Antioxidant activities of four superoxide dismutases in Metarhizium robertsii and their contributions to pest control potential.

The superoxide dismutase (SOD) family of Metarhizium robertsii, a fungal insect pathogen, comprises six members functionally unknown yet, including Cu/ZnSODs (Sod1/5/6), MnSODs (Sod2/3), and FeSOD (Sod4). Here, we show a mitochondrial localization of Sod3 and Sod4 and a requirement of either sod4 or sod6 for the fungal life as suggested by an inability to be deleted. We found remarked roles of Sod1, Sod2, and Sod3 in sustaining antioxidant activity and the fungal potential against insect pests but no role of Sod5 in all examined phenotypes. Intracellular SOD activity decreased by 49% in Δsod1 and 22% in either Δsod2 or Δsod3. The decreased SOD activities concurred with altered enzymographs, in which one of two SOD-active bands in wild-type and rescued strains disappeared in Δsod1 rather than in Δsod2 and another band disappeared in Δsod3. Consequently, maximal cell sensitivity to superoxide anions generated by oxidant menadione occurred in Δsod1, followed sequentially by Δsod3 and Δsod2. The latter two mutants were more sensitive than Δsod1 to oxidant H2O2. Transcriptional analysis revealed partial compensation of one or two partner genes upregulated for the absence of sod1, sod2, or sod3 and full compensation of three partners largely upregulated for the absence of sod5, as well as differential expression of most catalase genes in each Δsod mutant. The three mutants also suffered defects in conidial thermotolerance, UVB resistance, and virulence. These findings unveil that, to adapt to different host spectra and habitats, some major SODs in M. roberstii are functionally differentiated from those known previously in Beauveria bassiana, a classic insect mycopathogen lacking Sod6.

Lysyl-tRNA synthetase (Krs) acts a virulence factor of Beauveria bassiana by its vital role in conidial germination and dimorphic transition.

Krs is a class II lysyl-tRNA synthetase (KRS) that is involved in cytosolic protein synthesis in budding yeast but functionally has not been explored in filamentous fungi. Previous transcriptomic analysis has revealed that a Krs-coding gene is likely involved in pathogenesis of Beauveria bassiana, a classic insect pathogen as a global source of fungal insecticides. Here, we show that Krs is localized in the cytoplasm of hyphal cells and acts as a substantial virulence factor in B. bassiana. Deletion of krs resulted in 10-h delayed germination, decreased (15 %) thermotolerance, and lowered (46 %) UV-B resistance of aerial conidia despite limited impact on conidiation capacity and slight or inconspicuous influence on radial growth on rich and minimal media with different carbon (10 sugars/polyols) and nitrogen (17 amino acids) sources. The deletion mutant suffered 58 % reduction in submerged blastospore yield (an index of in vitro dimorphic transition rate) in a minimal medium, and the reduction increased to 71 % in another trehalose-based medium mimic to insect haemolymph. In standardized bioassays, median lethal actions of Δkrs against Galleria mellonella larvae through the infections passing and bypassing the insect cuticle were prolonged to 192 and 153 h from wild-type median lethal time (LT50) estimates of 119 and 109 h, respectively. Microscopic examination revealed 2-d delayed presence of in vivo formed hyphal bodies in the haemolymph of the larvae infected by Δkrs in either mode. These findings unveil a vital role of Krs in conidial germination and dimorphic transition and its contribution to the fungal potential against arthropod pests.

Differential Roles for Six P-Type Calcium ATPases in Sustaining Intracellular Ca2+ Homeostasis, Asexual Cycle and Environmental Fitness of Beauveria bassiana.

A global insight into the roles of multiple P-type calcium ATPase (CA) pumps in sustaining the life of a filamentous fungal pathogen is lacking. Here we elucidated the functions of five CA pumps (Eca1, Spf1 and PmcA/B/C) following previous characterization of Pmr1 in Beauveria bassiana, a fungal insect pathogen. The fungal CA pumps interacted at transcriptional level, at which singular deletions of five CA genes depressed eca1 expression by 76-98% and deletion of spf1 resulted in drastic upregulation of four CA genes by 36-50-fold. Intracellular Ca2+ concentration increased differentially in most deletion mutants exposed to the stresses of Ca2+, EDTA chelator, and/or endoplasmic reticulum and calcineurin inhibitors, accompanied with their changed sensitivities to not only the mentioned agents but also Fe2+, Cu2+ and Zn2+. Liquid culture acidification was delayed in the Δspf1, Δpmr1 and ΔpmcA mutants, coinciding well with altered levels of their extracellular lactic and oxalic acids. Moreover, all deletion mutants showed differential defects in conidial germination, vegetative growth, conidiation capacity, antioxidant activity, cell wall integrity, conidial UV-B resistance and/or virulence. Our results provide the first global insight into differential roles for six CA pumps in sustaining intracellular Ca2+ level, asexual cycle and environmental fitness of B. bassiana.

Discovery of a new intravacuolar protein required for the autophagy, development and virulence of Beauveria bassiana.

High proportions of hypothetical proteins exist in genomic databases of fungi, including putative secretory proteins (PSPs) likely involved in fungal invasion and virulence. Here we characterize one of many PSPs revealed in the previous transcriptome of Beauveria bassiana (a fungal insect pathogen) infecting a global lepidopteran pest and name it vacuole-localized protein 4 (VLP4) because this small, domain-lacking protein (22.96 kDa) was specifically localized in the vacuoles of hyphal cells. Deletion of VLP4 resulted in repression of almost all genes acting in autophagy and central development pathways. Consequently, the deletion mutant formed no autophagosome in hyphal vacuoles and displayed severe defects in aerial conidiation. conidial hydrophobicity to the insect surface, and secretion of cuticle-degrading Pr1 proteases required for normal cuticle infection. Blastospore formation was inhibited in the submerged mutant culture mimic to insect haemolymph, and formation of hyphal bodies in vivo was delayed. The fungal virulence was attenuated in the absence of VLP4. These phenotypic defects were well restored by targeted gene complementation. Our findings unveil a vital role of VLP4 in B. bassiana and call attention to many more PSPs for new insights into the interactions of fungal insect pathogens with insects.

Effect of vacuolar ATPase subunit H (VmaH) on cellular pH, asexual cycle, stress tolerance and virulence in Beauveria bassiana.

Vacuolar ATPase (V-ATPase) is a conserved multi-subunit protein complex that mediates intracellular acidification in fungi. Here we show functional diversity of V-ATPase subunit H (BbVmaH) in Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of BbvmaH resulted in elevated vacuolar pH, increased Ca2+ level in cytosol but not in vacuoles, accelerated culture acidification and reduced accumulation of extracellular ammonia. Aerial conidiation and submerged blastospore production were largely delayed and reduced in the deletion mutant, respectively, accompanied with a significant delay in conidial germination, alterations of conidia and blastospores in morphology, size and/or density, and severe growth defects in minimal media with different carbon and nitrogen sources. Despite null responses to osmotic, oxidative and cell wall perturbing stresses, the deletion mutant showed increased sensitivity to Ca2+, Zn2+ and Cu2+ during growth while its conidia were less tolerant to a wet-heat stress at 45°C and UV-B irradiation. Intracellular glycerol and mannitol contents also decreased significantly. Its virulence to Galleria mellonella larvae was significantly attenuated when conidia were topically applied for normal cuticle infection or injected into haemocoel for cuticle-bypassing infection. All phenotypic changes were restored by targeted gene complementation. Our results indicate that BbVmaH plays an important role in sustaining not only vacuolar acidification but also cytosolic calcium accumulation, ambient pH homeostasis, in vitro asexual cycle and virulence in B. bassiana.