Function Analysis Of Protein Phosphatase 2C,which Interacted With G Protein Alpha Subunit In Phytophthora Sojae

Root rot disease of soybean caused by Phytophthora sojae leads to extremely significant economic losses on soybean production worldwide every year.It is one of the major diseases on soybean.Therefore,how to control P.sojae effectively is an important task for researchers.At the sexual life cycle stage,P.sojae form Oospores.Oospores can survive for years in the soil or rot soybeans and wait for the right conditions to cause the disease occurrence and epidemic.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 and the production of Oospores is crucial to disease control.Screening for G protein a subuint binding proteins.G-Protein signaling pathways play a vital role in the infection process of P.sojae.PsGPA1 is a G protein alpha subunit(Ga)in Phytophthora sojae.The previous study reveals that,PsGPA1 in P.sojae contributes to zoospore swimming,chemotaxis and pathogenicity.Thus,screening for G protein α subuint binding proteins is important to help us to know the mechanism of G-protein signaling pathways in P.sojae.We screened for the PsGPA1 interacting proteins from P.sojae total protein with the GST Pull-down assay.We finally got 75 putative PsGPA1 binding proteins and choosed 4 proeins to do more reasches.GST Pull-down to verify the interactions between four putive candidates and PsGPAl.We choosed a ketol-acid reductoisomerase,a Guanylate-binding protein,a Phospholipase D-like protein and a Protein phosphatase 2C to interact with PsGPAI by GST Pull-down assay.We conformed that a ketol-acid reductoisomerase and a Protein phosphatase 2C can interact with PsGPA1.In the G-protein signaling pathways,there are active and inactive forms of Ga.We could add GTP or GDP to get active or inactive form of Ga in vitro.We confirmed that a PsPP2C can interact with PsGPAl whether added with GTP or GDP.In vitro phosphatase assay of PsPP2C.Prediction of protein encoded by PsPP2C is a kind of serine/threonine phosphatase,which can catalyze other proteins to be dephosphorylated.We measured the absorbance of a molybdate malachite green:phosphate complex to determine the amount of free phosphate generated in the reaction.However,the interaction with PsGPAl did not significantly change the activity of PsPP2C.PsPP2C is involved in the cell growth,process of sexual/asexual reproduction and pathogenicity in P.sojae.We did more researches to further learn the biological functions of PsPP2C,a PsGPAl-interacing protein in P.sojae.PsPP2C-silenced and PsPP2C-overexpressed mutants were generated using glycol PEG-mediated protoplast stable transformation.Phenotypic analysis showed that silencing of PsPP2C affected the growth rate,pathogenicity,production of oospore as well as production of Sporangia of P.sojae.PsPP2C is localized to the plasma membrane and the cytoplasm.The PsPP2C-overexpressed mutants show no Phenotypes different from the wide type.To sum up,PsPP2C is involved in the cell growth and process of sexual/asexual reproduction,which is vital important to survive.