Functional Characterization Of Proline Dehydrogenases,And Preliminary Insights Into The Anti-Oxidation Mechanism Mediated By Multiprotein Bridging Factor 1 In Beauveria Bassiana

Beauveria bassiana, an important fungal biocontrol agent, has wide host range and has been developed into a variety of preparations for biological control of insect pests. This paper mainly studies the function of Beauveria bassiana proline dehydrogenase BbPDH in fungal growth and development. At the same time, transcriptomic analysis was used to explore the potential antioxidant pathways regulated by multiprotein bridging factor 1(BbMBF1).Functional characterization of BbPDH In order to perform the phenotype and biochemical analysis of BbPDH, we constructed the single gene knockout strains and complement strains. Subcellular co-localization analyses of green fluorescent protein and MitoTraker fluorescent dyes indicated that BbPDH protein was localized in the mitochondria. BbPDH regulated growth and thermotolerance of B. bassiana. Except for BbPDH2, gene disruption of other three genes significanty reduced fungal vegetative growth on the defined media. After replacing the nitrogen source, the three knockout strains showed growth defects. When using NH4Cl as nitrogen source, α-ketoglutaric acid, succinic acid, citric acid and malic acid, respectively, as carbon source, ΔBbPDH1 and ΔBbPDH4 had recovery growth trend. While the growth rate of ΔBbPDH3 is still decline. After thermal stress, the growth rate of the wild-type, ΔBbPDH2 and ΔBbPDH3 strains decreased 11.17%,11.83% and 19.61%, respectively. However, ΔBbPDH1 and ΔBbPDH4 mutants were more sensitive to heat stress. There was no significant difference in conidia yield between the wild strain and ΔBbPDH2 or ΔBbPDH3 mutants. But gene disruption of BbPDH1 and BbPDH4 reduced conidial production in different degrees. There were no notable difference in blastospore yield between the wid type and ΔBbPDH1, ΔBbPDH2 or ΔBbPDH3 mutants, while the blastspore production of ABbPDH4 mutant significantly decreased. The blastospore size of ΔBbPDH2 mutant did not significantly change, while the other three mutant strains have bigger blastospore when compared with wild type strain. Gene disruption did not considerably affect conidial germination on rich media, but resulted in a notable reduction in germination rate of ΔBbPDH1 and ABbPDH4 mutants on oligotrophic condition. The bioassay indicated that three knockout strains (ΔBbPDH1, ΔBbPDH3 and ABbPDH4) exhibited the reduced virulence, but in different degrees. Thus, four BbPDH genes play different roles in growth, thermotolerance and pathogenicity of B. bassiana.Antioxidant pathway regulated by BbMBFl in B. bassiana Multiprotein bridging factor 1 (BbMBF1), an evolutionarily conserved transcriptional cofactor, plays an important role in development and stress tolerance of eukaryotic organisms. It has been established that the growth of ABbMBF1 strain is suppressed under menadione stress. RT-PCR showed the expression level of this gene increased under oxidative stress conditions in wild strains. To explore the down-stream targets regulated by BbMBF1 under menadione stress, a comparative trancriptome was performed on the wild type and the ΔBbMBF1 mutant strains. Transcriptomic analysis showed that the oxidation-response genes regulated by BbMBF1 were significantly enriched in the functional catalogues of metabolism, cell rescue and transportation. Moreover, bioinformatic analysis predicted a putative motif which distributed mainly over the promoters of genes associated with metabolism and detoxification. In order to obtain the putative transcription factor(s) interacted with BbMBF1p, we performed a co-immunoprecipitation experiment using BbMBF1 antibody to precipitate protein complex. The precipitated proteins were analyzed by mass spectrometry, and transcription factor BbAP-1 was considered as a putative transcription factor. Additionally, ΔBbAP-1 mutant strain also exhibited a significantly reduced resistance to oxidative stress. In conclusion, these results indicate that BbMBFl contributes to B. bassiana response to oxidative stress by mediating the potential transcription factors regulating pathway of metabolism and detoxification. These findings provide the new clues/targets for improving potency of entomopathogenic fungi by molecular manipulation.