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

Abstract

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 theircapability fornormal cuticle infection, in whichmost 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.

Roles of BrlA and AbaA in Mediating Asexual and Insect Pathogenic Lifecycles of Metarhizium robertsii

“…In a previous study to characterize the key transcription factor Msn2 downstream of the MAPK Hog1 signaling cascade, the counts of identified DEGs were 3% smaller and 12% greater in the Δ msn2 mutant’s responses of M. robertsii than of B. bassiana to oxidative stress and heat shock, respectively [ 49 ]. In addition, up to 1818 DEGs (1006:801) were identified from the deletion mutant’s transcriptome of cfp , a gene encoding small cysteine-free protein confirmed as a virulence factor in B. bassiana [ 50 ], while only 604 DEGs (251:353) were found in the deletion mutant’s transcriptome of a cfp homolog also acting as a virulence factor in M. robertsii [ 46 ]. In the present and previous studies, gene expression networks either controlled by BrlA and AbaA for asexual development and those controlled by Msn2 for stress responses or by CFP required for virulence are largely different between M. robertsii and B. bassiana .…”

“…A status of each strain’s hemocoel colonization vital for mycosis development and host mummification was examined as described previously [ 46 ]. Briefly, hemolymph samples were taken from surviving larvae 6 days post-NCI and 3 days post-CBI.…”

“…Four-day-old cultures of the Δ brlA , Δ abaA -1, and WT strains (three cultures per strain) grown on cellophane-overlaid PDA plates at the optimal regime were sent to Lianchuan BioTech Company (Hangzhou, China) for construction and analysis of brlA - and abaA -specific transcriptomes as described previously [ 31 , 46 ]. Clean tags generated by filtration of all raw reads from sequencing were mapped to the M. robertsii genome [ 39 ].…”

Essential Role of WetA, but No Role of VosA, in Asexual Development, Conidial Maturation and Insect Pathogenicity of Metarhizium robertsii

“…The quality properties of conidia collected from the 15-day-old cultures were assessed as indices of viability [GT 50 (h) at 25°C], hydrophobicity in an aqueous-organic system, and LD 50 (J/cm 2 ) for resistance to UVB irradiation (weighted wavelength: 312 nm), as described previously ( 16 , 17 ). SEM images of conidial surfaces were collected to show microstructures of hydrophobin rodlet bundles.…”

“…A small cysteine-free protein (CFP), as a high virulence factor, differentially regulates 1,818 genes in B. bassiana ( 16 ), whereas only 604 genes are differentially regulated by its homolog, also proven as a virulence factor in M. robertsii ( 17 ). Therefore, B.…”