Essential Role of COP9 Signalosome Subunit 5 (Csn5) in Insect Pathogenicity and Asexual Development of Beauveria bassiana

Abstract

Csn5 is a subunit ofthe COP9/signalosome complex in model fungi. Here, we report heavier accumulation of orthologous Csn5 in the nucleus than in the cytoplasm and its indispensability to insect pathogenicity and virulence-related cellular events of Beauveria bassiana. Deletion of csn5 led to a 68% increase in intracellular ubiquitin accumulation and the dysregulation of 18 genes encoding ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and ubiquitin-specific proteases, suggesting the role of Csn5 in balanced ubiquitination/deubiquitination. Consequently, the deletion mutant displayed abolished insect pathogenicity, marked reductions in conidial hydrophobicity and adherence to the insect cuticle, the abolished secretion of cuticle penetration-required enzymes, blocked haemocoel colonisation, and reduced conidiation capacity despite unaffected biomass accumulation. These phenotypes correlated well with sharply repressed or abolished expressions of key hydrophobin genes required for hydrophobin biosynthesis/assembly and of developmental activator genes essential for aerial conidiation and submerged blastospore production. In the mutant, increased sensitivities to heat shock and oxidative stress also correlated with reduced expression levels of several heat-responsive genes and decreased activities of antioxidant enzymes. Altogether, Csn5-reliant ubiquitination/deubiquitination balance coordinates the expression of those crucial genes and the quality control of functionally important enzymes, which are collectively essential for fungal pathogenicity, virulence-related cellular events, and asexual development.

Genome-Wide Insight into Profound Effect of Carbon Catabolite Repressor (Cre1) on the Insect-Pathogenic Lifecycle of Beauveriabassiana

“…As a result of qPCR analysis, either hyd1 or hyd2 expression was upregulated in ∆cre1 relative to the WT strain despite differential suppression of three other genes (hyd3-5) that encode hydrophobin-like proteins but remain functionally unknown yet (Figure 4F). For the mutant, no change in conidial hydrophobicity was consistent with an intact outermost rodlet-bundle layer of conidial coat, which showed a 'bold' phenotype when hyd1 and hyd2 were both deleted [39] or sharply repressed in the absence of a gene encoding the cysteine-free protein CFP [45], the cosubunit Ssr4 of chromatin-remodeling SWI/SNF and RSC complexes [50] or the fifth subunit Csn5 of COP9 signalosome complex [51]. The subtle change in the morphology of assembled rodlet bundles were likely associated with upregulated expression of hyd1 and hyd2 required for hydrophobin biosynthesis and assembly in B. bassiana.…”

“…For each strain, conidial hydrophobicity crucial for conidial adherence to insect cuticle [41] was assayed in a diphasic (aqueous-organic) system, followed by scanning electron microscopic (SEM) examination for the presence/absence of hydrophobin rodlet bundles in the outermost layer of conidial coat, as described previously [45,50,51]. To assess the antioxidant capability of conidia important for response to superoxide reactive species (ROS) generated by host immunity defense during NCI or CBI [52], 100 µL aliquots of a 10 6 conidia/mL suspension were spread evenly onto the plates of trehalose-peptone agar (TPA, i.e., CDA amended with 3% trehalose and 0.3% peptone as carbon and nitrogen sources respectively) alone (control) or supplemented with gradient concentrations of menadione (0.01-0.04 mM) and H 2 O 2 (1-4 mM), respectively, followed by an incubation at 25 • C. From 4 h incubation onwards, germinated and non-germinated conidia were counted hourly from three microscopic fields of each plate until no more germination change.…”

“…both deleted [39] or sharply repressed in the absence of a gene encoding the cysteine-free protein CFP [45], the cosubunit Ssr4 of chromatin-remodeling SWI/SNF and RSC complexes [50] or the fifth subunit Csn5 of COP9 signalosome complex [51]. The subtle change in the morphology of assembled rodlet bundles were likely associated with upregulated expression of hyd1 and hyd2 required for hydrophobin biosynthesis and assembly in B. bassiana.…”

Fungal insecticidal activity elevated by non‐risky markerless overexpression of an endogenous cysteine‐free protein gene in Beauveria bassiana

“…Several cellular events associated with NCI, hemocoel colonization and fungal response to host immune defense were examined for in-depth insight into a virulence difference between the strain OES3 and the parental WT as described previously. 38,39 First, as a trait crucial to NCI initiation, conidial adherence to insect cuticle was assayed on locust (Locusta migratoria) hind wings pre-treated as described previously. 40 Briefly, conidia were suspended in sterile water not containing any surfactant (to avoid a possible interference with conidial surface trait) through thorough vortex and standardized to 10 7 conidia mL −1 , followed by spotting 5-μL aliquots of the suspension on the central areas of hind wings attached to 0.7% water agar.…”

“…Several cellular events associated with NCI, hemocoel colonization and fungal response to host immune defense were examined for in‐depth insight into a virulence difference between the strain OES3 and the parental WT as described previously 38,39 . First, as a trait crucial to NCI initiation, conidial adherence to insect cuticle was assayed on locust ( Locusta migratoria ) hind wings pre‐treated as described previously 40 .…”