The Preliminary Identification Of Related Factors In The Process Of PsGK4 And PsIMPA1 Regulated Asexual Reproduction 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.In the field,zoospores identify the chemical signals secreted by host plants,directional swim and colonize to complete the infection.Therefore,exploreing the sporangium development,zoospore release and trend of asexual reproduction in the molecular mechanism,it will help us discover this kind of disease prevention and control of molecular targets,and lay the foundation for molecular design of disease resistance.In the preliminary study of the laboratory,the molecular mechanisms involved in the asexual reproduction of soybean were studied.We found the gene PsGPA1 which encodes G protein ? subunit in the G signal transduction pathway silenced significantly affected zoospore chemotaxis.The gene PsGPR11 which encodes a G protein coupled receptor silenced mutant showed germination rate decreased by about 40%compared with that of the wild type.MAPK encoding gene PsMPK7 in the MAPK signaling pathway affected the germ tube and defects of the resting cells were also decreased.PsMPK1 is involved in the regulation of the mycelium growth and spore formation of soybean.After silenced nucleo-cytoplasmic transport process related protein code gene PsIMPA1,the sporangia germination rate and pathogenicity decreased significantly.The silencing of SNARE protein family gene PsSYP6 significantly affected the yield and normal development of the spore capsule.In order to further identify more asexual reproduction related factors and to explore the relationship between them in P.sojae,this study based on the early identification of PsGK4 and PsIMPA1 to screened the related interaction protein and related genes in the transcription level.Screening for G protein coupled receptor PsGK4 binding proteins.GPCR-G Protein signaling pathways play a vital role in the infection process of P.sojae.PsGK4 is a GPCR protein in Phytophthora sojae.The previous study reveals that PsGK4 in P.sojae contributes to zoospore swimming,chcmotaxis and pathogenicity.Thus,screening for PsGK4 binding proteins is important to help us to know the mechanism of GPCR signaling pathways in P.sojae.We screened for the PsGK4 interacting proteins from P.sojae total protein with the co-immunoprecipitation assay.We finally got 70 putative PsGK4 binding proteins.Due to the difficulty in the expression of the full-length protein of PsGK4,we finally use the 57 amino acid domain(GPCR seven transmembrane domain of the first vesicle membrane)in the end of the N terminal.And we choosed 3 proteins to do more reasches.We choosed Ps330899?Ps359771?Ps247465 to interact with PsGK4 by GST Pull-down assay.The Ps359771 protein contains a functional domain of the enzyme.Ps247465 protein contains a Sec23 domain,and the protein is of type COP? vesicle coat protein complex group.Ps500558 protein contains a domain Rab-GTPase-TBC.Because of lower solubility,we can not identify whether Ps247465 can interact with PsGK4.We conformed that the Ps330899?Ps359771 can not interact with PsGK4.This experiment is to be further validated by optimizing the protein expression,the integrity of the protein expression or the use of other analysis methods.The other candidate proteins also need further analysis and validate.Screening the genes associated with asexual reproduction through the analysis of the PsIMPA1-silenced mutants transcriptional groups.Karyophilic proteins are a conserved family of proteins in eukaryotic cells which can transport macromolecular substance from cytoplasm to nucleus to regulate transcriptional expression of some genes.Bioinformatics predict that there is only one importina gene in genome of P.sojae.PsIMPA1-silenced transformants can not produce spores,zoospores and infect the ability of scavenger active oxygens,which can also induce the decline of pathogenic ability.In this study,RNA-seq was used to study the change of gene expression pattern of PsIMPA1 gene silencing mutants in the mycelium and spore stages(compared with the wild type).The results revealed that the silenced of PsIMPA1 significantly affected including G protein signaling pathway,MAPK signaling pathway and SNARE protein family genes were significantly down-regulated,which part of the gene function have been in preliminary laboratory studies were identified,which also from the side reflects the reliability of sequencing data.The differential expression of a large number of transcription factors was also obtained in the sequencing data,including the common transcription factors,such as bZIP,C2H2,HSF and so on.