Functional Characterization Of Protein Kinase Gene BbSNF1and α-glucoside Transporter Gene BbAGT1in Entomopathogenic Fungus Beauverua Bassiana

Beauveria bassiana, an insect pathogen, represents a model system in fungal development and pathogenesis research and has important applications in biological control of agricultural insect pests. In present study, we performed the functional analysis of AMPK kinase BbSNF1and α-glucoside transporter gene BbAGT1in B.bassiana.Functional analysis of BbSNF1AMPK protein kinases are highly conserved in fungi. In B. bassiana, BbSNF1open reading frame was determined to be22230bp long, with two introns in genomic sequence and encode702amino acids, with the PKinase domains presenting in all SNF1homologous proteins. To further identify the function of BbSNFl, we constructed disruption mutants and complement strains. Phenotype of three strains displayed as follows. Disruption mutants showed significantly reduced growth rate while growing on different carbon/nitrogen sources, which are based on CPZ media, as compared with wild type and complement type. When cultured in different carbon sources media based on CPZ media, especially for glucose and trehalose as sole canbon source, the culture broth of disruption mutant appeared lower pH values. We found that disruption strains produced higher concentration of lactic acid, pyruvic acid and citric acid, but lower content of oxalic acid. In addition, disruption mutant appeared the significant reduction in conidia yield on the different carbon/nitrogen sources as well as the formation of conidiation structure was significantly delayed in ABbSNF1mutant. Insect virulence bioassay indicated the ablation of BbSNFl greatly reduced the fungal virulence against Greater Waxmoth, i.e. lethal time (LT50) of disruption mutant prolonged by75%and25%in topical and intrahemocoel injecction bioassay, respectively. qRT-PCR was used to analyze the effect of disruption oof BbSNF1on expression of gene encoding chitinase, protease, carboxylate transporter and sugar transporter. Among tested17chitinase genes,13genes were repressed in in disruption strains, and18of48genes encoding proteinase were significantly repressed. For14genes encoding monocarboxylic acid transporter,9and8genes were enhanced in disruption mutant in trehalose and sucrose as single carbon source, respectively. In addition,31of forty sugar transporter related genes of mutant strains have higher expressing level when cultured on glucose carbon sources media, and28of forty on trehalose carbon sources media.B. bassiana blastospore was another form of infectious propagules as well as an important development stage in insect hemocoel. We studied the regulation of BbSNFl in blastospores gowth and development in different carbon/nitrogen sources. As compared with wild type strain,△BbSNF1mutant showed significant reduction in blastospores yield and mycelial biomass, and blastospore size became smaller in media containing organic nitrogen. We also found that formation of blastospore-producing structure was obviously delayed in disruption strains but not in wild strains and complement strains. Blastospore development was also delayed in insect hemocoel and△BbSNFl mutant was easily melanized by host phagocytes. Thus, BbSNFl plays an important role in the regulation of growth, development, nutrient metabolism and especially the virulence of B. bassiana.a-glucoside transporter gene BbAGTl contributes to carbohydrate utilization, growth, conidiation and virulence Carbohydrate transporters are critical players mediating nutrient uptake during saprophytic and pathogenic growth for most filamentous fungi. For entomopathogenic fungi, such as B. bassiana, assimilation of a-glucosides, in particular, trehalose, the major carbohydrate constituent of the insect haemolymph, has been hypothesized to represent an important ability for infectious growth within the insect hemocoel. In this study, a B. bassiana a-glucoside transporter homolog was identified and genetically characterized via generation of a targeted gene disruption mutant ABbAGT1and gene complementation strain△BbAGT1::BbAGT1. Disruption of the gene BbAGTl resulted in decreased conidial germination, growth, and yield on various carbohydrates (a-glucosides, monosaccharides and polyols) as compared to the wild-type strain. Insect bioassays revealed decreased mean lethal mortality time using both topical and intrahemocoel injection assays, although final mortality levels were comparable in both the mutant and wild type. Gene expression profiles showed altered expression of other putative transporters in the knockout mutant as compared to the wild type. These results highlighted complex sugar utilization and responsiveness in B. bassiana and the potential role for trehalose assimilation during fungal pathogenesis of insects.