BbWor1, a Regulator of Morphological Transition, Is Involved in Conidium-Hypha Switching, Blastospore Propagation, and Virulence in Beauveria bassiana

Abstract

Morphological transition is an important adaptive mechanism in the host invasion process. Wor1 is a conserved fungal regulatory protein that controls the phenotypic switching and pathogenicity of Candida albicans. By modulating growth conditions, we simulated three models of Beauveria bassiana morphological transitions, including CTH (conidia to hyphae), HTC (hyphae to conidia), and BTB (blastospore to blastospore). Disruption of BbWor1 (an ortholog of Wor1) resulted in a distinct reduction in the time required for conidial germination (CTH), a significant increase in hyphal growth, and a decrease in the yield of conidia (HTC), indicating that BbWor1 positively controls conidium production and negatively regulates hyphal growth in conidium-hypha switching. Moreover, ΔBbWor1 prominently decreased blastospore yield, shortened the G₀/G₁ phase, and prolonged the G₂/M phase under the BTB model. Importantly, BbWor1 contributed to conidium-hypha switching and blastospore propagation via different genetic pathways, and yeast one-hybrid testing demonstrated the necessity of BbWor1 to control the transcription of an allergen-like protein gene (BBA_02580) and a conidial wall protein gene (BBA_09998). Moreover, the dramatically weakened virulence of ΔBbWor1 was examined by immersion and injection methods. Our findings indicate that BbWor1 is a vital participant in morphological transition and pathogenicity in entomopathogenic fungi. IMPORTANCE As a well-known entomopathogenic fungus, Beauveria bassiana has a complex life cycle and involves transformations among single-cell conidia, blastospores, and filamentous hyphae. This study provides new insight into the regulation of the fungal cell morphological transitions by simulating three models. Our research identified BbWor1 as a core transcription factor of morphological differentiation that positively regulates the production of conidia and blastospores but negatively regulates hyphal growth. More importantly, BbWor1 affects fungal pathogenicity and the global transcription profiles within three models of growth stage transformation. The present study lays a foundation for the exploration of the transition mechanism of entomopathogenic fungi and provides material for the morphological study of fungi.

Keywords: Beauveria bassiana; Wor1; blastospore; conidia; germination; morphological transition; virulence.

FIG 1

Disruption of BbWor1 affects fungal vegetative growth and asexual development of B. bassiana. (A) Images of fungal colonies at 6 days and 14 days of growth under the temperature condition of 25°C on SDAY medium spotted with 1-μl aliquots of conidial suspension (scale bar: 20 mm). (B) Colony size of fungal colonies from 6 days to 14 days of culture on SDAY at 25°C. (C) Biomass and conidial yield of the fungal strains on SDAY plates for 6 days at 25°C in the HTC model (bottom). In the model of conidium-hypha switching by BbWor1 regulation, arrows and bars represent positive regulation and negative regulation, respectively (top). (D) Time until 50% conidial germination (GT₅₀) in GB broth. (E) Blastospore yields after 4 days of culture in NLB medium at 25°C. (F) Cell cycle (G₀/G₁, S and G₂/M phases) of unicellular blastospores determined by DNA content profiles with FACS analysis. The asterisk (*) denotes significant differences (Tukey’s HSD, P < 0.05). Error bars, standard deviations (SDs) of three replicates.

FIG 2

Effects of BbWor1 loss on the virulence of B. bassiana. (A and B) Mortalities in immersion and intrahemoceol injection assays of ΔBbWor1 (DM) and WT or ΔBbWor1/BbWor1 (CM) were recorded. (C) The mean lethal time (LT₅₀) for immersion and intrahemoceol injection tests were analyzed with Probit analysis. (D) Microscopy images (scale: 20 μm) obtained by LSCM for the blastospores in the hemolymph samples of surviving larvae at 3 days postinjection. Black arrows indicate blastospores, and white arrows indicate host hemocytes. (E) Images of fungal outgrowths at the surface of cadavers 3 days post-death through immersion (left) or injection (right) bioassays. The asterisk (*) denotes significant differences (Tukey’s HSD, P < 0.05). Error bars, SDs of three replicates.

FIG 3

Disruption of BbWor1 affects the transcription profile of B. bassiana in the three models. (A) DEGs whose expression was upregulated and downregulated were determined in the ΔBbWor1 strain via comparison with WT in the CTH, HTC, and BTB models. (B) Venn diagram showing the number of DEGs between the WT and ΔBbWor1 strains in the three models. (C to E) GO classification of DEGs into three main categories: biological processes, cellular components, and molecular functions. (F and G) GO enrichment analysis for DEGs whose expression was upregulated and downregulated between WT and ΔBbWor1 in the HTC and BTB models. The top 5 terms are displayed (Q < 0.05).

FIG 4

Identification of BbWor1 downstream genes and determination of the expression of target genes. (A) The binding site data of Wor1 (21, 22) were used to identify consensus binding sites in promoter regions of 4 genes selected from 37 common repressed DEGs in three models. (B) Yeast one-hybrid assay of the interaction of BbWor1 and 4 gene motifs. All motifs were introduced to autoactivation testing with 250 ng/ml aureobasidin A (AbA) on SD/−Ura medium, and physical interactions were measured on SD/−Leu medium with 250 ng/ml AbA. PC, positive control (yeast cells transformed with the pGADT7-Rec-p53 vector and p53-AbAi). NC, negative control (yeast cells transformed with pGADT7-Rec-BbWor1 and a blank vector [pAbAi]). (C and D) qRT-PCR validation of BBA_02580 and BBA_0998 expression. The relative transcript levels (RTLs) of the two genes are presented as the ratio of WT in the CTH model. Different lowercase letters denote significant differences (Tukey’s HSD, P < 0.05).