Identification And Functional Study Of Small Secretory Proteins And Expansin Proteins From Phytophthora Capsici

Phytophthora capsici is a very harmful plant pathogenic oomycete,which can infect hundreds of vegetables and other horticultural crops.Phytophthora capsici has a fast spreading speed,a high frequency of mutation,and is prone to outbreaks and disasters,causing huge losses to social and economic activities every year.In recent decades,the incidence and severity of the disease have increased significantly,and there is an urgent need to crack its pathogenic mechanism to provide a theoretical and practical basis for the prevention and control of P.capsici.In the zigzag model of the interaction between plants and pathogens,plant cells can resist pathogen infection and effector interference by identifying pathogen-related pattern molecules PAMP to trigger PTI(PAMP-triggered immunity)and identifying effector to trigger ETI(Effector-Triggered Immunity)response,and pathogens will evolve new effectors can evade recognition or interfere with immune defense responses and successfully invade plants.In this long arms race,the effector proteins of pathogens have undoubtedly become the best research object for us to understand the interaction mechanism between plants and pathogens.In this study,we screened small secretory proteins and expansin proteins that play an important role in the interaction between P.capsici and plants,and conducted preliminary studies on their functions.This work can provide a theoretical basis for revealing the pathogenic mechanism of Phytophthora,and will lay the foundation for our understanding of the interaction mechanism between pathogens and plants.The specific work of this research is as follows:Identification and function study of Phytophthora capsica small secretory proteins:In individual studies,it has been confirmed that some small proteins secreted by plant pathogens are key virulence factors.As far as we know,the whole genome secretion of small proteins of Phytophthora pathogens has not been publicly reported,and many small proteins related to the disease process have yet to be determined.Therefore,we hope to identify the small secreted proteins hidden in the genome of P.capsici that are involved in the pathogenic process,to explore the function and mechanism of pathogenicity-related small proteins in Phytophthora.In the early stage,the laboratory used bioinformatics analysis technology to predict in the P.capsici LT263 genome based on whether it contains signal peptides,transmembrane domains,and conservation.169 small secreted proteins with a coding length of less than 250 amino acids were obtained.On this basis,we use the induction of N.benthamiana cell death as a screening indicator,two genes Pc SSP4 and Pc SSP6 that can induce cell death of N.benthamiana were successfully identified,suggesting that these two genes may play an important role in the interaction between P.capsici and plants.After analyzing the expression patterns of genes Pc SSP4 and Pc SSP6 in the infection stage of Phytophthora capsici,we found that the gene Pc SSP4 showed a significant up-regulated expression trend in the early stage of infection,and the expression multiple is about 7 times.while the gene Pc SSP6 showed a lower overall expression during the infection stage.Finally,the two genes were knocked out separately,and it was found that the knockout of Pc SSP4 gene reduced the pathogenicity of P.capsici,indicating that Pc SSP4 may be involved in the infection process of P.capsici as a new virulence factor.In the Pc SSP6 gene knockout experiment,it was found that the knockout of this gene had no effect on the pathogenicity of P.capsici,but accelerated the growth rate of P.capsici.This gene may play an important role in the growth and development of P.capsici,and its function in the process of interaction between P.capsici and plants requires further study.Identification and function study of phytophthora capsici expansin proteins: The expansin-like genes of microorganisms such as bacteria and fungi are obtained from plants through horizontal gene transfer(HGT).The expansin-like proteins in pathogens such as bacteria,fungi and nematodes can participate in the pathogenic process and induce plant resistance.However,this type of gene has not been reported in detail in oomycetes,so we conducted related studies on the expansin-like gene involved in the pathogenic process of P.capsici.In the early stage,our laboratory used bioinformatics analysis technology to predict in the genome of P.capsici LT263,and obtained 12 genes containing expansin domains.On this basis,using the induction of N.benthamiana cell death as a screening indicator,we successfully identified a gene Pc EXLX11 that can induce cell death of N.benthamiana.It is implied that this gene may play an important role in the interaction between P.capsici and plants.Next,we analyzed the expression pattern of the gene Pc EXLX11 during the infection stage of P.capsici and found that the gene has slightly up-regulated express trends in the early stage of infection,and the expression multiple is less than 2 times.Finally,we knocked out and overexpressed the gene Pc EXLX11,and found that the knockout strain of this gene enhanced the pathogenicity of P.capsici,while the overexpression strain reduced the pathogenicity,indicating that Pc EXLX11 participates in the interaction process between P.capsici and plants,and can stimulate plant immune response.In summary,we have identified for the first time that the small protein genes Pc SSP4 and Pc SSP6 and the expansin-like gene Pc EXLX11 of P.capsici can induce cell death of N.benthamiana and we have respectively revealed the roles of these three genes in the pathogenic process.The research results help to reveal the interaction mechanism between Phytophthora and host plants,provide a theoretical basis for revealing the pathogenic mechanism of Phytophthora,and also have important significance for the formulation of green disease prevention and control technologies.