Screening And Functional Analysis Of G Protein Alpha Subunit In Phytophthora Sojae

Phytophthora sojae as one of the most important plant pathogenic oomycete, cause a devastating disease, soybean root rot disease in soybean production. At the asexual life cycle stage, P. sojae form sporangium on the tips of flooded mycelia and release zoospores. In the field, zoospores identify the chemical signals secreted by host plants, directional swim and colonize to complete the infection. Therefore, understanding the molecular mechanism of chemotaxis of zoosopore is crucial to disease control.Screening for Ga binding proteins. The preliminary study in our lab shows that, P. sojae PsGPA1silencing severely affect zoospore behavior including chemotaxis to the soybean isoflavones daidzein. G-protein signaling pathways have a certain role in sporangium formation and zoospore behavior. In order to understand how does Ga regulate the G-protein signaling pathways, we use the yeast two-hybrid to screen for the G alpha subunit interacting proteins from P. sojae cDNA library. We got a putative Ga binding protein guanine nucleotide-binding protein subunit beta--like protein.In the G protein signaling pathway, there are active and inactive forms of Ga subunit. Mutations of a concerved arginine (R117) into a histidine and of a conserved glutamine (Q203) into a leucine are known to disrupt the GTPase activities of the Ga subunit and lock the Ga protein in its active state. In order to verify whether PsRACK1are able to interact with two forms of Ga proteins, we use yeast two-hybrid and GST Pull-down to verify the interaction between PsRACK1and PsGPA1, PsGPAl(R177H,Q203L). We get consistent results that PsRACK1can indeed interact with PsGPA1while can not interact with psGpA1(R177H,Q203L)In order to understand the function of PsRACK1in P. sojae, we use the PEG-mediated transformation to silence PsRACK1gene in this paper, and get two silent mutants. We test phenotypes such as vegetative growth, sexual reproduction, stress factor tolerance and virulence change in PsRACK1mutants, then find that oospores production decreased significantly in silent mutants compared with wild-type. These results indicate that PsRACK1may involved in sexual reproduction regulatory pathways in P. sojae.