Functional Study And Comparative Transcriptome Analysis Of Genes Related To Asexual Development Of Phytophthora Sojae

Phytophthora sojae is a plant pathogen,which mainly causes Phytophthora root rot in soybean and causes serious harm to global soybean production.P.sojae is an oomycete belonging to the Stramenopiles.It is similar to diatoms and brown algae in evolution,but far away from fungi.There are significant differences in cell wall composition,physiological and biochemical metabolism,reproduction mode and genetic variation mechanism between them,which also makes P.sojae have a series of unique pathogenic mechanisms.Therefore,the study on the growth and pathogenic process of P.sojae is helpful to analyze the pathogenic mechanism of P.sojae and find potential drug targets.In this study,the Leucinerich repeat(LRR)-containing protein family obtained was used as the research object by P.sojae.In view of the important biological characteristics of producing zoospores in the asexual stage of P.sojae,seven candidate genes were functionally screened for genes related to asexual development through gene knockout and phenotypic analysis,and one of the key genes for flagellar development of zoospores,Ps FRP1,was functionally analyzed.In addition,transcriptome analysis was performed on the multiple stages of P.sojae life history and the knock-out mutants of six identified genes related to zoospore development,such as Ps FRP1,to reveal the specific regulatory genes and functional characteristics at different development stages,as well as the signaling pathways regulating zoospore development.The main research contents are as follows:Bioinformatics analysis of LRR protein family in P.sojae: It was found that the number of LRR-containing genes was expanding with the evolution of species by identifying the number of LRR-containing genes in 26 representative species of fungi,oomycetes,algae,plants and bacteria.It is suggested that LRR-containing genes differentiate and participate in more biological functions with the evolution of species.Phylogenetic tree construction of153 LRR-containing genes in P.sojae showed that LRR-containing genes in P.sojae were similar to Arabidopsis thaliana and could be divided into two groups.One group contained LRR motifs,namely LRR-only gene,which accounted for 58% of the LRR gene family in P.sojae.Other contains LRR motif and other functional motifs or domains(LRR-kinase,LRRothers),among which LRR-others account for 23% of The LRR gene family in P.sojae.Then,functional domains of LRR-containing genes specifically upregulated at the five stages of mycelium,zoospore,cyst germination,infection 3 h and infection 48 h were analyzed.Potential genes with functions at each stage of development were identified through comparative analysis of functional domains.Functional screening and analysis of candidate genes of LRR protein Ps FR P1 in P.sojae: Bioinformatics analysis showed that there were about 153 LRR gen es in the genome of P.sojae,of which 24 LRR proteins were similar to plant rece ptor protein kinases,including signal peptide,LRR structure,transmembrane domain and intracellular kinase domain.In this study,we focused on 120 genes,and selecte d 7 genes up-regulated in specific stages of asexual reproduction for gene function screening.Using CRISPR/Cas9 gene editing technology,seven gene replacement mut ants were obtained.Phenotypic analysis showed that the zoospores of Ps FRP1 muta nt could not move normally and tend to host.Scanning electron microscopy showedthat the zoospores of Ps FRP1 mutant were deformed and lost flagella,and the ger mination rate and pathogenicity of zoospores were also significantly reduced.After Ps FRP1 was supplemented in the mutant,the morphology of zoospores produced bythe transformants recovered to normal,and the phenotypes such as pathogenicity al so recovered.Compared with the other six LRR-only genes,Ps FRP1 contains not o nly 6 LRR domains at the N-terminal,but also a structural maintenance of chromos ome protein(SMC)domain at the C-terminal.The research further promoted the pr ogress on flagellar formation of zoospores.Comparative transcriptome analysis of Ps FRP1 and other five asexual-related genes of P.sojae: P.sojae formed sporangia in the asexual reproduction stage,then released zoospores and tended to the host,completed the rest within one minute,and then germinated into the infection stage.Therefore,studying the molecular mechanism of P.sojae in the sporangium and zoospore stage is an important scientific problem in the development of disease prevention and control.Based on the knock-out or silencing mutants of the six genes related to zoospore development(Ps FRP1,Ps GK4,Ps GPA1,Ps Hint1,Ps GPCR1 and Ps DRS2)identified in this study by previous laboratory members,the sporangia,zoospores stages samples were obtained,respectively,and RNA sample extraction and transcriptome sequencing were performed to study the molecular mechanisms regulating the interaction with host tissues during the sporangia stage,zoospore development and initial infection of P.sojae.The results showed that a large number of transcription factors,polysaccharide lyase,protein kinases and RNA-related gene families were involved in the development of sporangia and zoospores,which provided more clues for the study of signaling pathways in sporangia and zoospores development.