Functional Analysis Of Rho GTPase Activation Proteins(RhoGAPs) In Magnaporthe Oryzae

Rice blast is one of the most devastated diseases on rice,one of the world’s major food crops.The cAMP,MAPK,and Ca2+ signaling pathways are involved in the regulations of appressorial development and infection process of rice blast fungus Magnaporthe oryzae.Acting as a molecular switch,Rho family proteins,belonging to Ras superfamily,play an important role in the signal transmission through cycling between an inactive GDP-bound state and an active GTP-bound state.During this cycling process,RhoGAP,as an negative regulator,accelerates the intrinsic GTP-hydrolysis activity of the GTPases.Therefore,to study the role of RhoGAP in the growth,development and pathogenesis of M.oryzae,we identified and analyzed the biological functions of candidate RhoGAPs in M.oryzae.Firstly,we identified eight candidate RhoGAPs in M.oryzae through bioinformatical analysis,and they were MGG₀4377（Molrg1）,MGG₀4186（Morga1）,MGG₀6390（MoRga2）,MGG₀3048（MoRga3）,MGG₀9531（MoRga4）,MGG₀9303（MoRga5）,MGG₀9275（MoRga6）and MGG₀4006（MoRga7）.All of them contain conserved RhoGAP domain and arginine site.qRT-PCR results showed that different RhoGAPs possessed diverse transcript levels at different life stages,including vegetative growth,conidiation,appressorial development and infection process.Among them,Molrgl possessed dramatically high transcript levels during appressorial development and infection process than other stages and other RhoGAPs.Then,to study the biological functions,using functional genomics strategy,we generated the single deletion mutants for all the eight RhoGAPs,RhoGAP domain deletion mutants for MoRgal and MoLrg1,and also R1032A site-directed mutagenesis in Morga1.Pheotypic analysis results showed that,compared with wild type strain,the growth rate and conidiation of MoLrgl mutants were dramatically decreased,accompanying with severe defect in pathogenicity.Deletion of MoRgal resulted in abnormal conidiation and appressorial formation.Moreover,deletion of either RhoGAP domain or all three LIM domains in either MoRgal or MoLrg1 or,and R1032A site-directed mutagenesis in MoRgal could cause similar phenotypic defects just as the whole gene deletion mutants,suggesting that RhoGAP domain,at least one of the three LIM domains and arginine residue in the RhoGAP domain were essential for the normal RhoGAP functions involved in conidiation and appressorial development.Other six RhoGAPs were proved to be dispensable for growth,conidiation and pathogenicity,suggesting that,to some extent,there was a functional redundancy among eight RhoGAPs.Besides,cell wall sensitivity assay results exhibited that,different chemical compounds could cause growth defects at diverse levels in the deletion mutants of eight RhoGAP genes,and among them,Molrg1 deletion mutants possessed the lowest sensitivity to all tested chemicals.Moreover,a complicated interactive network was identified using yeast two-hybrid system and/or pull-down assay.Morgal and Molrgl could interact with both Rac1-CA and Cdc42-CA respectively,and other RhoGAPs also possessed specific interactive Rho proteins.Taking together,Molrgl and Morgal are two key members among eight RhoGAPs and play important roles in development and infectious processes of M.oryzae through coordinated but independent modulation of Rho GTPases.