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.

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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 HO. 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.

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.

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