Functional Analysis Of Five Phosphatidylinositol 3-kinase Coding Genes In Phytophthora Sojae

Phytophthora sojae is a kind of pathogenic oomycetes.Root rot caused by P.sojae is a serious disease in soybean production.Previous studies showed that the RxLR effector of P.sojae increased affinity with the host by binding to phosphatidylinositol 3-phosphate(PtdIns(3)P)on the host cell membrane.Besides part of PtdIns(3)P in host,P.sojae also can synthesize PtdIns(3)P to promote infection.Phosphatidylinositol 3-kinase(PI3K)are key genes in the PtdIns(3)P synthesis pathway.Five PI3K-coding genes were identified in P.sojae and named as PsPI3K1～5,among which PsPI3Kl and PsPI3K2 belong to class Ⅲand PsPI3K 3～5 belong to class I.PI3K cancreates PtdIns(3)P to regulate a variety of basic metabolic pathways in cells.However,the function of PI3Ks in P.sojae is largely unknown.This study proposed to use genetic transformation method to analyze the functions of the PI3Ks in P.sojae,uncover the key role of these genes in P.sojae growth and development,and expect to elucidate the function diversity among the PsPI3Ks.The detailedresearch content as follows:Prediction of PI3K protein structure and acquisition of PI3Ks knockout or silencing mutants in Phytophthora sojae.This study further analyzed the protein domain of PsPI3K1～5 and found that PsPI3K1,a homology gene of VPS34 in yeast,contained three functional domains of PI3K_C2,PI3Ka and PI3_PI4_kinase.PsPI3K2 contains a FYVE domain that has been shown to bind specifically to PtdIns(3)P.PsPI3K4 and PsPI3K5 contain conserved PI3Ka,PI3_PI4_kinase,PI3K_C2,PH and PI3K_RBD domains in PI3K.PsPI3K3 contains only two functional domains,PI3Ka and PI3_PI4～kinase.The results showed that the protein structure of PI3K in P.sojae was obviously differentiated.In order to investigate the specific functions of PsPI3K1～5,Using the CRISPR/Cas9 system,sgRNA(single guide RNA)and HDR(homology-directed repair)knockout vectors are designed according to the protein structure of PsPI3K1～5,and PsP13K1～5 were knocked out by genetic transformation method.Through three rounds of genome PCR verification,knockout mutants of PsPI3K1,PsPI3K3,PsPI3K4 and PsPI3K5 and silencing mutants of PsPI3K2 were successfully obtained,which laid the foundation of genetic materials for subsequent studies.Phenotypic analysis of PsPI3Ks knockout or silencing mutants.In order to further explore the biological functions of PsPI3K1～5 in growth development and pathogenesis of P.sojae,this study analyzed four phenotypic indexes:growth rate,adversity tolerance,pathogenicity and sporulation.The growth rate tests found that PsPI3K3 knock out mutants on the V8 plate significantly greater than the wild type strain,PsPI3K5 knock out mutants grown significantly smaller than the wild type,PsPI3K1,PsPI3K4 knockout mutants and PsPI3K2 gene-silencing mutants similar with wild type,the results show that PsPI3K3 and PsPI3K5 participated in the vegetative growth of P.sojae regulation.PsPI3K4 knockout mutants was extremely sensitive to stress treatment and PsPI3K5 knockout mutant was not sensitive to stress treatment under H2O2,metalaxyl and fluoropylamine stress,PsPI3K3-～transformants are consistent with the wild type and the control group under stress treatment.The virulence detection found that the lession area of PsPI3K1,PsPI3K2 and PsPI3K5 mutants were significantly reduced after inoculation compared with control group,and the plaque area was no significant difference between PsPI3K3/4 mutants and control stains.The results indicated that PsPI3K1,PsPI3K2 and PsPI3K5 were the key genes in virulence regulating.The count of zoospores showed that the number of zoospores of PsPI3K1,PsPI3K2,PsPI3K4 and PsPI3K5 mutants were significantly reduced.There was no difference in the production of zoospores between PsPI3K3 and control group.The results showed that PsPI3K1,PsPI3K2,PsPI3K4 and PsPI3K5 were involved in the production of Phytophthora sojae.In summary,we found that PsPI3K1 and PsPI3K2 are key genes affecting the pathogenesis.PsPI3K3 and PsPI3K5 are involved in regulating growth of mycelia.PsPI3K4 is a key regulator under stress tolarence.This study laid foundations for the study on the PI3K signaling pathway of P.sojae and explored a new way for the prevention and treatment of soybean root rot.