Functional Characterization Of Gelsolin Gene BbGEL1 And Analysis Of Related Genes Regulated By Protein Kinase Gene BbSNF1 During Conidiation In Entomopathogenic Fungus Beauveria Bassiana

Beauveria bassiana is not only a classic entomopathogenic fungus, but also an important model for fungal growth and development. In this study, we use B. bassiana as a model of fungal pathogen to explore the function of BbGEL1 in fungal metabolism, development and pathogenisis. Meanwhile, RNA-sequencing technique is used to reveal the downstream genes regulated by BbSNF1 during conidiationof B. bassiana.Functional analysis of BbGEL1 BbGEL1 open reading frame is determined to 1185bp long, without introns, encoding 394 amino acids. BbGEL1 protein belongs to CapG protein in gelsolin superfamily and contains three conserved gelsolin-like domains. To explore the BbGEL1 function, the gene disruption mutant and complement starins were constructed by strategy of homologous recombination and ectopic insertation, respectively. In B. bassiana, BbGEL1 protein does not only co-localize with actin, but also exsit in the other forms (e.g., in free state). Disruption of BbGELl could cause less formation of actin patches in mutant. Phenotypic analysis showed that BbGEL1 is required for fungal metabolism and response to thermal stress. Among seven different carbon sources, ablation of BbGELl resulted in defective growth of disruption mutant on media containing glucose and sodium acetate as single carbon source. Compared with wild type strain and complement strain; the disruption mutant appeared significant reduction in vetative growth on the mediua using gelatin, bovine serum albumin (BS A), casein as a nitrogen source, colony diameters reducing 33.33%,46.15% and 44.82% respectively. BbGEL1 is also required for fungal recovery growth at 25℃ after thermal stress at 40℃. However, the elevated temperatures for recovery growth could eliminate the difference in recovery growth between wild type and disruption mutant. BbGELl contributes to maintain the metabolism and extracelluar acidification ΔBbGELl mutants appeared to have the towered activity of extracelluar proteases and the increased ability to acidify the media. Mineral salts in Czapek’s medium plus BS A were supplemented with glucose, sucrose, trehalose or glycerol as the single carbon source. The disruption mutants have a significantly enhanced oxalic acid production whether extracellar or intracellular. This enhancement was especially obvious when sucrose as a carbon source, disruption mutants have a 15.4-fold increase when compared with wild type strain. BbGEL1 plays an important role in development fungal spores (conidia and blastospores). Wild strains and complement strains began to produce conidia on the forth day, and conidial yield almost reached the maximum on the eighth day. However, the disruption strains could not produce conidia until the eighth day, and conidial yield was only 6% of the wild-type strain, on the ninth day, reaching 30%. Compared with wild type strains, ΔBbGEL1 mutant produced very few blastospores and blastospore production fell by about 90%. Microscopic view showed the disruption of BbGELl resulted in significant reduced thickness in cell walls of mycelia (aerial and submerged) and spores (conidia and blastospores). Two bioassay methods, intrahemocoel injection and topical application, were used to evaluate the fungal pathogenicity against greater waxmoth. As compared with wild type, the disruption mutants appeared a 1.3-d delay in median lethal time (LT50) in intrahemocoel assays, In topical assays, the LT50 of disruption mutants was 8.3 days, which was significantly longer than that of wild type strain (4.2 days). Thus, BbGELl plays an important role in metabolism, spore development and pathogenicity of B. bassiana.Downstream genes of BbSNFl during conidiation Conidiation promotes fungal dispersal and survival in the environment, and is a determinant for the biocontrol potential of Beauveria bassiana. The SNF1/AMPK protein kinases function as an important regulator of fungal development and energy metabolism, and play a crucial role in conidiation of the filamentous fungi. In previous study, it has been established that the B. bassiana homo log (BbSNF1) controls conidial production This study showed that the ΔBbSNFl mutants displayed a delayed development of mycelia and conidia, but the conidiophore morphogenesis was not significantly changed in the mutants. Ablation of BbSNF1 significantly changed the metabolic homeostasis of intracellular amino acids during conidiation, and caused a notable reduction in the contents of seven amino acids (i.e., arginine, alanine, valine, phenylalanine, lysine, leucine, and glutamic acid). All above amino acids could recover conidiation of the mutants in different extents (ranging from 43.3-300%). Transcriptomic analysis revealed many putative target genes regulated by BbSNFl and associated withconidial development, and these genes were primarily involved in metabolism, cell rescue, and transport. Particularly, four categories related to the amino acid degradation were over-represented in the up-regulated genes, and three categories related to the amino acid biosynthesis were over-represented in the down-regulated genes. Moreover, the ΔBbSNF1 mutants displayed reduced expression level of the upstream and central regulators of conidiation, as well as the other regulator and cytoskeleton genes. Our data indicate that SNF1 kinase contributes to B. bassiana conidiation by regulating the metabolism and the central regulators of conidiation.