Comprehensive Insights into the Remarkable Function and Regulatory Mechanism of FluG during Asexual Development in Beauveria bassiana

Abstract

Asexual development is the main propagation and transmission mode of Beauveria bassiana and the basis of its pathogenicity. The regulation mechanism of conidiation and the key gene resources for utilization are key links to improving the conidia yield and quality of Beauveria bassiana. Their clarification may promote the industrialization of fungal pesticides. Here, we compared the regulation of morphology, resistance to external stress, virulence, and nutrient utilization capacity between the upstream developmental regulatory gene fluG and the key genes brlA, abaA, and wetA in the central growth and development pathway. The results showed that the ΔbrlA and ΔabaA mutants completely lost the capacity to conidiate and that the ΔwetA mutant had seriously reduced conidiation capacity. Although the deletion of fluG did not reduce the conidiation ability as much as deletions of brlA, abaA, and wetA, it significantly reduced the fungal response to external stress, virulence, and nutrient utilization, while the deletion of the three other genes had little effect. Via transcriptome analysis and screening the yeast nuclear system library, we found that the differentially expressed genes in the ΔfluG mutants were concentrated in the signaling pathways of ABC transporters, propionate metabolism, tryptophan metabolism, DNA replication, mismatch repair, and fatty acid metabolism. FluG directly acted on 40 proteins that were involved in various signaling pathways such as metabolism, oxidative stress, and cell homeostasis. The analysis indicated that the regulatory function of fluG was mainly involved in DNA replication, cell homeostasis, fungal growth and metabolism, and the response to external stress. Our results revealed the biological function of fluG in asexual development and the responses to several environmental stresses as well as its influence on the asexual development regulatory network in B. bassiana.

Keywords: Beauveria bassiana; asexual development; protein interaction; signaling pathway; upstream developmental regulatory protein; virulence.

Figure 1

Effects of fluG, brlA, abaA, and wetA on vegetative growth, aerial conidiation, and blastospore production of B. bassiana. (A) Top (row 1), bottom (row 2), and side (row 3) views of fungal colonies initiated with hyphal mass plugs (5 mm diameter) and cultivated for 10 days on SDAY at 25 °C. (B) Conidial yields from SDAY cultures initiated with 100 μL of hyphal suspension per plate and grown for 7 days at 25 °C with a 12:12 h light/dark cycle. (C) Blastospore yields in the submerged SDB cultures over 7 days of incubation at 25 °C and 150 rpm.

Figure 2

Contributions of fluG, brlA, abaA, and wetA to multiple stress responses and virulence of B. bassiana during growth. (A–F): EC₅₀ values of chemical stressors required to suppress radial growth by 50% after 7 days of cultivation on CZA plates at 25 °C with 12:12 h light/dark cycle. (G) LT₅₀ (no. of days) for hyphal virulence to G. mellonella larvae inoculated via topical application (immersed). Note: different letters on the bars denote significant differences in each group (Tukey’s HSD, p < 0.05). Error bars: SDs from three replicates.

Figure 3

Effects of fluG, brlA, abaA, and wetA on nutrient utilization of B. bassiana. (A–D) Relative colony sizes of B. bassiana strains initiated with hyphal mass plugs with 5 mm diameters, measured as the ratio of the mutant strain’s growth area to the wild strain’s growth area after 7 days of cultivation on various media at 25 °C. Note: different letters on the bars denote significant differences in each group (Tukey’s HSD, p < 0.05). Error bars: SDs from three replicates.

Figure 4

Overview of RNA-Seq data. (A) Histogram of differentially expressed genes (DEGs) from the ΔfluG mutant compared with the WT. Red and blue colored bars represent significantly upregulated and downregulated genes, respectively. (B) Gene ontology (GO) classification of DEGs from the ΔfluG mutant compared with the WT. The enriched GO terms are along the vertical axis, and the horizontal axis indicates the percentage of DEGs in a given term. (C,D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of up- and downregulated DEGs from the ΔfluG mutant compared with the WT. The ordinate represents the pathway name, the abscissa represents the enrichment factor, and the point size represents the number of DEGs in that pathway, while the point colors denote the differing Q-value ranges.

Figure 5

Heat map analysis of DEGs in key signaling pathways. (A,B) The relative transcript levels of DEGs in eight important signaling pathways were analyzed and are shown using a heat map.

Figure 6

GO classification and KEGG pathway enrichment analysis of proteins that FluG interacted with. (A,B): GO classification and KEGG pathway enrichment analysis of proteins that were selected by yeast two-hybrid library screening.

Figure 7

Genetic model diagram for upstream and central regulators in B. bassiana.