title: TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.
abstract: Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Vitamin D Research Institute, Shannxi University of Technology, Hanzhong 723001, Shannxi Province, China.
authors.forename: Youshan
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Huawei
authors.initials: H
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Huawei
authors.initials: H
authors.lastname: He
authors.affiliation: School of Management, Shannxi University of Technology, Hanzhong 723001, Shannxi Province, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
authors.forename: Zhonghuai
authors.initials: Z
authors.lastname: Xiang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Electronic address: xiaqy@swu.edu.cn.
authors.forename: Qingyou
authors.initials: Q
authors.lastname: Xia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D052982:Beauveria / Q000201:enzymology*; D012831:Bombyx / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007814:Larva / Q000382:microbiology; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D055550:Protein Stability; D011815:R Factors
chemical_list: D005656:Fungal Proteins; D019476:Insect Proteins; D011480:Protease Inhibitors
keywords: Invasive fungi; Protease inhibitors; Resistance factors; Silkworm defenses