BbCrel, A Carbon Catabolite Repressor, Influences The Growth, Conidial Yield And Virulence Of Beauveria Bassiana

Beauveria bassiana infects insect pests of commercial importance, and consequently has been extensively studied as a biological control agent or mycoparasite. Like many fungal insect pathogens, B. bassiana is a facultative saprophyte with both free-living (saprophytic) and insect pathogenic life-stages. The gene expression patterns in response to the different lifestyles mainly depend on carbon and nitrogen sources. To understand the molecular mechanism of life-stages transit, it is necessary to elucidate the gene expression profiles of B. bassiana in pathogenicity.Carbon catabolite repression (CCR) plays important roles in the shift between parasitical and saprophytic life-stages. However, the mechanism of CCR in B. bassiana is essentially unknown. To elucidate the function of CCR in B. bassiana, BbCre1, a homolog of Aspergillus nidulans CCR-related gene CreA, was isolated from this fungal pathogen.BbCre1 contained an open reading frame of 1263 bp, encoding a 420-aa protein with a calculated molecular mass of 46.0 kDa and a pI of 9.91. Like other fungal CreA proteins, the deduced BbCre1 protein had two C₂H₂ zinc finger regions and a nuclear localization signal. The BbCre1 protein shared identities of 73%, 51%, 54%, 54% and 49% with CreA proteins from Metarhizium anisopliae, A. nidulans, Botrytis cinerea, Sclerotinia sclerotiorum and Cochliobolus carbonum, respectively.Southern analysis indicated that BbCre1 was present as a single copy in B. bassiana genome. Two putative CREA (carbon-response regulator)-binding sites were found in upstream sequence of this gene. Consistently, Real-Time PCR showed that the expression of BbCre1 was repressed by glucose, suggesting that BbCre1 was involved in carbon metabolism in B. bassiana.The transformants expressing sense and anti-sense BbCre1 in B. bassiana were used to investigate the biological functions of this gene. The main results are as followings:1. BbCre1 affected the utilization of different carbon sources in B. bassiana. With xylose or ethanol as sole carbon source, colony growth rate and conidial yield of antisense transformants anti-cre-28 and anti-cre-54 were all higher than that of the wide-type strain; but colony growth rate and conidial yield of the overexpressing transformants O-cre-01 and O-cre-07 had reduced. However, no distinguishable difference in colony growth rate and conidial yield was found among these transformants and wild-type B. bassiana strain, when glucose was used as sole carbon source.2. BbCre1 significantly inhabited the expression of cuticle-degrading hydrolyase of B. bassiana. With glucose as sole carbon source, the expression level of Pr1-like protease was repressed in the wild-type strain; on the contrast, the level was significantly increased in the antisense-BbCre1 transformants.3. BbCre1 was relative to the virulence of B. bassiana against Myzus persicae. The median lethal time (LT₅₀) of B. bassiana was shortened 10-11 hours by antisense expressing BbCre1 and prolonged 6-8 hours by overexpressing BbCre1 over that of the control, with the concentration of 1×10⁷ conidia /ml.The results in this study suggested that BbCre1 gene was involved in carbon catabolite and virulence in Beauveria bassiana.