Unveiling a Novel Role of Cdc42 in Pyruvate Metabolism Pathway to Mediate Insecticidal Activity of Beauveria bassiana

Abstract

The small GTPase Cdc42 acts as a molecular switch essential for cell cycles and polar growth in model yeast, but has not been explored in Beaurveria bassiana, an insect-pathogenic fungus serving as a main source of fungal formulations against arthropod pests. Here, we show the indispensability of Cdc42 for fungal insecticidal activity. Deletion of cdc42 in B. bassiana resulted in a great loss of virulence to Galleria mellonella, a model insect, via normal cuticle infection as well as defects in conidial germination, radial growth, aerial conidiation, and conidial tolerance to heat and UVB irradiation. The deleted mutant's hyphae formed fewer or more septa and produced unicellular blastospores with disturbed cell cycles under submerged-culture conditions. Transcriptomic analysis revealed differential expression of 746 genes and dysregulation of pyruvate metabolism and related pathways, which were validated by marked changes in intracellular pyruvate content, ATP content, related enzyme activities, and in extracellular beauvericin content and Pr1 protease activity vital for fungal virulence. These findings uncover a novel role for Cdc42 in the pathways of pyruvate metabolism and the pyruvate-involved tricarboxylic acid cycle (TCA cycle) and a linkage of the novel role with its indispensability for the biological control potential of B. bassiana against arthropod pests.

Keywords: Pr1 family proteases; TCA cycle; beauvericin; cell cycle; entomopathogenic fungi; gene expression and regulation; pyruvate metabolism; virulence.

Figure 1

Role of cdc42 in the asexual cycle in vitro of B. bassiana. (A) Diameters of SDAY colonies incubated for 7 days after initiated with ~10³ conidia at the optimal regime. (B) Conidial yields measured from the SDAY cultures during a 7 day incubation after initiated by spreading 100 μL aliquots of a 10⁷ conidia/mL suspension at the optimal regime. (C–E) GT₅₀ (h), LT₅₀ (min), and LD₅₀ (J/cm²) estimates as the respective indices of conidial quality for the time length of 50% germination at 25 °C, tolerance to a wet-heat stress at 45 °C and resistance to UVB irradiation. * p < 0.05 in Tukey’s HSD tests. Error bars: SDs from three replicates.

Figure 2

Impact of cdc42 deletion on the cell cycle of blastospores and the formation of hyphal septa in B. bassiana. (A) Cell-cycle patterns of unicellular blastospores stained with the DNA-specific dye propidium iodide after collection from the 3-day-old SDBY colonies incubated at optimal 25 °C. Different cell-cycle phases are shown as standard deviations of the means from three samples per strain (2 × 10⁴ stained spores per sample). (B) LSCM images (scales: 5 μm) of hyphal septum patterns and morphology. The presented hyphae were stained with the cell-wall-specific dye calcofluor white after collection from the 3-day-old SDBY cultures. Note that two opposite types of abnormal septum patterns are present in the Δcdc42 mutant’s culture.

Figure 3

Essential role of cdc42 in insect-pathogenic lifestyle of B. bassiana. (A) Survival percentages of G. mellonella larvae after topical application (immersion) of a 10⁷ conidia/mL suspension for normal cuticle infection and LT₅₀ (d) estimates made by modeling analysis of time-mortality trends. (B) Microscopic images (scale: 20 μm) for the status of hyphal bodies (arrowed) and insect hemocytes (HC) in the hemolymph samples taken from surviving larvae 5 d post-infection. (C) Concentrations of hyphal bodies (HBs) from the hemolymph samples. * p < 0.05 in Tukey’s HSD tests. Error bars: SDs from three independent replicates (A) or three surviving larvae infected per strain (C).

Figure 4

Transcriptomic analysis of Δcdc42 versus WT strains in B. bassiana. (A) Heat map of differentially expressed genes (DEGs). (B) Counts of DEGs enriched to GO terms of three function classes. (C) Counts of DEGs enriched to KEGG pathways at different levels of p values.

Figure 5

Diagram for involvements of identified DEGs in the pyruvate metabolism and related pathways of the Δcdc42 mutant versus the WT strain. Red and green denotes up- and downregulated genes, respectively. Each gene with its tag locus in the B. bassiana genome is illustrated with its expression status in Δcdc42 (red or green) as compared to WT.

Figure 6

Impacts of cdc42 deletion on pyruvate metabolism, energy production, and virulence-related beauvericin and Pr1 proteases in B. bassiana. (A–C) Pyruvate contents, activities of TCA cycle-related citrate synthase (CS) and fumarate dehydrogenase (FDH), and ATP contents quantified from the extracts of 5-day-old SDBY cultures incubated at 25 °C, respectively. (D) Beauvericin contents measured from the supernatants of the 5-day-old SDBY cultures by HPLC. (E) Total activities of Pr1 family proteases quantified from the supernatants of the 5-day-old SDBY cultures. p < 0.05 *, 0.01 ** or 0.001 *** in Student’s t tests. Error bars: SDs from three independent samples analyzed.