Function Analysis Of Phytophthora Sojae PsBAGs And PsMYB16

Root rot caused by Phytophthora sojae is one of the most serious diseases on soybean,causing huge economic losses,seriously endangering food security.Due to the high field variability of P.sojae,it is very important to continuously and deeply study the pathogenic mechanism of P.sojae and to find suitable control targets.BAG（B-cell lymphoma2-associated athanogene）proteins are widely distributed in multicellular species and plays an important role in the growth,development and stress response of organisms;MYB transcription factors are key factors,in the gene expression regulatory network,are important for the growth and pathogenicity of the pathogen.However,there are few studies about BAG proteins and MYB transcription factors in oomecetes.In this study,the BAG and MYB genes of P.sojae were knocked out,the phenotype of the knock-out mutants were determined,and the functions of P.sojae BAG and MYB were preliminarily explored to provide a theoretical basis for clarifying the pathogenic mechanism of Phytophthora sojae.The specific research content is as follows:Knockout of PsBAGs and phenotypic analysis of PsBAGs knock-out mutants:Through bioinformatics analysis,three BAG protein-coding genes were identified in the P.sojae genome.The transcription pattern of PsBAGs was analyzed.It was found that the three PsBAGs proteins were differentially expressed during the growth and infection process of P.sojae.At the time of cyst germination and the early and late stages of the infection stage,the expression level of PsBAGs is higher.The triple knock-out mutant of PsBAGs（T13,T14,T27）is obtained through the CRISPR/Cas9 system,the biologically phenotypic of knock-out mutans showed the PsBAGs knock-out mutants had no significant difference in sporangia and zoospore number compared with wild type,but the germination rate of cysts was significantly lower.Soybean hypocotyl inoculation experiments showed that the PsBAGs knock-out mutants had significantly reduced pathogenicity.The response of the knock-out mutant to abiotic stress was determined,and it was found that knock-out of the BAG gene does not affect the sensitivity of P.sojae to oxidative stress,osmotic stress,and salt stress,but the mutants was significantly sensitive to endoplasmic reticulum stress caused by Tunicamycin and DTT.These results indicate that PsBAGs are involved in the germination,infection,pathogenic process and the response process to endoplasmic reticulum stress of P.sojae.Knockout of MYB transcription factor PsMYB16 and phenotypic analysis of PsMYB16 knock-out mutants:In the previous research,a P.sojae leucine-rich repeat receptor like kinase Ps LRR-RLK8 was identified.Ps LRR-RLK8 knock-out mutants showed a decrease in the number of zoospores,a decrease in virulence,and an increase in the sensitivity to metalaxyl and H₂O₂.The PsMYB16 transcription level was found to be significantly down-regulated in the Ps LRR-RLK8 knock-out mutant.It has been reported that MYB transcription factor is involved in the regulation of zoospore development of P.sojae.To verify whether PsMYB16 is involved in the zoospore development and infection process of P.sojae,we use the CRISPR/Cas9 system to knocked out PsMYB16,and found that the knock-out mutants has no significant difference in growth rate,sporangia and zoospore formation,and pathogenicity to soybean.The susceptibility to H₂O₂ and metalaxyl was tested,and it was found that knocking out PsMYB16 did not affect the sensitivity of P.sojae to H₂O₂and metalaxyl.These results indicated that PsMYB16 has no effect on the vegetative growth,asexual reproduction,infection process and stress response of P.sojae.In conclusion,the results show that PsBAGs is a key factor in the endoplasmic reticulum stress response of and involved in regulating the zoospores germination and infection process,whereas PsMYB16 does not affect the hyphae growth and zoospore formation,stress response,and pathogenic processes of P.sojae.