Functional Analysis Of PP2A Regulatory Subunit PsPP2Ab1 And Clathrin Light Chain PsCLC1 In Phytophthora Sojae

Soybean is a kind of food and oil crops,which have important significance in human agricultural activities.Phytophthora sojae infects soybeans and caused annual losses in soybean production worldwide.In the life cycle of P.sojae,sporangia are important organs in the asexual reproduction stage.The large number of zoospores released by sporangium can help pathogens spread.Oospores that survived from harsh external environment should germinate and product sporangium first,then release zoospores to infect host plants.However,the research on molecular regulation mechanism of sporangium stage of P.sojae is still very limited.This study is based on transcriptome data obtained in earlier research in our lab,finding genes that are specifically expressed during the sporangium stage of P.sojae.Among the candidate gene coding products,protein phosphatase 2A(PP2A)is highly conserved among species and involved in regulating growth and asexual reproduction in multiple species,its regulatory subunits can regulate the activity of PP2A,Clathrin ligh chain is one of the important substances that mediate endocytosis.In the transcription data of sporangium and zoospore stages,the two candidates corresponding to the abovementioned proteins have significant specific expression.With the update of gene function research technology of P.sojae,the early RNA interference/gene silencing was gradually replaced by CRISPR/Cas9 gene editing technology.This technology has higher efficiency and accuracy in gene knock out,becoming the main techniques for studying gene function.In this study,CRISPR/Cas9 gene editing technology was used to knock out PsPP2Ab1,which encodes a phosphatase regulatory subunit,and PsCLCl,which encodes a clathrin light chain,and explored in-situ complementation of the knocked out genes for the first time.The main research contents are as follows:Function analysis of protein phosphatase 2A regulatory subunit PsPP2Ab1:Protein phosphatase 2A(PP2A)is a subgroup of widely conserved serine/threonine phosphatases and play diverse roles in transcription,translation,cell cycle and many other cell activities,its regulatory subunit can regulate the function of PP2A.In this study,a regulatory subunit of PP2A(PsPP2Ab1)was identified in P.sojae,its protein domain has been forecasted.Evolutionary relationship has been analyzed.Using CRISPR/Cas9 gene editing techology to knock-out and complement PsPP2Ab1.Functional analysis showed that the deletion of PsPP2Ab1 affect the hyphae growth rate,sporangium production and pathogenicity of P.sojae.The results indicate that PsPP2Ab1 involved in growth,development of sporangium and pathogenicity of P.sojae.Function analysis of Clathrin light chain PsCLC1:Clathrin-mediated endocytosis is one of the important channels for cells to absorb outside substances.In this chapter,we do a family analysis of PsCL C1,which encoding the clathrin light chain in P.sojae and found that CLC is a highly conserved family among species.The transcript level of PsCLC1 was significantly reduced at zoospore stage.The specific protein and evolutionary relationship are in line with the evolution of species.PsCLC1 was successfully knocked out using CRISPR/Cas9 gene editing technology and mutations The phenotypic analysis results showed that the deletion of PsCLC1 decreased the pathogenicity significantly,the ability to adapt to salt stress environment was significantly reduced,but it did not affect the hyphae growth rate and sporangium development of sojae.The results indicate that PsCLC1 involved in response of salt stress and pathogenicity of P.sojae.In this study,PsPP2Abl and PsCLC1 with significant changes in expression levels in sporangium and zoospore stages.were selected to do evolutionary relationship between the gene families,CRISPR/Cas9 gene knockout was performed on them.Phenotypic analysis revealed that PsPP2Ab1 has involved in regulating the growth of vegetative mycelium and sporangium development of P.sojae.Both of PsPP2Abl and PsCLC1 are involved in regulating the infection process of P.sojae.This study provided new research ideas for the regulation mechanism of sporangium development of P.sojae and provided a theoretical basis for the control of soybean root rot.