Study On The Function Of PiGK5 And Phosphoproteomics Analysis Of Sexual Reproduction Induced By Hormone α1 In Phytophthora Infestans

Potato late blight caused by Phytophthora infestans is considered to be one of the most destructive plant diseases.The pathogen can infect the leaves,stems,and tubers of potato and destroy the plants rapidly and even cause the death of plants in a few hours,seriously harming the development of potato industry.Sexual reproduction of Phytophthora plays an important role in the evolution and continuation of the species and is regulated by hormones.A1 and A2 mating type strains of Phytophthora can produce hormone α1 and α2 respectively.When the A1 and A2 mating type strains are cultured together,hormone α1 and α2 induce the differentiation of the female and male gametophyte of the opposite mating type strain respectively,so as to complete the sexual reproduction and produce oospores.However,the mechanism of sexual reproduction of Phytophthora is still not revealed.In this study,the long-term preservation method of Phytophthora infestans is established.The growth curve of several isolates of Phytophthora infestans was plotted to determine their optimal incubation time and their mating type was measured by different methods.The coding region of Pi GK5 c DNA of the A1 mating type strain HQK8-3 was cloned and sequenced.The subcellular localization of Pi GK5 protein was determined by technology of fluorescent protein fusion expression.The function of Pi GK5 in asexual and sexual reproduction and pathogenicity was studied through RNA interference technology.Differential phosphoprotein profiles of the A2 mating type strain induced by hormone α1at different time points were analyzed through i TRAQ technology.The results showed that the four preservation methods used in this study could maintain the viability of the isolates of P.infestans better and had no significant influence on the growth and morphological characteristics of the isolates,but the influence of the four methods on pathogenicity of P.infestans was isolate-specific.The optimal incubationtime of the isolate HQK8-3,YF3,2PO-D,2PO82001,USA1,and P7723 was 12,13,11,9,18,and 7 days respectively.The isolate HQK8-3,2PO-D,and USA1 was A1 mating type and the isolate 2PO82001 and P7723 was A2 mating type.The isolate YF3 was A1self-fertile type.These work laid the foundation for the following study.The coding region of Pi GK5 c DNA of the isolate HQK8-3 was 2763 bp long,encoding 920 amino acids.The sequence similarity between the c DNA of the isolate HQK8-3 and the c DNA published by NCBI was 99.07%,while the amino acid sequence similarity was 93.42%.The structure prediction of Pi GK5 of the isolate HQK8-3 was consistent with the structure prediction of Pi GK5 published by NCBI,suggesting that their functions could also be consistent.Pi GK5 had the highest relative expression level in zoospores in the A1 mating type,followed by cyst,and relatively less in vegetative mycelia,sporangia and germinating cysts.Pi GK5 is located in the protoplast membrane and some vesicular structures in the cells.The Pi GK5 gene is involved in the formation of zoospores and oospores in the A1 mating type and therefore is a very important gene for the asexual and sexual reproduction in P.infestans.Pi GK5 may be the receptor of hormone α2.Furthermore,the Pi GK5 gene is involved in the pathogenicity of the A1 mating type strain,which may be an important pathogenic factor for P.infestans.Through phosphproteomics analysis on the A2 mating type strain induced by hormone α1,6355 phosphorylated peptides were obtained and 2508 phosphoproteins were identified.One way anova analysis showed that 453 differential phosphoproteins were screened in hormone α1 group,of which 249 proteins were detected only in hormoneα1 group compared with the control.After analysis of the GO function annotation at level2 and KEGG pathway,eight phosphoproteins with significant differences were screened,including PITG₀1203T0,PITG₀6415T0,PITG₀7286T0,PITG₀7289T0,PITG₁0062T0,PITG₁9213T0,PITG₁9890T0,and PITG₂2434T0.Analysis of T Test showed that 185,43,189 and 43 differential phosphoproteins were screened respectively in the A2 mating type strain induced by hormone α1 for 1h,3h,8h and 24 h.After analysis of the GO function annotation at level 2 and KEGG pathway,ninephosphoproteins with significant differences were screened,including PITG₀0397T0,PITG₀0845T0,PITG₀1947T0,PITG₀4443T0,PITG₀4663T0,PITG₀7567T0,PITG₁0083T0,PITG₁6183T0,and PITG₁7706T0.The 17 proteins are highly likely to be involved in the sexual reproduction process in P.infestans.Their functions need to be further validated in the future.In this study,the function of Pi GK5 gene in sexual and asexual reproduction and pathogenicity of P.infestans were studied for the first time.Differential phosphoprotein profiles of the A2 mating type strain induced by hormone α1 were analyzed by phosphoproteomics method and a group of proteins closely related to the sexual reproduction of P.infestans were screened.The completion of this work has important significance for the discovery of molecular mechanism of sexual reproduction in P.infestans and the effective prevention and control of Phytophthora diseases,such as potato late blight.