Function Of Phytophthora Sojae Phosphatidylinositol Kinase PI3K4 And Arabidopsis Thaliana BAG7

Phytophthora sojae belongs to Oomycetes,which is widely distributed all over the world.Soybean root rot caused by pathogens such as P.sojae is the most serious root disease in the growth process of soybeans,which will lead to a significant decline in yield and quality,causing huge economic losses worldwide.Therefore,analysis of the basic biological characteristics and pathogenic mechanism of P.sojae is of great significance for exploring the target genes to control P.sojae scientifically and efficiently.When pathogens infect host plants,they secrete a series of effectors to suppress the immune defense response of host plant.Among them,Rx LR effectors are one type of the weapons used by P.sojae to attack the host.Previous studies have shown that Rx LR effectors can bind to phosphatidylinositol3-phosphate[Ptd Ins（3）P]on the cell membrane of host plants to increase the affinity with the host for better cell entry.Ptd Ins（3）P is mainly produced by phosphorylation of Phosphatidylinositol（Ptd Ins）by class II and class III phosphatidylinositol 3-kinase（PI3K）.Five PI3K-coding genes were identified in P.sojae（Ps PI3K1～5）.However,the specific function of PI3Ks in P.sojae is largely unknown.In our previous study,we obtained knockout mutants of Ps PI3K1/3/4/5 and silencing mutant of Ps PI3K2 via CRISPR-cas9system.In this study,we further analyzed the performance of Ps PI3K1～5 genes in response to reactive oxygen stress and endoplasmic reticulum（ER）stress response in P.sojae,and explored the specific functions in the regulation of adversity.In addition,previous studies have found that plant B-cell lymphoma2-associated athanogene 7（BAG7）can be transported from the ER to the nucleus under heat stress,thereby involved in the regulation of heat-induced resistance to Phytophthora,but the specific mechanism of its interaction with Phytophthora was unknown.In this study,we found that the subcellular localization of BAG7 plays a dual role in regulating Arabidopsis thaliana resistance to Phytophthora.At the same time,we found that the Rx LR effector Avh262 secreted by P.sojae can block BAG7 from the ER to the nucleus through Binding immunoglobulin protein（Bi P）,thereby promoting self-infection.The specific research contents of the above two parts are as follows:1.PI3K4 plays a key role in the response of P.sojae to various stressesIn this study,we firstly re-analyzed the evolution of the five PI3K proteins encoded by the P.sojae genome,and found that Ps PI3K1 belongs to Class III PI3K,Ps PI3K3/4/5 belong to Class I PI3K,Ps PI3K2 is unique to Oomycetes and belongs to the Class Unique type.The domain prediction of Ps PI3K1～5 showed that the Ps PI3K2 protein contained FYVE domain,while the evolutionarily homologous Ps PI3K4 and Ps PI3K5 both contained the Pleckstrin Homolgy domain.We tested the expression of five PI3Ks genes under the treatment of H₂O₂,ER stress inducer tunicamycin（TM）and DL-Dithiothreitol（DTT）,and found that the expression of Ps PI3K4 gene was significantly upregulated.Subsequently,we tested the growth rate of the knockout or silenced transformants of Ps PI3K1～5 genes under the treatment of PI3K inhibitors,TM and biocontrol bacteria.We found that Ps PI3K4transformant were more sensitive to various adversity treatments than other transformants.We also observed that the hyphae morphology of Ps PI3K4 transformant under the treatment of H₂O₂ and TM was obviously abnormal.In addition,the normal production of Ps PI3K4sporangium and zoospores was significantly inhibited by TM treatment.Based on the above results,we believe that PI3K4 is a key gene of P.sojae in response to multiple stresses such as reactive oxygen species,ER stress and biocontrol bacteria.2.Study on the Function of Arabidopsis thaliana BAG7 in Phytophthora-Plant InteractionBAG7 is the molecular chaperone of Bi P protein and plays a key role in maintaining the ER unfolded protein response（UPR）.In this study,we found that BAG7 negatively regulates the Arabidopsis thaliana resistance to Phytophthora by inhibiting the cell death mediated by ER stress.When plants sense the infection signal of Phytophthora,BAG7 is transported from the ER to the nucleus,and positively regulates Arabidopsis thaliana resistance to Phytophthora by enhancing ER stress-mediated cell death.This process is inhibited by the accumulation of Bi P mediated by Phytophthora effector Avh262,thus promoting self-infection.At the same time,this study found that there is a conserved M1region in the Immunoglobulin/albumin-binding（IAB）domain of Avh262,and the Alpha helix in this region is a key secondary structure for Avh262 to stabilize the Bi P protein.After destroying the secondary structure,the localization of Avh262 in cells and the ability of Avh262 to stabilize Bi P are affected.In addition,we found that the virulence of Avh262depends on BAG7 through inoculation experiments.In summary,we found that the P.sojae effector Avh262 can retain BAG7 in the ER by increasing the accumulation of Bi P and hinder the transport of BAG7,thereby inhibiting the resistance of Arabidopsis thaliana to Phytophthora and promoting the infection of Phytophthora.