Genetic And Molecular Basis For Adaptation To Ambient Ph Change And Intracellular PH Maintenance Contributes To Biological Control Potential In Beauveria Bassiana

Beauveria bassania is a filamentous fungal entomopathogen that has been widely applied for biological control of arthropod pests in agriculture and forestry. The fungal potential against arthropod pests is dependent on noly only virulence but also cellular tolerance to outdoor stresses, such as high temperature, UV irradiation, applied agrochemicals and environment pH. These stresses affect the persistence and efficacy of a fungal insecticide after field applicaton, This study sought to elucidat how B. bassiana adapts to environmental pH change by characterizing the functions of seven proteins in Pal signaling pathway, Na+/H+ antiporter (Nhx1) and vaucuolar ATPase subunit H (VmaH). The results are summarized below.The Pal pathway required for ambient pH adaptation regulates B. bassiana growth, conidiation and osmotolerance in a pH-dependent manner. The Pal/Rim pathway essential for fungal adaptation to ambient pH has been unexplored in B. bassiana, a classic fungal entomopathogen. Here, we show the characterized Pal pathway comprising transcription factor PacC and upstream six Pal partners (PalA/B/C/F/H/I) in B. bassiana. Their coding genes were all transcribed most abundantly in standard wild-type culture under the alkaline condition of pH 9. Deletion of pacC or each pal gene resulted in a significant delay of culture acidification in a minimal broth (initial pH= 7.3). This delay concurred with altered accumulation levels of intra/extracellular organic acids and drastically depressed expression of some enzyme genes required for the syntheses of oxalic and lactic acids. All deletion mutants except Apall showed growth defects and hypersensitivity to NaCl, KC1, LiCl or sorbitol at pH 9, an alkaline condition leading to fragmented vacuoles in their hyphal cells exposed to osmotic stress. In these mutants, conidiation was significantly facilitated at pH 3 more than at pH 7 but suppressed slightly at pH 9. Mild virulence defects also occurred in the absence of pacC or each pal gene. These changes were restored by targeted gene complementation. Taken together, PacC and Pal partners regulate the growth, conidiation and osmotolerance of B. bassiana in a pH-dependent manner, highlighting their vitality for the fungal pH response.The Na+/H+antiporter Nhxl controls vacuolar fusion/fission indispensible for life cycles in vitro and in vivo in B. bassiana. The sole Na+/H+ antiporter Nhx1 has been generally unexplored in filamentous fungi. The Nhx1 orthologue in B. bassiana was characterized in this study. An eGFP-tagged Nhxl fusion accumulated in small punctuate structures, presumably endosomal and trans-Golgi network compartments, between septum and tubular vacuole of each wild-type cell stained with a vacuole-specific dye. Deletion of nhxl accelerated vacuolar acidification, abolished vacuolar fusion due to the presence of many small, irregular vacuoles in the mutant cells and of only a few large or tubular vacuoles in wild-type cells, suppressed drastically aerial conidiation and submerged blastospore production, and caused more severe defects in vegetative growth than in conidial germination on rich and minimal media. The deletion also reduced cellular tolerance to high osmolarity, heat shock and several metal ions but increased conidial UV-B resistance. Intriguingly, Δnhxl was unable to infect a model insect(Galleria mellonella) through cuticle penetration or intrahaemocoel injection. Its conidia produced much less biomass and extracellular cuticle-degrading enzymes during incubation in a minimal broth and failed to form blastospores in the insect haemolymph. All changes were restored by nhxl complementation. These results provide novel insight into an indispensability of Nhx1 for not only pH homeostasis and fusion of vacuoles but life cycle in vitro and in vivo of the fungal insect pathogen.The vacuolar ATPase subunit H (VamH) required for intra/extracellular pH homeostasis contributes to biological control potential of B. bassiana. VmaH is one of many subunits of a vacuolar ATPase complex required for fungal pH homeostasis. Expression of eGFP-tagged VmaH fusion in B. bassiana demonstrated that VmaH was localized in the fungal cytoplasm rather than in the fungal vacuoles. Deletion of vmaH results in a significant increase of vacuolar pH but facilitated acidification of liquid culture in a minimal broth, accompanied with a drastic reduction (87%) in ammonia secretion to the culture from hyphal cells. Perhaps due to the disturbed pH homeostasis, the deletion mutant exhibited growth defects in a rich medium or in a minimal medium with altered carbon/nitrogen sources or with pH adjusted to 3 to 7. Compared with wild-type, the deletion mutant suffered a great delay in aerial conidiation and severe defect in submerged blastospore production. The mutant conidia and blastospores showed significant reductions in size and density. In stardardized bioassays, the deletion mutant lost more virulence to G. mellonella larvae infected through normal cuticle penetration than through intrahaemocoel infection. Conidial thermotolerance and UV-B resistance were also significantly lower in the deletion mutant than in the wild-type strain. All these changes were well restored by targeted vmaH complementation. All tegother, VmaH acts as an important mediator of intra/extracellular pH homeostasis and hence contributes significantly to the biological control potential of B. bassiana.