Functional Research And Analysis Of Cell Surface Protein Gene Bbcsp1 In Beauveria Bassiana

Beauveria bassiana is an important entomopathogenic fungus that widely distributes in nature and acts as a parasite with a broad host range.It could lead to the wild insects infections and epidemics,and some strains also cause silkworm white muscardine disease.The silkworm cuticle,as the first natural barrier to prevent the infection,plays an important role in the immune defense against the invasion of entomopathogenic fungus.In the process of adhesion and penetration to the epidermis of silkworm,a series of physiological,pathological and immune responses occurred in the silkworm body,then B.bassiana also took corresponding gene-specific strategies.Therefore,the search and identification of the relevant genes and their functions involved in this process is significance not only to further reveal the pathogenic mechanism of B.bassiana,but also to lay a foundation for finding new strategies to control white muscardine disease of silkworm,Bombyx mori.B.bassiana GXsk1011 is a hyper-virulent strain isolated from Bombyx mori by our laboratory group.In our previous study by B.bassiana GXsk1011 transcriptomic analysis in response to the epidermis of three different insects,the cell surface protein gene Bbcsp1（BBA＿09174）was annotated and showed up-regulated expression significantly in response to the epidermis of Bombyx mori,comparing with that in response to the epidermis of both Helicoverpa armigera and Clanis bilineata.Meanwhile,the cell surface proteins are different from the cell wall proteins in B.bassiana from some extent,whose structure and function are also rarely reported.In this study,Bbcsp1 as a test object was cloned and analyzed by bioinformatics,and its mutant strains were constructed by genetic engineering.Then by comparing the biological phenotypic differences of wild strains and mutant strains,the possible biological function of Bbcsp1 could be investigated.The main results are as follows:1.The coding sequences of the cell surface protein gene Bbcsp1 were PCR-amplified from B.bassiana GXsk1011.The bioinformatics analysis showed that the ORF of Bbcsp1 was 1053 bp long and encoded 311 amino acids,which were rich in glycine and alanine.And Bbcsp1 was predicted a hydrophilic protein with no obvious signal peptide,no transmembrane domain and glycosylation site.The predicted conserved domain was DUF3129（domains of unknown function 3129）,whose definite properties and functions have not been clearly identified in the database.2.Bbcsp1 knockout and overexpression vector were constructed and transformed into B.bassiana GXsk1011 by ATMT.Based on homologous replacement and screening by PCR and RT-PCR,the Bbcsp1 knockout mutant strain（ΔBbcsp1）and overexpression mutant strain（OE-Bbcsp1）were successfully obtained to follow-up experiments.3.After knock out of Bbcsp1,the growth and development of B.bassiana were affected,whose formation of sporulation structure was delayed and aerial mycelia became thinner.The conidia germination rate,tolerance to hyperosmotic stress and the ability to resist heat of conidia and hyphae were all decreased.In the insect bioassays,when the insect was topically infected with 10⁶ conidia/mL conidia coating the Bombyx mori epidermis,the LT₅₀ value ofΔBbcsp1,that was 154.38±5.92 h,was significant longer than that of WT which was 108.39±8.19 h.While the conidia were injected into blood cavity,LT₅₀ values indicated that no obvious difference was observed between WT andΔBbcsp1,the LT₅₀ value was 54.12±0.77 h and 55.36±2.50 h,respectively.4.When Bbcsp1 was overexpressed in B.bassiana,the aerial mycelia and sporulation structure were more abundant.The germination rate and heat tolerance of conidia were enhanced but not significantly,and the heat tolerance of mycelia showed a decreasing trend,while the decline was less than that ofΔBbcsp1.In the insect bioassays,LT₅₀ value of OE-Bbcsp1 which was 85.74±1.10 h was significantly shortened compared with the wild strain by topical infection.Likewise,there was no obvious difference between mutants OE-Bbcsp1 and WT during injection assays,and the LT₅₀ value of them was 52.94±1.18 h and 55.36±2.50 h,respectively.5.By analysis of the experimental results comprehensively,Bbcsp1 was involved in the conidia germination,permeability and heat resistance of B.bassiana.The overexpression of Bbcsp1 had no significant improved effect on the germination rate of conidia,indicating that Bbcsp1 could not be up-regulated to improve the germination rate to enhance virulence.In the insect injection assays,no virulence difference was observed between WT and mutants,however,a significant difference was evident between WT and mutants when inoculation method was instead of natural infection to the insect surface.That indicated Bbcsp1 of B.bassiana could take action during the interaction between conidia and host epidermis.In addition,Bbcsp1 was predicted non-enzymatic that could not participate in degradation of cuticle to enhance virulence.In combination with our previous results,Bbcsp1 showed a higher expression in response to silkworm cuticle than other insects cuticle,it was speculated that Bbcsp1 of B.bassiana GXsk1011 might be a adhesive protein with certain host recognition function.