Essential Roles of Two FRQ Proteins (Frq1 and Frq2) in Beauveria bassiana's Virulence, Infection Cycle, and Calcofluor-Specific Signaling.

Two FRQ proteins (Frq1 and Frq2) distinct in molecular mass and structure coexist in Beauveria bassiana, an asexual insect-pathogenic fungus. Frq1 and Frq2 have been proven to have opposite nuclear rhythms that can persistently activate developmental activator genes and hence orchestrate nonrhythmic conidiation in vitro under light or in darkness. Here, we report the essentiality of either FRQ, but Frq2 being more important than Frq1, for the fungal virulence and infection cycle. The fungal virulence was attenuated significantly more in the absence of frq2 than in the absence of frq1 through either normal cuticle infection or cuticle-bypassing infection by intrahemocoel injection, accompanied by differentially reduced secretion of Pr1 proteases required for the cuticle infection and delayed development of hyphal bodies in vivo, which usually propagate by yeast-like budding in the host hemocoel to accelerate insect death from mycosis. Despite insignificant changes in radial growth under normal, oxidative, and hyperosmotic culture conditions, conidial yields of the Δfrq1 and Δfrq2 mutants on insect cadavers were sharply reduced, and the reduction increased with shortening daylight length on day 9 or 12 after death, indicating that both Frq1 and Frq2 are required for the fungal infection cycle in host habitats. Intriguingly, the Δfrq1 and Δfrq2 mutants showed hypersensitivity and high resistance to cell wall-perturbing calcofluor white, coinciding respectively with the calcofluor-triggered cells' hypo- and hyperphosphorylated signals of Slt2, a mitogen-activated protein kinase (MAPK) required for mediation of cell wall integrity. This finding offers a novel insight into opposite roles of Frq1 and Frq2 in calcofluor-specific signal transduction via the fungal Slt2 cascade.IMPORTANCE Opposite nuclear rhythms of two distinct FRQ proteins (Frq1 and Frq2) coexisting in an asexual fungal insect pathogen have been shown to orchestrate the fungal nonrhythmic conidiation in vitro in a circadian day independent of photoperiod change. This paper reports essential roles of both Frq1 and Frq2, but a greater role for Frq2, in sustaining the fungal virulence and infection cycle since either frq1 or frq2 deletion led to marked delay of lethal action against a model insect and drastic reduction of conidial yield on insect cadavers. Moreover, the frq1 and frq2 mutants display hypersensitivity and high resistance to cell wall perturbation and have hypo- and hyperphosphorylated MAPK/Slt2 in calcofluor white-triggered cells, respectively. These findings uncover a requirement of Frq1 and Frq2 for the fungal infection cycle in host habitats and provide a novel insight into their opposite roles in calcofluor-specific signal transduction through the MAPK/Slt2 cascade.

The DUF1996 and WSC domain-containing protein Wsc1I acts as a novel sensor of multiple stress cues in Beauveria bassiana.

Wsc1I homologues featuring both an N-terminal DUF1996 (domain of unknown function 1996) and a C-terminal WSC (cell wall stress-responsive component) domain exist in filamentous fungi but have never been functionally characterized. Here, Wsc1I is shown to localize in the vacuoles and cell wall/membrane of the insect mycopathogen Beauveria bassiana and hence linked to cell membrane- and vacuole-related cellular events. In B. bassiana, deletion of Wsc1I resulted in marked increases of hyphal and conidial sensitivities to hyperosmotic agents, oxidants, cell wall perturbing chemicals, and metal cations (Cu2+ , Zn2+ , Fe2+ , and Mg2+ ) despite slight impact on normal growth and conidiation. Conidia produced by the deletion mutant showed not only reduced tolerance to both 45°C heat and UVB irradiation but also attenuated virulence to a susceptible insect through normal cuticle infection or cuticle-bypassing infection. Importantly, phosphorylation of the mitogen-activated protein kinase Hog1 was largely attenuated or nearly abolished in the Wsc1I-free cells triggered with hyperosmotic, oxidative, or cell wall perturbing stress. All changes were well restored by targeted gene complementation. Our findings highlight a novel role of Wsc1I in sensing multiple stress cues upstream of the Hog1 signalling pathway and its pleiotropic effects in B. bassiana.

P-type Na+/K+ ATPases essential and nonessential for cellular homeostasis and insect pathogenicity of Beauveria bassiana.

ENA1 and ENA2 are P-type IID/ENA Na+/K+-ATPases required for cellular homeostasis in yeasts but remain poorly understood in filamentous fungal insect pathogens. Here, we characterized seven genes encoding five ENA1/2 homologues (ENA1a-c and ENA2a/b) and two P-type IIC/NK Na+/K+-ATPases (NK1/2) in Beauveria bassiana, an insect-pathogenic fungus serving as a main source of fungal insecticides worldwide. Most of these genes were highly responsive to alkaline pH and Na+/K+ cues at transcription level. Cellular Na+, K+ and H+ homeostasis was disturbed only in the absence of ena1a or ena2b. The disturbed homeostasis featured acceleration of vacuolar acidification, elevation of cytosolic Na+/K+ level at pH 5.0 to 9.0, and stabilization of extracellular H+ level to initial pH 7.5 during a 5-day period of submerged incubation. Despite little defect in hyphal growth and asexual development, the Δena1a and Δena2b mutants were less tolerant to metal cations (Na+, K+, Li+, Zn2+, Mn2+ and Fe3+), cell wall perturbation, oxidation, non-cation hyperosmolarity and UVB irradiation, severely compromised in insect pathogenicity via normal cuticle infection, and attenuated in virulence via hemocoel injection. The deletion mutants of five other ENA and NK genes showed little change in vacuolar pH and all examined phenotypes. Therefore, only ENA1a and ENA2b evidently involved in both transmembrane and vacuolar activities are essential for cellular cation homeostasis, insect pathogenicity and multiple stress tolerance in B. bassiana. These findings provide a novel insight into ENA1a- and ENA2b-dependent vacuolar pH stability, cation-homeostatic process and fungal fitness to host insect and environment.

Subtilisin-like Pr1 proteases marking the evolution of pathogenicity in a wide-spectrum insect-pathogenic fungus.

Subtilisin-like Pr1 proteases of insect-pathogenic fungi are a large family of extracellular cuticle-degrading enzymes that presumably determine a capability of hyphal invasion into insect hemocoel through normal cuticle infection, but remain poorly understood although often considered as virulence factors for genetic improvement of fungal potential against pests. Here, we report that not all of 11 Pr1 family members necessarily function in Beauveria bassiana, an ancient wide-spectrum pathogen evolved insect pathogenicity ~200 million years ago. These Pr1 proteases are phylogenetically similar to or distinct from 11 homologues (Pr1A-K) early named in Metarhizium anisopliae complex, a young entomopathogen lineage undergoing molecular evolution toward Pr1 diversification, and hence renamed Pr1A1/A2, Pr1B1-B3, Pr1 C, Pr1F1-F4,4 and Pr1 G, respectively. Multiple analyses of all single gene-deleted and rescued mutants led to the recognition of five conserved members (Pr1C, Pr1G, Pr1A2, Pr1B1, and Pr1B2) contributing significantly to the fungal pathogenicity to insect. The conserved Pr1 proteases were proven to function only in cuticle degradation, individually contribute 19-29% to virulence, but play no role in post-infection cellular events critical for fungal killing action. Six other Pr1 proteases were not functional at all in either cuticle degradation during host infection or virulence-related cellular events post-infection. Therefore, only the five conserved proteases are collectively required for, and hence mark evolution of, insect pathogenicity in B. bassiana. These findings provide the first referable base for insight into the evolution of Pr1 family members in different lineages of fungal insect pathogens.