Identification Of A Beauveria Bassiana Binding Protein CI8A From Silkworm, Bombyx Mori

The entomopathogenic fungus Beauveria bassiana is being developed as an important bioinsecticide with broad host ranges, which depended on its high efficacy against insect pests and low virulence on non-target animals. B. bassiana invades insects mainly through cuticle by mechanical penetration and enzymolysis. Once the germ tubes get into the host hemocoel, they proliferate in the hemocoel releasing extracellular enzymes and bioactive metabolites with insecticidal properties. Coevolved with B. bassiana, the insects develop effective innate immune systems to fight off the fungi, containing cell-mediated response and humoral reponse including phagocytosis, nodulation, encapsulation, antimicrobial peptide syntheses via Toll or Imd pathway, and melanization of pathogen surfaces. Although the silkworm (Bombyx mori) draft genome sequence was completed in 2004, the mechanism of silkworm against fungal invasion is still unclear.In the present research, we identified four fungus-binding proteins via binding assay and MALDI-TOF-MS analysis. Previous studies showed that three of them including aβ-1,3-glucan recognition protein ((3GRP), a low molecular lipoprotein 30k (6G1) and an antimicrobial peptide have been reported to interact with fungi. We then focus our study on CI8A, which is a silkworm serpin and identified as a fungus-binding protein for the first time in this study. To understand the interaction between fungi and CI8A, we produced the CI8A fusion protein and antiCI8A antibody. The expressions of CI8A on both of the mRNA and protein levels were investigated by real-time RT-PCR and Western blotting, respectively, using the materials obtained from silkworm larvae challenged by B. bassiana. The results showed that the mRNA transcripts of CI8A were increased, while the CI8A proteins showed little variation.In addition, the binding of CI8 A protein with the major components of fungi cell wall containing chitin, curdlan and cellulose was proved by in vitro binding assay and Western blotting. This result indicated that CI8A might be a recognition factor in silkworm immune response. Finally, the weak inhibition of CI8A fusion protein on the growth of B. bassiana spores was observed by spore germination rate assay.