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Summary: pH is one of the important environmental factors that affect the growth, development and pathogenicity of postharvest pathogen. The transcription factor PacC dominates the pH signal pathway. PacC in Trichothecium roseum showed three typical conserved zinc finger domains and closest homology to Fusarium graminearum. T. roseum increased the environmental pH both in vitro and in vivo. Expression patterns of TrpacC under different pH showed that at increasing pH from 3 to 5, the wild‐type (WT) strain induced the expression of TrPacC in parallel to increased fungal growth; however, TrPacC expression decline at higer pH than 5, while fungal growth continued to increase. Development of a ΔTrPacC mutant down‐regulated the expression of TrbrlA, TrabaA and TrwetA, reduced sporulation and delayed spore germination, resulting in smaller spores and sparse hyphae. ΔTrPacC mutant was sensitive to ionic stress, oxidative stress and cell wall integrity stress compared to the WT strain, especially the ionic stress. In addition, ∆TrPacC mutant showed reduced pathogenicity to muskmelon and tomato fruits. Taken together, T. roseum is an alkalinizing fungus, and the acidic environment could induce TrPacC expression. TrPacC positively regulates fungal growth and development as well as pathogenicity showing effect on fungal response to different stresses. [ABSTRACT FROM AUTHOR]

Summary: Adaptation to low‐oxygen (LO) environment in host tissues is crucial for microbial pathogens, particularly fungi, to successfully infect target hosts. However, the underlying mechanisms responsible for hypoxia tolerance in most pathogens are poorly understood. A mitochondrial protein, BbOhmm, is demonstrated to limit oxidative stress resistance and virulence in the insect fungal pathogen, Beauveria bassiana. Here, we found that BbOhmm negatively affected hypoxic adaptation in the insect haemocoel while regulating respiration‐related events, heme synthesis and mitochondrial iron homeostasis. A homologue of the mammalian sterol regulatory element‐binding proteins (SREBPs), BbSre1, was shown to be involved in BbOhmm‐mediated LO adaptation. Inactivation of BbSre1 resulted in a significant increase in sensitivity to hypoxic and oxidative stress. Similar to ΔBbOhmm, ΔBbSre1 or the ΔBbOhmmΔBbSre1 double mutant accumulated high levels of heme and mitochondrial iron, regulating the similar pathways during hypoxic stress. BbSre1 transcriptional activity and nuclear import were repressed in ΔBbOhmm cells and affected by intracellular reactive oxygen species (ROS) and oxygen levels. These findings have led to a new model in which BbOhmm affects ROS homeostasis in combination with available oxygen to control the transcriptional activity of BbSre1, which in turn mediates LO adaptation by regulating mitochondrial iron homeostasis, heme synthesis and respiration‐implicated genes. [ABSTRACT FROM AUTHOR]

Summary: The ability to withstand heat and cell wall stress is critical for successful infection of target hosts by many pathogenic fungi. We report on the characterization of BbThm1 (transcription factor for heat and membrane integrity), a Zn(II)2Cys6 (Gal4‐like) family member, which regulates resistance to heat (32°C) and specific detergents in the insect pathogenic fungus, Beauveria bassiana. BbThm1 gene knock‐out mutants showed severely impaired growth/resistance at 32°C and to SDS, but mild to moderate phenotypic responses to other stresses including oxidative, osmotic and cell‐wall targeting compounds, or to various fungicides. Shifts in cell wall properties, adhesion and decreased viability were noted for the ΔBbThm1 mutant. Susceptibility to SDS but not heat could be rescued by exogenous ergosterol. Insect bioassays revealed decreased virulence of the ΔBbThm1 mutant against Galleria mellonella both topically and via intrahemocoel injection assays that correlated with impaired in insecta hyphal body formation and altered host immune prophenol oxidase (PO) activation. Transcriptomic analyses of the wild‐type and ΔBbThm1 mutants revealed up‐regulation of hydrolases but down‐regulation of a wide range of basic metabolic processes. These data reveal fine tune aspects of the transcription factor network that regulates specific stress responses to contribute to the overall pathogenic process. [ABSTRACT FROM AUTHOR]

Summary: Gcn5 is a core histone acetyltransferase that catalyzes histone H3 acetylation on N‐terminal lysine residues in yeasts and was reported to catalyze H3K9/K14 acetylation required for activating asexual development in Aspergillus. Here, we report a localization of Gcn5 ortholog in the nucleus and cytoplasm of Beauveria bassiana, a fungal insect pathogen. Deletion of gcn5 led to hypoacetylated H3 at K9/14/18/27 and 97% reduction in conidiation capacity as well as severe defects in colony growth and conidial thermotolerance. Two master conidiation genes, namely brlA and abaA, were transcriptionally repressed to undetectable level in Δgcn5, but sharply upregulated in wild‐type, at the beginning time of conidiation. Based on chromatin immunoprecipitation, both DNA and acetylation levels of the distal and proximal fragments of the brlA promoter bound by acetylated H3K14 alone were upregulated in wild‐type, but not in Δgcn5, at the mentioned time. In Δgcn5, normal cuticle infection was abolished while virulence through cuticle‐bypassing infection was greatly attenuated, accompanied by drastically reduced activities of putative cuticle‐degrading enzymes, retarded dimorphic transition and transcriptional repression of associated genes. These findings unveil a novel mechanism by which Gcn5 activates asexual development pathway by acetylating H3K14 and regulates the virulence‐related cellular events in B. bassiana. [ABSTRACT FROM AUTHOR]

Genomic data have identified a class of fungal specific transcription factors (FsTFs) that are thought to regulate unique aspects of fungal gene expression, although the functions of many of these proteins remain unknown. Here, a novel FsTF (BbStf1), which features a leucine zipper dimerization domain and a fungal transcription factor regulatory middle homology region, was characterized in Beauveria bassiana, a filamentous insect fungal pathogen. Transcriptional activation and nuclear localization were experimentally confirmed for BbStf1. Disruption of Bbstf1 resulted in increased tolerance to oxidative stress and cell wall perturbation, accompanied by increased peroxidase (POD) and superoxide dismutase (SOD) activities and ratio of reduced/oxidized glutathione (GSH/GSSG), and by thickened cell wall and altered composition. Gene expression profile analysis revealed that transcription patterns of antioxidant enzyme and cell wall integrity-involved genes were altered in the ∆Bbstf1, including some BbStf1-targeted genes clarified with evidence. The ∆Bbstf1 strain displayed greater virulence to Galleria mellonella in the bioassays through both topical infection and intrahaemocoel injection due to more rapid proliferation in the haemocoel as compared to the wild-type strain. Altogether, BbStf1 acts as a negative regulator of antioxidant response, cell wall integrity and virulence in B. bassiana., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

High-osmolarity glycerol (HOG) pathway required for yeast osmoregulation relies upon the mitogen-activated protein kinase (MAPK) Hog1 cascade that comprise the MAPKKKs Ssk2/Ssk22 and Ste11 converging on the MAPKK Pbs2. Here we show a Hog1 cascade with the unique MAPKKK Ssk2 acting in Beauveria bassiana. Hypersensitivity to high osmolarity and high resistance to fludioxonil fungicide appeared in Δ ssk2, Δ pbs2 and Δ hog1 mutants whereas the two hallmark phenotypes were reversed in Δ ste11. Increased sensitivity to heat shock and decreased sensitivity to cell wall perturbation also occurred in the three mutants but not in Δ ste11 although antioxidant phenotypes were different in all deletion mutants. Intriguingly, signals of Hog1 phosphorylation induced by osmotic, oxidative and thermal cues were present in Δ ste11 but absent in Δ ssk2 and Δ pbs2. Moreover, vegetative growth on minimal media with different carbon/nitrogen sources was much more suppressed in Δ ste11 and Δ ssk2 than in Δ pbs2 and Δ hog1 although all mutants suffered similar, but severe, conidiation defects on a standard medium. Normal host infection was abolished in Δ ste11 while virulence was differentially attenuated in other mutants. Our findings exclude Ste11 from the Hog1 cascade that regulates multiple stress responses and environmental adaptation of B. bassiana and perhaps other filamentous fungi. [ABSTRACT FROM AUTHOR]

The PacC transcription factor is an important component of the fungal ambient pH-responsive regulatory system. Loss of pacC in the insect pathogenic fungus Beauveria bassiana resulted in an alkaline pH-dependent decrease in growth and pH-dependent increased susceptibility to osmotic (salt, sorbitol) stress and SDS. Extreme susceptibility to Congo Red was noted irrespective of pH, and ΔBbpacC conidia showed subtle increases in UV susceptibility. The ΔBbPacC mutant showed a reduced ability to acidify media during growth due to failure to produce oxalic acid. The ΔBbPacC mutant also did not produce the insecticidal compound dipicolinic acid, however, production of a yellow-colored compound was noted. The compound, named bassianolone B, was purified and its structure determined. Despite defects in growth, stress resistance, and oxalate/insecticidal compound production, only a small decrease in virulence was seen for the ΔBbpacC strain in topical insect bioassays using larvae from the greater waxmoth, Galleria mellonella or adults of the beetle, Tenebrio molitor. However, slightly more pronounced decreases were seen in virulence via intrahemcoel injection assays ( G. mellonella) and in assays using T. molitor larvae. These data suggest important roles for BbpacC in mediating growth at alkaline pH, regulating secondary metabolite production, and in targeting specific insect stages. [ABSTRACT FROM AUTHOR]

Mas5, a yeast heat shock protein classified to the Hsp40 group, is homologous to bacterial archetype DnaJ but functionally unexplored in filamentous fungi. Here we identify a Mas5 homologue (46.86 kDa) in B eauveria bassiana and show its indispensability for host infection and environmental adaptation of the fungal insect pathogen. The deletion of mas5 caused severe defects in aerial conidiation, conidial germination and submerged blastospore production (mimic to host haemocoel). The deletion mutant lost 100% virulence to G alleria mellonella larvae through normal cuticular penetration (topical inoculation) and 50% through cuticle-bypassing infection (intrahaemocoel injection). It formed no blastospore in vivo after inoculation or only a very few after injection. Its extracellular (cuticle degrading) enzymes and virulence-relating Pr1 proteases were 62% and 32% less active respectively. It became more sensitive to high osmolarity, oxidation, cell-wall perturbation, heat shock and UV- B irradiation. These concurred with reduced contents of intracellular mannitol and trehalose, decreased activities of antioxidant enzymes, impaired cell walls and suppressed transcripts of stress-responsive and virulence-relating genes. All the changes were restored by targeted mas5 complementation. All together, Mas5 is indispensable for the in vitro and in vivo life cycle of B . bassiana by targeting many sets of enzymes/proteins at transcriptional and post-transcriptional levels. [ABSTRACT FROM AUTHOR]

The Hog1 mitogen-activated protein (MAP) kinase regulates environmental stress responses and virulence in the entomopathogenic fungus Beauveria bassiana. To further characterize this pathway, we constructed a subtraction library enriched for genes regulated by Hog1. One targeted gene, encoding a novel membrane protein, Ohmm (oxidative homeostasis membrane-protein-mitochondria), was uniquely identified as being downregulated in the Hog1 background during growth under non-stress and osmotic stress conditions, but upregulated under oxidative stress. Ohmm was an experimentally validated flavinbinding protein and targeted to the mitochondria. Deletion of Ohmm resulted in increased oxidative stress resistance, whereas overexpression caused an opposite phenotype. The Ohmm showed accumulation of reactive oxygen species with alterations in cell wall composition and compatible solute accumulation evident as compared with the wild type parent. Conidiation was reduced > 80%; however, conidia produced by the Ohmm strain germinated significantly faster than wild type cells. Insect bioassays using the greater wax moth revealed increased virulence for the Ohmm strain in both topical and intrahemocoel injection assays, indicating a negative effect of the presence of Ohmm with respect to pathogenesis. As predicted from our characterization, deletion of Ohmm in a Hog1 background rescued its oxidative sensitivity phenotype, confirming that Ohmm acts downstream of the Hog1 MAP-kinase. [ABSTRACT FROM AUTHOR]

For most organisms, carbon and nitrogen uptake are essential for growth, development and, where applicable, pathogenesis. The role of the carbon catabolite repressor transcription factor homologue BbcreA in the entomopathogenic fungus B eauveria bassiana was investigated. Deletion of BbcreA resulted in pleiotropic effects, including nutrient toxicity, leading to a novel cell lytic phenotype. Fungal growth in rich media and minimal media containing select amino acids/peptides was severely compromised, with microscopic examination revealing conidial-base germ tube degeneration and cell lysis occurring during growth, a phenomenon exacerbated at higher temperatures (32°C). Depending upon nutrient conditions, growth, pigment and aerial mycelium production, sporulation and dimorphic transition to blastospore production were also impaired in the Δ BbcreA strain. Although loss of BbcreA resulted in de-repression of secreted protease and lipase, enzymes critical in mediating pathogenesis, insect bioassays indicated severe defects in virulence using both topical and intra-haemocoel injection assays, with eruption and subsequent sporulation on host cadavers greatly reduced in the mutant. These data suggest that BbcreA functions as more than a carbon repressor and plays important roles in nutrient utilization, cell homeostasis and virulence. In particular, BbcreA is required for proper assimilation of select amino acids and peptides, including asparagine, arginine and proline. [ABSTRACT FROM AUTHOR]

In fungi, G-protein coupled receptors ( GPCRs) link ligand/nutrient sensing to growth, mating, developmental/life-stage activation and pathogenesis. A GPCR was characterized from the entomopathogenic fungus, Beauveria bassiana ( BbGPCR3), which links nutrient sensing to stress response and development. Δ BbGPCR3 mutants grew slower on various carbohydrates and displayed increased sensitivity to osmotic, oxidative and cell wall stresses. Gene expression profiling revealed a set of heat-shock and antioxidant factors that failed to be induced under oxidative stress and aberrant regulation of compatible solute-forming enzymes and cell wall biosynthesis/remodelling proteins in Δ BbGPCR3 after osmotic stress. Glucose-specific developmental defects included reduced (> 90%) conidiation and reduced dimorphic transition to the production of yeast-like blastospores, effects suppressed in media containing trehalose or glycerol, but not by addition of cyclic AMP. Insect bioassays revealed reduced virulence in topical assays but no effect in intrahaemoceol injection assays, indicating that BbGPCR3 was important in sensing signals during the initial interaction with the host but dispensable for post-penetration events. Comparative gene expression profiling of Δ BbGPCR3 mutants grown in glucose media compared with wild-type/glucose and Δ BbGPCR3/trehalose grown cells revealed sets of genes misregulated and recovered, respectively. These data link BbGPCR3 to broad developmental and genetic networks that include the major MAP kinase pathways. [ABSTRACT FROM AUTHOR]

The aim of this paper was to understand whether the endobacterium identified as Candidatus Glomeribacter gigasporarum has an effect on the biology of its host, the arbuscular mycorrhizal fungus Gigaspora margarita, through the study of the modifications induced on the fungal proteome and lipid profile. The availability of G. margarita cured spores (i.e. spores that do not contain bacteria), represented a crucial tool to enable the comparison between two fungal homogeneous populations in the presence and the absence of the bacterial components. Our results demonstrate that the endobacterial presence leads to a modulation of fungal protein expression in all the different conditions we tested (quiescent, germinating and strigolactone-elicited germinating spores), and in particular after treatment with a strigolactone analogue. The fungal fatty acid profile resulted to be modified both quantitatively and qualitatively in the absence of endobacteria, being fatty acids less abundant in the cured spores. The results offer one of the first comparative metabolic studies of an AM fungus investigated under different physiological conditions, reveal that endobacteria have an important impact on the host fungal activity, influencing both protein expression and lipid profile, and suggest that the bacterial absence is perceived by G. margarita as a stimulus which activates stress-responsive proteins. [ABSTRACT FROM AUTHOR]