Host-dependent contributions of the Cfcdp1 protease gene to virulence in the entomopathogenic fungus Cordyceps fumosorosea.

BACKGROUND: The genomes of broad host range insect pathogenic fungi, including Cordyceps fumosorosea, encode for a suite of secreted proteases implicated in targeting, penetration, and degradation of the host exoskeleton or cuticle. These cuticle-degrading proteases act as critical virulence factors, but their functions within the biological context, particularly in relation to host specificity, remain poorly characterized. RESULT: A C. fumosorosea protease gene, Cfcdp1, was identified and a targeted gene-knockout strain constructed. Minor growth defects were observed for the ΔCfcdp1 strain when compared to the wild-type parent and complemented (ΔCfcdp1::Cfcdp1) strains, with delayed and decreased sporulation noted for the mutant. Decreased subtilisin-like protease activity was seen for the ΔCfcdp1 strain, although total secreted protease activity was similar between the mutant and wild-type strains. Insect bioassays using whitefly, Bemisia tabaci, and cabbageworm, Pieris rapae, showed decreased infectivity, i.e. 2.4-3.4-fold increase in lethal dose (LC50 ) and an increased time to death (LT50 ), for the ΔCfcdp1 strain. In contrast, insect bioassays using the diamondback moth, Plutella xylostella, or the brown planthopper, Nilaparvata lugens, showed increased infectivity, i.e. a 3-5-fold decrease in LC50 , and a decreased LT50 . Differential effects were also seen on the fecundity of B. tabaci infected by the different fungal strains. CONCLUSION: These data reveal host-dependent effects of a protease implicated in cuticle degradation on C. fumosorosea virulence. The implications of these findings in suggesting context-dependent requirements of cuticle-degrading enzymes and their potentially differential roles in mediating virulence towards different hosts are discussed. © 2019 Society of Chemical Industry.

The Ifchit1 chitinase gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea.

The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The ΔIfchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 °C compared to the wild type and complemented (ΔIfchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 °C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the ΔIfchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC 50 (threefold to fourfold) and a significantly delayed time to death, LT 50 from 3 to 6 days, for the ΔIfchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.