The Elongator Subunit Elp3 Regulates Development, Stress Tolerance, Cell Cycle, and Virulence in the Entomopathogenic Fungus Beauveria bassiana

Abstract

Transcriptional activity is mediated by chromatin remodeling, which in turn is affected by post-translational modifications, including histone acetylation. Histone acetyltransferases (HATs) are capable of promoting euchromatin formation and then activating gene transcription. Here, we characterize the Elp3 GNAT family HAT, which is also a subunit of Elongator complex, in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. BbElp3 showed high localization levels to mitochondria, with some nuclear and cytoplasmic localization also apparent. Targeted gene knockout of BbElp3 resulted in impaired asexual development and morphogenesis, reduced tolerances to multiple stress conditions, reduced the ability of the fungus to utilize various carbon/nitrogen sources, increased susceptibility to rapamycin, and attenuated virulence in bioassays using the greater wax moth, Galleria mellonella. The ΔBbElp3 mutant also showed disrupted cell cycle, abnormal hyphal septation patterns, and enlarged autophagosomes in vegetative hyphae. Transcriptome analyses revealed differential expression of 775 genes (DEGs), including 336 downregulated and 438 upregulated genes in the ΔBbElp3 strain as compared to the wild type. Downregulated genes were mainly enriched in pathways involved in DNA processing and transcription, cell cycle control, cellular transportation, cell defense, and virulence, including hydrophobins, cellular transporters (ABC and MFS multidrug transporters), and insect cuticular degrading enzymes, while upregulated genes were mainly enriched in carbohydrate metabolism and amino acid metabolism. These data indicate pleiotropic effects of BbElp3 in impacting specific cellular processes related to asexual development, cell cycle, autophagy, and virulence.

Keywords: asexual development; autophagy; cell cycle; gene transcription; histone acetyltransferase; multidrug transporter.

Figure 1

Characterization of B. bassiana Elp3: subcellular localization, histone acetyltransferase activity, and contribution to conidiation. (A) Representative images of cellular localization monitoring the BbElp3-GFP fusion protein. Scale bars: 10 µm. Hyphal cells were harvested from 3 d SDB cultures and costained with the mitochondria-specific dye (Mito-tracker Red) and nuclei-specific dye (DAPI). (B) Western blot analyses of histone H3 acetylation. (C) Scanning electron microscopy (SEM) images of aerial hyphae and sporophore of cells harvested from SDAY plats grown for 4 d. Scale bars: upper panels, 20 µm; lower panels, 5 µm. (D) Conidial yields assessed for each strain over a 9 d time course of growth on SDAY plates. (E) Conidial hydrophobicity indexes. (F,G) Conidial germination times (GT50) and conidial tolerances to heat stress and UV-B irradiation determined as detailed in the Methods section. (H) FACS analysis for cell size (FSc) and density (SSc) of aerial conidia grown for 7 d on SDAY. Asterisk indicates significant difference (p < 0.05) from unmarked (Tukey’s HSD). Error bars: SD from three technical replicates.

Figure 2

Contribution of BbElp3 to multi-stress tolerances. (A,B) Representative images and quantification of relative growth inhibition (RGI) of fungal colonies grown at 25 °C for 8 d on CZA supplemented with NaCl (0.4 M), KCl (0.4 M), H₂O₂ (2 mM), menadione (MND; 0.02 mM), rapamycin (RAP, 1 μg mL⁻¹), SDS (100 μg mL⁻¹), Congo Red (CGR; 10 μg mL⁻¹), hydroxyurea (HU; 10 mM), methyl methanesulfonate (MMS, 0.05%), carbendazim (CBD, 10 μg mL⁻¹), and iprodione metabolite (IPM, 10 μg mL⁻¹). All colonies were initiated by spotting 1 μL of 1 × 10⁶ conidia/mL suspension on the plates. All experiments were performed three times, with each containing three technical replicate plates. Error bars = ± SD. Asterisk indicate significant difference (p < 0.05) from unmarked (Tukey’s HSD). Error bars: SD from three technical replicates.

Figure 3

Impact of loss of Elp3 on cell cycle, hyphal septation, and autophagosome formation in B. bassiana. (A) Representative microscopic of hyphal cells (3 d SDB cultures) stained with calcofluor white. Scale bars = 20 µm. (B) Quantification of mean hyphal cell length and width. (C) FACS analyses of blastospore (harvested from NLB cultures grown for 3 d) cell size (FSc) and cell density (SSc). (D,E) FACS analysis for blastospore cell cycle progression. (F) Representative TEM images of hyphal cells (3 d SDB cultures) treated with 10 μg mL⁻¹ rapamycin for 12 h. Scale bars = 0.5 µm. (G) Quantification of the average numbers and diameters of autophagosomes in each rapamycin-treated hyphal cell. Asterisk indicates significant difference (p < 0.05) from unmarked (Tukey’s HSD). Error bars: SD from three technical replicates.

Figure 4

Impact of Elp3 on B. bassiana virulence and virulence-related properties. (A,B) Insect bioassay survival curves and calculated LT₅₀ values using the greater wax moth (G. mellonella) larvae as hosts after topical application (immersion) and intra-hemocoel injection, respectively. (C) Representative images of fungal outgrowth on the surfaces of cadavers 4 d post death. (D,E) Quantification of fungal biomass, total extracellular protease (ECEs), and Pr1 protease activities after 3 d growth in CZB-BSA. (F) Representative microscopic images of submerged hyphae and blastospores after 3 d growth in CZB and TPB. Scale bars = 20 μm. (G,H) Fungal biomass levels and blastospore yields quantified after 3 d growth in CZB and TPB. Asterisk indicates significant difference (p < 0.05) from unmarked (Tukey’s HSD). Error bars: SD from three technical replicates.

Figure 5

Comparative transcriptomic analyses of ΔBbElp3 and wild-type B. bassiana. (A) transcriptome. (A) Distributions of p-values and ratios for the genes identified to be downregulated, upregulated, or not differentially regulated (ND) in ΔBbElp3 versus the wild type. (B) Cluster analysis of the 775 differentially expressed genes (DEGs) found in the transcriptomic analyses of the ΔBbElp3 mutant versus wild-type strains. (C) FunCat annotation into 15 functional categories of significantly regulated genes in ΔBbElp3 versus wild type. A, metabolism; B, protein with binding function or cofactor requirement; C, cell rescue, defense, and virulence; D, cellular transportation; E, protein fate; F, transcription; G, cell cycle and DNA processing; H, biogenesis of cellular components; I, cellular communication/signal transduction mechanism; J, regulation of metabolism and protein function; K, energy; L, cell type differentiation; M, protein synthesis; N, systemic interaction with the environment; O, cell fate. (D) Counts and confidence intervals of differentially expressed genes enriched into the top 20 KEGG pathways in ΔBbElp3 versus the wild type.