Characterization Of PH-responsive Transcription Factor, BbPacC, In The Entomopathogenic Fungus Beauveria Bassiana

In nature, alterations in the ambient pH usually have an important influence on the growth, development or secondary metabolism of fungi. To survive and proliferate, fungi must be able to tailor gene expression to the particular pH of their growth environment. Thus, elucidating how fungi adapt to pH alteration is an important aspect of understanding the mechanism of their life activities. In this study, a pH-responsive transcription factor gene BbPacC, was cloned from the entomopathogecic fungus Beauveria bassiana. Functions of the gene were elucidated using gene expression pattern, gene disruption and overexpression, complementation experiments. The main results are as follows:1. Gene BbPacC cloning and expression patternA 3990 bp DNA sequence, including the coding and flanking regulatory regions of BbPacC (GeneBank no.:JX145340), was obtained through homologous cloning. The 1890 bp coding region includes two introns, and the 1773 bp ORF encodes a protein consisting of 590 amino acids. The protein was predicted to has a molecular weight of 63.5 kDa, an isoelectric point of 8.2, and include a conserverd domain known as arsenite-resistance protein (ARS2). Amino acid sequences alignment showed that its homology was 85% with PacC in Cordyceps militaris. These results suggest that the gene is a PacC in Beauveria bassiana. In addition, quantitative RT-PCR results showed that the expression of BbPacC was very low in the early growth stage, but dramatically elevated to eightfold in later or secondary metabolic stage than that in the early stage. Furthermore, the expression of BbPacC was suppressed by acidic conditions, but highly induced by salt stress or alkaline conditions.2. BbPacC is involved in stress toleranceOn CZA medium, growth of gene knockout strain were restrained under alkaline conditions and salt stress. DNA sequence analysis showed that the promoter of Na+/K+P-type ATPase gene had as many as 5 PacC consensus binding sites. The qRT-PCR also verified that the expression of this P-type ATPase gene was regulated by BbPacC. These data suggest that BbPacC may be negatively involved in hyphal growth, and that the gene may influence acid-alkali adaptability and salt tolerance, through regulation of the transcription of Na+/K+P-type ATPase.3. BbPacC is involved in growth and development of B. bassiana BbPacCinfluences hypha growthBiomass determination showed that ablation strain resulted in faster growth than wild-type in minimum medium (CZB), but overexpression strain led to slower growth.BbPacC is involved in sporulationSporulation test showed that the conidial yield of ablation strain was increased by 107.34%, while overexpressing strain declined by 17.80%, compared with that of the wild-type. DNA sequence analysis showed that the promoter of the key sporulation related gene fluG contained PacC consensus binding sites. Moreover, the expression level of fluG in ablation strain increased remarkably, and near to fourfold higher than that in wild-type. These results suggest that BbPacC might influence sporulation by the negative regulation of fluG transcription.BbPacC influences conidial germination and appressorium formationGermination test showed that the conidial half-germination time of gene knockout strain (10.67 h) was 1.03 h longer than that of wild-type (9.64 h). Further test revealed that the appressorium formation rate of ablation strain was decreased by 46.96%, compared with that of wild-type. These results indicate that BbPacC might involve in conidial germination and appressorium formation.4. BbPacC is involved in virulence of the fungus BbPacC influences lethalityBioassay showed that the half lethal time (LT50) of ablation strain (123.99 h) to 3-instar Galleria mellonella larval was delayed by 26.66%, when compared to wild-type (97.89 h). However, there was no obvious difference between overexpression strain and wild-type. These results indicated that BbPacC might participate in virulence in B. bassiana.BbPacC regulates oxalic acid accumulationQuantification analysis revealed that, in fermentation broth (SDB), the production of oxalic acid in gene knockout strain was 11.09% less than that in wild-type, while overexpression strain was increased by 125.00%. Using bromcresol purple as indicator to detect the effect metabolites on media pH, results showed that, either 1/4 SDB or CZB media, cultured with ablation strain, appeared blue color. The results of pH determination confirmed that the pH value of gene knockout strain cultured in CZB medium was neutral, but that with wild-type or overexpression strain were acidic. DNA sequence analysis revealed that the upstream sequences of OAH and PYC, two vital genes for oxalic acid biosynthesis, contained PacC consensus binding sites. The qRT-PCR revealed that, the expression levels of OAH and PYC in BbPacC overexpression strain were significantly elevated, approximately 2-6 fold higher than those in wild-type. These results suggested that BbPacC participated in oxalic acid biosynthesis by regulating the expression of OAH and PYC.BbPacC regulates the expression of alkaline serine protease geneThe halo ring of gene knockout strain on CZA containing skim milk was obviously larger than that of wild-type, suggesting that loss of BbPacC induced secreted protease gene expression by changing ambient pH. DNA sequence analysis showed that the promoter of alkaline serine protease gene Serp contained six PacC consensus binding sites with a 900 bp promoter region. The qRT-PCR showed that the expression of this gene was significantly elevated in ablation strain, but decreased in overexpression. These data suggested that BbPacC controled the expression of Serp.