| Pythium guiyangense is a entomopathogenic oomycete found by professor Su from Guiyang Medical College of China.It has long-lasting mosquito killing period,wide range of mosquitoes killing,easy preparation of strains and safety to non-target organisms and can be a potential species for mosquito biological control.Previous studies have found that P.guiyangense could be infected hosts through cuticle and intestinal tract.It could secrete degrading enzymes,such as protease,chitinase and esterase,to degrade insect cuticle and peritrophic membrane during infection.In addition to protease,chitinase is also involved in many pathogenic bacteria in some studies,and is the key factor to affect the virulence of pathogenic bacteria.In plants,host plants can induce the production of chitinase when infected by pathogen.Chitinase can enhance the disease resistance of plants through suppressing the germination of fungal spores.Seed germination can induce chitinase releasing into the surrounding media to inhibit the growth and reproduction of fungi.In insects,chitinase can affect insect molting,growth and development.Chitinase can catalyze chitin to be N-acetylglucosamine to assist the infection of pathogens.In the process of pathogen infection,in order to avoid the recognition and successfully invade,pathogens will secrete effectors to interfere with the immune response of hosts.In recent years,major breakthroughs have been made in researches of intracellular effectors of oomycetes,including RxLR and CRN.CRN effectors are one of the few highly conserved proteins,which are widely existed in oomycetes such as Pythium,Peronospora and Albuginaceae.CRN effectors were first found in Phytophthora infestans and named for its role in cell crinkling and necrosis.The studies on CRN effectors mainly concentrate in plant pathogenic oomycetes,and less in mosquito pathogenic oomycetes.However,the genome-wide distribution,evolution and biological functions of chitinases are rarely reported in oomycetes.In this study,we systematically investigated GH18 chitinases of the mosquito pathogenic oomycete,P.guiyangense using bioinformatics and experimental assays.Totally 3 pairs of GH18 chitinase genes distributed in three distinct phylogenic clusters were identified from the genome of P.guiyangense which is consistent with that in plant pathogenic oomycetes.Further transcriptional analysis revealed that Pgchil/2 was highly expressed at the development stage,while Pgchi3/4 and Pgchi5/6 were up-regulated at the infection stages.The biological function analysis of chitinase genes using genetic transformation silencing method showed that silencing of Pgchil/2 resulted in the alteration of mycelial morphology and reduced zoospore production,without affecting on virulence.However,attenuation of Pgchi3/4 and Pgchi5/6 genes not only regulated oxidative stress responses,but also led to decreased infection rates to mosquito larvae.Taken together,this study provides a comprehensive overview of P.guiyangense chitinase family and reveals their diverse roles in the development,stress response,and virulence,which would provide insightful information on the molecular mechanism of chitinase in entomopathogenic pathogens.Twenty-eight CRN effectors were identified in P.guiyangense through bioinformatics analysis.Our laboratory has established a toxicity evaluation system in Sf9 cell line in the early stage,and screened out 5 CRN effectors(PgCRN31,PgCRN33,PgCRN37,PgCRN40 and PgCRN44)which had significant toxicity.In this study,we established a toxicity screening system in C6/36 cell line to verify the toxicity of CRN effectors.In order to further study the mechanism of CRN effectors,PgCRN31 was selected as the next research object after the comprehensive analysis.GFP-PgCRN31 was expressed in C6/36 cells,and 8 candidate genes were selected through immunoprecipitation,non-gelatinized identification and bioinformatics analysis. |
PDF Full Text Request