A Small Cysteine-Free Protein Acts as a Novel Regulator of Fungal Insect-Pathogenic Lifecycle and Genomic Expression.

Small secreted proteins (SSPs), particularly cysteine-rich proteins secreted during fungal infection, comprise virulence effectors in plant-pathogenic fungi but remain unknown in insect-pathogenic fungi. We report here that only a small cysteine-free protein (CFP) is indispensable for insect pathogenicity of among 10 studied SSPs (99 to 274 amino acids [aa]), including seven hypothetical proteins containing 0 to 12 Cys residues. CFP (120 aa) features an N-terminal signal peptide (residues 1 to 17), a nuclear localization signal motif (residues 24 to 57), and no predictable domain. Its homologs exist exclusively in insect-pathogenic Cordycipitaceae and Clavicipitaceae. Fluorescence-tagged CFP fusion protein was localized in the nucleus but extracellularly undetectable, suggesting an inability for CFP to be secreted out. Disruption of resulted in abolished pathogenicity via normal cuticle infection, attenuated virulence via hemocoel injection, compromised conidiation capacity versus little growth defect, impaired conidial coat, blocked secretion of cuticle-degrading enzymes, impeded proliferation , disturbed cell cycle, reduced stress tolerance, and 1,818 dysregulated genes (genomic 17.54%). Hundreds of those genes correlated with phenotypic changes observed in the disruption mutant. Intriguingly, nearly 40% of those dysregulated genes encode hypothetical or unknown proteins, and another 13% encode transcription factors and enzymes or proteins collectively involved in genome-wide gene regulation. However, purified CFP showed no DNA-binding activity in an electrophoretic mobility shift assay. These findings unveil that CFP is a novel regulator of fungal insect-pathogenic life cycle and genomic expression and that cysteine richness is dispensable for distinguishing virulence effectors from putative SSPs in Small cysteine-rich proteins secreted during plant-pathogenic fungal infection comprise virulence effectors. Our study confirms that only a cysteine-free protein (CFP) is determinant to insect-pathogenic fungal virulence among 10 small putatively secreted proteins containing 0 to 12 Cys residues. Disruption of abolished insect pathogenicity and caused not only a series of compromised cellular events associated with host infection and disease development but also dysregulation of 1,818 genes, although no DNA-binding activity was detected in purified CFP samples. Nearly 13% of those genes encode transcription factors and enzymes or proteins collectively involved in transcriptional regulation. Altogether, CFP serves as a novel regulator of the fungal insect-pathogenic life cycle and genomic expression. Cysteine richness is dispensable for distinguishing virulence effectors from the fungal SSPs.