Screening And Functional Research Of Chemotaxis Related Genes In Hytophthora Sojae

The causative Phytophthora sojae of soybean root rot is a devastating plant pathogen that infects soybeans and causes serious hazards to the production of the global soybean industry.In the disease cycle of soybean root rot,the asexual stages of P.sojae involve the development of sporangia and zoospores.Zoospores released by mature sporangia were armed with two flagella,which can assist zoospores move to new hosts in response to signals from soybean roots and initiate new infections.Thus,zoospore chemotaxis plays a crucial role during disease cycles.However,little is known about the molecular mechanisms of chemotaxis.On one hand,in the perspective of protein level,this study starts from G protein signals which is known involved in chemotaxis,the downstream effectors were screened by mass spectrometry.On the other hand,transcriptional perspective was taken and focusing on zoospore of P.sojae,the candidate genes related to chemotaxis were identified by transcriptome sequencing.Finally,the biological function of these chemotaxis related candidates were investigated by CRISPR/Cas9 mediated gene knockout system.Whole-genome transcriptional analysis of zoospore chemotaxis in Phytophthora sojae:In Phytophthora sojae,zoospores can specifically recognize isoflavones,which is secreted by soybean roots,and apply chemotactic migration to find new hosts and initiate the infection process.Therefore,zoospores chemotaxis is a key point in the infection cycle of P.sojae.In order to identify more chemotaxis related genes in P.sojae,we treated zoospores with soybean root hairs or daidzein.Then we extracted total RNA of these treated zoospore samples for transcriptome sequencing.Through transcriptional analysis,we identified 2,308 and 714 differentially expressed genes in root hair and daidzein treatments,among which 74%and 79%were up-regulated,indicating that most of the candidate genes were activated during chemotactic response.Subsequently,we performed qRT-PCR analysis on candidate genes,which is up-regulated after chemoattractant induction.In combination with the transcription patterns of candidate genes in different stages of P sojae,we selected 8 genes for functional study.Using the CRISPR/Cas9 mediated knockout technology,we were succeed in knocking out five genes of them(PsCBP1,PsHK1,PsMP1,PsDRS2 and PsGPCR1)and phenotypic analyses were conducted among these mutants.It was found that the knockout of PsCBPl did not affect chemotaxis and virulence;PsHK1 and PsMP1 knockout mutants displayed significant reduced virulence;zoospores ofPsDRS2 knockout-mutants lost the ability to move,exhibit depressed ratio of germinated cyst,elevated ratio of abnormal germ tubes,and attenuated virulence on soybean hypocotyls;The knockout of PsGPCR1 significantly affected zoospore chemotaxis and virulence on soybean hypocotyls.Screening and functional research of downstream effectors of G protein signal in Phytophthora sojae:The G protein pathway utilizes its downstream effectors to regulate signal processing,integration and coordination,which exerts important roles in organismal signal sensing and transduction.Chemotaxis is a major approach for the spread of Phytophthora species in the field.Previously,it was demonstrated that G protein a subunit is involved in regulation of chemotaxis and virulence in P.sojae,indicating that G protein signal play an important role in chemotaxis.However,little is known about its downstream effectors how G protein signal involves in the regulation of chemotaxis.In order to reveal the molecular mechanisms of G protein signal in chemotactic movement,we took Ga protein as the entry point,combining in vivo co-precipitation and mass spectrometry methods,and identified a series of candidate proteins of G protein signal in P.sojae.For example,protein kinase and protein phosphatase,small G proteins,calcium signal protein,etc.Based on the functional prediction and transcription patterns of candidate proteins,six candidate proteins were selected for following functional study.We found that the protein kinases PsSNF1 and calmodulin PsCAM1 could not been mutated and the mutants of protein phosphatase PsPPEFl and calcium channel protein PsCACC1 exhibit no obvious changes in virulence.Besides,the protein kinase PsYPK1 and small G protein PsRABB1 may involve in the regulation of virulence of P.sojae.Functional research of PsGPAl and PsYPKl in regulation of sporangia formation and virulence in Phytophthora sojae:Previously,we identified a PsGPA1 interaction candidate,PsYPK1,by in vivo immunoprecipitation,and found that this gene is essential for pathogenic process of P.sojae.In order to further investigate the biological relationship and regulatory mechanism between PsGPAl and PsYPK1,we confirmed the physical interaction between PsGPA1 and PsYPK1 through in vitro and in vivo methods,and the key region of interaction is the N-terminal region of PsYPK1.Subsequently,we conducted comprehensive phenotypic analyses of Ps YPK1 knockout mutant,and found that the production of sporangium and oospore are significantly repressed.Besides,PsYPK1 knockout mutant displays obvious defective mycelia growth and virulence.In addition,subcellular localization experiments showed that the N-terminal region of PsYPK1 affects its nuclear localization,which is necessary for sporangium formation.Interestingly,PsGPA1 can interfere with the normal localization pattern of PsYPK1 by interacting with its N-terminal region,thus inhibiting sporangium formation.This study identified an interaction protein of PsGPAl and revealed their molecular mechanism in co-regulation of sporangium development in P.sojae.This study identified a series of chemotaxis related genes through transcriptional analysis.Based on CRISPR/Cas9 mediated gene knockout system,two genes were proved to involve in regulating zoospore chemotaxis,which will provide important clues for the molecular mechanism underlying zoospore chemotaxis and help explain early interactions between phytophthora pathogens with the hosts,thus provide theoretical basis for diseases control.On the other hand,this study identified lots of downstream candidate proteins of G protein signal in P.sojae by co-precipitation and mass spectrometry analyses.Based on gene knockout and supplementary system,an indepth research was conduct in one of the candidates,protein kinase PsYPK1,which revealing that PsGPA1 interact with PsYPK1 to interfere with its the nuclear localization,thus inhibiting the expression levels of downstream sporangium formation related gene in P.sojae.This experimental system has broadened the depth of functional research and takes an important new step in phytophthora spieces.