Functional characterization of BbEaf6, a homolog of Eaf6 in Beauveria bassiana: Implications for fungal virulence and stress response

Figures & data

Figure 1. Subcellular localization of BbEaf6 and its role on conidiation and conidial properties in B. bassiana.

(A) Laser scanning confocal microscopy (LSCM) images (scale bars: 10 µm) depicting the subcellular localization of GFP-tagged Eaf6 fusion protein expressed in 3-day-old wild type (WT) cells.(B) Quantification of conidial yields during 4 to 9-day cultivation on SDAY plates.(C) Flow cytometry analysis for cell size (FSc) and density (SSc) of aerial conidia collected from 7-day-old SDAY cultures.(D) Assessment of conidial viability indicated by the time required for 50% germination (GT₅₀).(E) Microscopic visualization of growing of the sporophores (pointed by blue arrows) and conidia (pointed by purple arrows) on SDAY plates during 3–6 day cultivation (scale bars = 20 µm).

All experiments were performed in triplicate. (Tukey’s honestly significant difference test, p < 0.05).

Fig. 2. Impact of BbEaf6 deletion on multi-stress tolerance in B. bassiana.

(A, B) Representative images and relative growth inhibition (RGI) of fungal colonies grown at 25°C for 8 days on CDA media supplemented with various stress-inducing agents: NaCl (0.4 M), KCl (0.4 M), CuCl₂ (2 mM), MnCl₂ (4 mM), FeCl₂ (4 mM), H₂O₂ (2 mM), menadione (MND; 0.02 mM), sodium dodecyl sulfate (SDS, 100 μg/ml), Congo red (CGR; 10 μg/ml), hydroxyurea (HU; 10mM), and methyl methanesulfonate (MMS; 0.05%).

(Tukey’s honestly significant difference test, p < 0.05).

Fig. 3. Contributions of Eaf6 in hyphal septation and cell cycle control in B. bassiana.

(A) Microscopic images (scale bars: 20 µm) showing hyphal cells collected from 72-hour-old SDB cultures and stained with calcofluor white (CFW).(B) Flow cytometry analysis for cell size (FSc) and density (SSc) of blastospores collected from 3-day-old nitrogen-limited broth (NLB) cultures.(C) Mean cell length and width of hyphal cells stained with calcofluor white from each strain.(D, E) Flow cytometry analysis for the distribution of cell cycle phases of blastospores collected from 3-day-old nitrogen-limited broth (NLB) cultures.

(Tukey’s honestly significant difference test, p < 0.05).

Fig. 4. Impact of Eaf6 deletion on fungal virulence upon insect host in B. bassiana.

(A-D) Survival trends and median lethal time (LT₅₀) estimates of Galleria mellonella larvae after topical application (immersion) and intrahemocoel injection, respectively.(E, F) Biomass levels and blastospore yields quantified respectively from the 3-day-old submerged cultures in Czapek-Dox broth (CZB) and trehalose-peptone broth (TPB).(G) Images depicting fungal outgrowths on the surfaces of cadavers 4 days post-death.(H) Microscopic images (scale: 20 μm) showing the submerged hyphae and blastospores from the 3-day-old cultures in CZB and TPB, respectively.

(Tukey’s honestly significant difference test, p < 0.05).

Fig. 5. The global regulatory role of BbEaf6 in gene transcription B. bassiana.

(A) Distributions of p values and ratios for the genes significantly up-regulated, down-regulated and not differentially regulated (No-Diff) in ΔEaf6 versus wild type.(B) Cluster analysis of differentially expressed genes (DEGs) in ΔEaf6 versus wild type.(C) Top 20 Gene Ontology (GO) terms enriched based on the differentially expressed genes (DEGs) in ΔEaf6 versus WT.(D, E) Functional annotation for the top 20 functional categories of significantly up- or down-regulated genes in ΔEaf6 versus WT using the FunCat database.