Gene Cloning And Functional Analysis Of A Beauveria Bassiana Catalase (BbcatA)

As a classic entomopathogenic hyphomycete, Beauveria bassiana has been widely applied for arthropod pest control due to easy production, formulation and environmental safety. The virulence and field persistence of a fungal formulation are known to be affected by multiple stresses, which are barriers to the application of fungal insecticides. Catalases are important antioxidant enzymes in eukaryotic cells and constitute a primary source of cellular defenses against cell damages by reactive oxygen species (ROS) often induced under an environmental stress. However, little is known about the catalases of fungal entomopathogens. This study sought to elucidate the structure, subcellular localization and function of a Beauvria bassiana catalase (BbcatA) by gene cloning and knockout. The results are summarized below.Cloning and analysis of BbcatA gene. The full-length sequence of BbcatA cloned from the genome of the wild-type B. bassiana strain Bb2860 consists of a 2175-bp ORF and a 60-bp intron, encoding a protein sequence of 724 amino acids (80.7 kDa) with no more than 72% sequence identity to the catA members known from other fungi. The deduced protein sequence was phynogenetically most close to that of Cladosporium fulvum catA (xx%) and characteristic with the haem binding site and the concerved amino acid residues in the catalytic triad of fungal catalases. Based on maximum likelihood analysis of amino acid sequences, BbcatA is classified to large-subunit GroupⅡin the mo no functional catalase family.The transcriptional expression of BbcatA was at a maximal level on the second day of normal colony growth on SDAY plates but drastically declined on day 3, followed by a second peak on day 6. In SDB cultures (B. bassiana mycelia) under the oxidative stress of 50 mM H2O2, the transcriptional expression of BbcatA was enhanced by 2.5 and 4.5 fold after 1 and 3 h stress, respectively. The results indicate that BbcatA plays an important role in B. bassiana response to the oxidative stress of H2O2,.Effects of BbcatA knockout on phenotypic features of B. bassiana. A BbcatA disrupted mutant of Bb2860, namely△BbcatA, was constructed via Agrobacterium mediated fungal transformation The conidia of ABbcatA became more sensitive to the stress of H2O2. Median lethal responses of△BbcatA for conidial tolerance to H2O2 (LC50), UV-B irradiation (LD50), and the heat stress of 45℃(LT50) were reduced by,42.2%,51.7% and 37%, respectively, compared with the estimates (4.69 mM,0.42 J/cm2 and 52.2 min) for the wild-type strain. Moreover, The final mortalities of the second-instar larvae of Spodoptera litura caused by△BbcatA and Bb2860 were 59.8% and 86.9% under the sam spray, respectively. Probit analysis indicated that the median letha time (LT50) of△BbcatA (6.4 days) was 28% longer than that of Bb2860 (5.0 days).Conclusively, BbcatA contributes significantly to the stress tolerance and virulence of B. bassiana.