Three Small Cysteine-Free Proteins (CFP1–3) Are Required for Insect-Pathogenic Lifestyle of Metarhizium robertsii

Unique CFP (cysteine-free protein; 120 aa) has been identified as an extraordinary virulence factor in Beauveria bassiana (Cordycipitaceae), a main source of wide-spectrum fungal insecticides. Its homologs exclusively exist in wide-spectrum insect pathogens of Hypocreales, suggesting their importance for a fungal insect-pathogenic lifestyle. In this study, all three CFP homologs (CFP1–3, 128–145 aa) were proven essential virulence factors in Metarhizium robertsii (Clavicipitaceae). Despite limited effects on asexual cycles in vitro, knockout mutants of cfp1, cfp2 and cfp3 were severely compromised in their capability for normal cuticle infection, in which most tested Galleria mellonella larvae survived. The blocked cuticle infection concurred with reduced secretion of extracellular enzymes, including Pr1 proteases required cuticle penetration. Cuticle-bypassing infection by intrahemocoel injection of ~250 conidia per larva resulted in a greater reduction in virulence in the mutant of cfp1 (82%) than of cfp2 (21%) or cfp3 (25%) versus the parental wild-type. Transcriptomic analysis revealed dysregulation of 604 genes (up/down ratio: 251:353) in the Δcfp1 mutant. Many of them were involved in virulence-related cellular processes and events aside from 154 functionally unknown genes (up/down ratio: 56:98). These results reinforce the essential roles of small CFP homologs in hypocrealean fungal adaptation to insect-pathogenic lifestyle and their exploitability for the genetic improvement of fungal insecticidal activity.