Function Analysis Of Beauveria Bassiana Alpha-glucoside Transporter (BbATG1) And Identification Of Heat Stress Responsive Genes

Environmental stresses are crucial factors affecting the field persistence and efficacy of various Beauveria bassiana formulations against arthropod pests. This study sought to investigate the function of B. bassiana a-glucoside transporter (BbAGT1) and to isolate heat-stress responsive genes by means of suppression subtractive hybridization (SSH). The results are summarized below.Gene cloning, knockout and functional analysis of BbAGTl. The full-length gene BbAGT1 cloned from the genome of a wild-type B. bassiana strain (Bb2860) was a 1883-bp sequence with three introns and four exons, encoding for a 565 amino acid protein with the predicted molecular weight of 63.65 kDa and the isoelectronic point of 5.52. The deduced BbAGT1 showed 90-99% sequence identity to 21 a-glucoside transporters known from other fungi and special substrate binding sites for transporting proteins in the major facilitator superfamily. The gene BbAGTl was disrupted via Agrobacterium-mediated transformation, yielding a BbAGT1-absent mutant, namely ABbAGT1, which was confirmed in PCR and Southern blotting analyses. Several phenotypic features were then compared between ABbAGT1 and Bb2860 to explore the role of BbAGT1 in carbon exploitation, stress resistance and virulence. Compared to Bb2860, the mutant ABbAGT1 grown on the plates of Czapek's medium containing 3% trehalose as mere carbon source showed 66,64 and 90% reductions in the rates of spore germination and colony growth and the capacity of conidial production, respectively. The conidia of this mutant displayed 27% reduction in antioxidative capability,22% decrease in tolerance to the wet-heat stress of 48℃, and 28% decrease in resistance to UV-B irradiation, based on the median lethal responses of both strains to the different stresses. Moreover, the median lethal time (LT50) of ABbAGT1 against the apterous adults of Myzus persicae was 14% longer than the LT50 of Bb2860. These data indicate that the BbAGTl gene contribute significantly to the examined phenotypic features.Identification of heat stress responsive genes in B. bassiana. A suppression subtractive cDNA library was constructed using the SSH method in order to isolate heat stress responsive genes from B. bassiana based on their differential expression in response to a heat stress at 40℃. Twenty-eight putative heat-responsive genes were found from the SSH library consisting of 528 positive clones. These genes were homologues of known genes involved in various cellular processes including substance transport, metabolism and stress response. Of those,12 genes were examined for their expression patterns over the time (15,30,60 and 120 min) of heat stress at 40℃using quantitative real-time PCR (qRT-PCR). As a result, the expressions of all the examined genes were heat-inducible and expression patterns over the time of the heat stress differed significantly among the examined genes, suggesting the involvements of different genes in the time course of B. bassiana responses to the heat stress.Conclusively, BbAGT1 plays an important role in the mediation of carbon exploitation, multi-stress resistance and virulence of B. bassiana. The heat stress responsive genes identified from the cDNA library are important targets for future studies on molecular mechanisms involved in B. bassiana thermotolerance and for use in genetic transformation of the fungal bioctontro candidate strains for mycoinsecticides better resisting high summer temperatures.