGFl deletion mutant was nonpathogenic even inoculating the mycelial plugs on wounded leaves of rice and barley. The complementary mutants suppressed all these defects of deletion mutants indicating that MoRGF1 is truly responsible for these phenotypes. In order to confirm whether MoRgfl could interact with specific Rho GTPase, we assayed the interaction of the RhoGEF domain of MoRgfl with Rho family proteins using yeast two-hybrid approach. The results were not the sam as what we expected, because RhoGEF domain is not specific to bind with Rho1 protein. We will further to detect MoRgfl-Rhol interaction by in vitro co-IP. Taken together, our study based on MoRgfl will be in favor of better understanding the function of Rhol and their regulatory mechanism underlying fungal development and pathogenicity, and further study would help to find an ideal molecular target for controling the damage caused by Magnaporthe oryzae.