Hypothetical protein MAA_07646 is required for stress resistance and pathogenicity in Metarhizium robertsii.

Metarhizium robertsii, a vital entomopathogenic fungus for pest management, relies on various virulence-related proteins for infection. Identifying these proteins, especially those with unknown functions, can illuminate the fungus's virulence mechanisms. Through RNA-seq, we discovered that the hypothetical protein MAA_07646 was significantly upregulated during appressorium formation in M. robertsii. In this study, we characterized MAA_07646, finding its presence in both the nucleus and cytoplasm. Surprisingly, it did not affect vegetative growth, conidiation, or chemical tolerance. However, it played a role in heat and UV radiation sensitivity. Notably, ΔMAA_07646 exhibited reduced virulence in Galleria mellonella larvae due to impaired appressorium formation and decreased expression of virulence-related genes. In conclusion, MAA_07646 contributes to thermotolerance, UV resistance, and virulence in M. robertsii. Understanding its function sheds light on the insecticidal potential of M. robertsii's hypothetical proteins.

The homeobox transcription factor MrHOX7 contributes to stress tolerance and virulence in the entomopathogenic fungus Metarhizium robertsii.

Entomopathogenic fungi, including Metarhizium species, represent promising environmentally friendly biopesticides. Understanding the molecular mechanisms governing their infection processes is vital for enhancing their effectiveness. Transcription factors (TFs) play critical roles in gene regulation, yet the functions of many TFs in M. robertsii remain unknown. Homeobox transcription factors, implicated in diverse cellular processes, have received limited attention in entomopathogenic fungi. Here, we identify and characterize, a homeobox TF, MrHOX7, in the model entomopathogenic fungus M. robertsii. Subcellular localization and transcriptional profiling revealed MrHOX7's nuclear localization and high expression during conidia and appressoria formation. Deletion of Mrhox7 (ΔMrhox7) enhanced conidial tolerance to heat and UV-B stress, accompanying with upregulated stress-related gene expression. Intriguingly, ΔMrhox7 exhibits inhibited virulence exclusively through topical inoculation. Further investigations unveiled reduced conidial adhesion and appressorium formation, with downregulation of the adhesion gene Mad1 and appressorium-related genes, as the underlying causes of the reduced fungal virulence. Our findings illuminate the role of MrHOX7 in stress tolerance and virulence, providing insights into the molecular basis of fungal biopesticides.

Metarhizium robertsii MrAbaA affects conidial pigmentation via regulating MrPks1 and MrMlac1 expression.

Pigments of conidia play a crucial role in fungal defense against environmental stressors such as UV radiation. The molecular basis of conidial pigmentation has been studied in the entomopathogenic fungus Metarhizium robertsii, while limited information been reported on function mechanisms transcription factors governing conidial pigmentation. Here, we identified transcription factor MrAbaA binding to the promoter regions of both MrPks1 and MrMlac1 in M. robertsii using yeast one-hybrid technology. Chromatin immunoprecipitation quantitative PCR assays further confirmed the interaction. Furthermore, overexpression of MrAbaA in M. robertsii resulted in increased conidial pigment accumulation and enhanced tolerances to UV stress by upregulated the MrPks1 and MrMlac1 expression. Taken together, MrAbaA affects conidial pigmentation by interacting with the promoter regions of both MrPks1 and MrMlac1 in M. robertsii. This work advances the understanding of the regulation mechanism for conidial pigmentation in entomopathogenic fungi.

Role of Two G-Protein α Subunits in Vegetative Growth, Cell Wall Integrity, and Virulence of the Entomopathogenic Fungus Metarhizium robertsii.

Heterotrimeric G-proteins are crucial for fungal growth and differentiation. The α subunits of heterotrimeric G-proteins play an essential role in controlling signal transduction. However, the function of G-protein α subunits in entomopathogenic fungi remains poorly understood. Two group II Gα subunits (MrGPA2 and MrGPA4) were characterized in the entomopathogenic fungus, Metarhizium robertsii. Bioinformatics analysis showed that the relationship between MrGPA2 and MrGPA4 was closer than that of other MrGPAs. Both green fluorescent protein (GFP)-tagged MrGPA2 and MrGPA4 were localized at the cytoplasm. Furthermore, ∆MrGpa2∆MrGpa4 double mutants showed remarkably reduced vegetative growth compared to the wild-type and single-mutant strains, which was accompanied by the downregulation of several growth-related genes, such as ssk2, pbs2, stuA, hog1, and ac. Only the ∆MrGpa2∆MrGpa4 double mutant was sensitive to Congo red stress. The insect bioassay demonstrated significantly attenuated virulence for the ∆MrGpa2∆MrGpa4 double mutant compared to the wild-type and single-mutant strains. Further analysis indicated that double deletion of MrGpa2 and MrGpa4 had no effect on appressorium formation but suppressed the expression levels of several virulence-related genes in the insect hemocoel. These findings demonstrate that MrGpa2 and MrGpa4 exhibit functional redundancy and contribute to the vegetative growth, stress tolerance, and pest control potential in M. robertsii.

Mr-AbaA Regulates Conidiation by Interacting with the Promoter Regions of Both Mr-veA and Mr-wetA in Metarhizium robertsii.

Conidiation is a pivotal strategy for fungi to resist adverse environments and disperse to new habitats, which is especially important for entomopathogenic fungi whose conidia are infective as fungal pesticide propagules. However, the molecular mechanism for regulating conidiation in entomopathogenic fungi is not fully understood. Here, we characterized the regulatory mechanism of the key developmental transcription factor Mr-AbaA. Bioinformatic analysis, transcriptional profiles, and subcellular localization of Mr-abaA indicated that AbaA functioned as a transcription factor in the conidiophore development and conidium stages. Microscopic examination showed that the null mutant of Mr-abaA differentiated into defective phialides to produce an abacus structure instead of conidia. Loss of Mr-abaA resulted in the inhibition of submerged blastospore separation in vitro. Moreover, yeast (Saccharomyces cerevisiae) one-hybrid assays of interactions between genes and deletion of Mr-veA showed that Mr-AbaA regulates conidiation by interacting with the promoter regions of Mr-veA and Mr-wetA. These results demonstrate that Mr-AbaA positively regulates conidiation in Metarhizium robertsii by regulating the velvet family ortholog gene Mr-veA and contributes to the separation of blastospores in submerged culture. IMPORTANCE Metarhizium robertsii is an emerging model entomopathogenic fungus for developing biopesticides; therefore, a comprehensive understanding of its conidiation is very important for its application. In this study, we revealed that the transcription factor Mr-AbaA is involved in the control of aerial conidiation and blastospore separation in submerged culture. Further yeast one-hybrid assays demonstrated that Mr-AbaA interacts with the promoter regions of Mr-veA and Mr-wetA, which code for proteins involved in the control of conidiation. This finding provides new insight into the regulation of the conidiation of this important entomopathogenic fungi.

G-Protein Subunit Gαi in Mitochondria, MrGPA1, Affects Conidiation, Stress Resistance, and Virulence of Entomopathogenic Fungus Metarhizium robertsii.

G proteins are critical modulators or transducers in various transmembrane signaling systems. They play key roles in numerous biological processes in fungi, including vegetative growth, development of infection-related structures, asexual conidiation, and virulence. However, functions of G proteins in entomopathogenic fungi remain unclear. Here, we characterized the roles of MrGPA1, a G-protein subunit Gαi, in conidiation, stress resistance, and virulence in Metarhizium robertsii. MrGPA1 was localized in the mitochondria. MrGpa1 deletion resulted in a significant reduction (47%) in the conidiation capacity, and reduced expression of several key conidiation-related genes, including fluG, flbD, brlA, wetA, phiA, and stuA. Further, MrGpa1 disruption resulted in decreased fungal sensitivity to UV irradiation and thermal stress, as determined based on conidial germination of ΔMrGpa1 and wild-type (WT) strains. Chemical stress analysis indicated that MrGpa1 contributes to fungal antioxidant capacity and cell wall integrity, but is not involved in tolerance to antifungal drug and osmotic stress. Importantly, insect bioassays involving (topical inoculation and injection) of Galleria mellonella larvae revealed decreased virulence of ΔMrGpa1 strain after cuticle infection. This was accompanied by decreased rates of appressorium formation and reduced expression of several cuticle penetration-related genes. Further assays showed that MrGpa1 regulated intracellular cyclic AMP (cAMP) levels, but feeding with cAMP could not recover the appressorium formation rate of ΔMrGpa1. These observations suggest that MrGpa1 contributes to the regulation of conidiation, UV irradiation, thermal stress response, antioxidant capacity, and cell wall integrity in M. robertsii. This gene is also involved in insect cuticle penetration during infection. These findings raise the possibility of designing powerful strategies for genetic improvement of M. robertsii conidiation capacity and virulence for killing pests.