The Zinc Finger Transcription Factor BbCmr1 Regulates Conidium Maturation in Beauveria bassiana

Abstract

The entomopathogenic fungus Beauveria bassiana is a typical filamentous fungus and has been used for pest biocontrol. Conidia are the main active agents of fungal pesticides; however, we know little about conidial developmental mechanisms and less about maturation mechanisms. We found that a Zn₂Cys₆ transcription factor of B. bassiana (named BbCmr1) was mainly expressed in late-stage conidia and was involved in conidium maturation regulation. Deletion of Bbcmr1 impaired the conidial cell wall and resulted in a lower conidial germination rate under UV (UV), heat shock, H₂O₂, Congo red (CR) and SDS stresses compared to the wild type. Transcription levels of the genes associated with conidial wall components and trehalose synthase were significantly reduced in the ΔBbcmr1 mutant. Further analysis found that BbCmr1 functions by upregulating BbWetA, a well-known transcription factor in the central development of BrlA-AbaA-WetA. The expression of Bbcmr1 was positively regulated by BbBrlA. These results indicated that BbCmr1 played important roles in conidium maturation by interacting with the central development pathway, which provided insight into the conidial development networks in B. bassiana. IMPORTANCE Conidium maturation is a pivotal event in conidial development and affects fungal survival ability under various biotic/abiotic stresses. Although many transcription factors have been reported to regulate conidial development, we know little about the molecular mechanism of conidium maturation. Here, we demonstrated that the transcription factor BbCmr1 of B. bassiana was involved in conidium maturation, regulating cell wall structure, the expression of cell wall-related proteins, and trehalose synthesis. BbCmr1 orchestrated conidium maturation by interplaying with the central development pathway BrlA-AbaA-WetA. BbBrlA positively regulated the expression of Bbcmr1, and the latter positively regulated BbwetA expression, which forms a regulatory network mediating conidial development. This finding was critical to understand the molecular regulatory networks of conidial development in B. bassiana and provided avenues to engineer insect fungal pathogens with high-quality conidia.

Keywords: BbCmr1; BbWetA; Beauveria bassiana; conidium maturation.

FIG 1

Expressed profile of Bbcmr1 in WT. (A) The relative transcript level of Bbcmr1 in WT incubated in PDB for 4 d and on PDA for 5-15d. (B) DIC and fluorescence microscopic images of P_Bbcmr1::eGFP strain in liquid medium (PDB) for 12–96 h and on solid medium (PDA) for 12–360 h. GFP fluorescence was observed in germinated conidia, hyphae, blastospores, conidiophore, newly formed conidia, and mature conidia. pb3::eGFP strain with constructive promoter pgpdA (pb3) was used as a positive control (Scale bars, 2 μm).

FIG 2

Conidial germination rates of various strains under different stresses. The treatments, including standardized condition (A), UV-B irradiation (B), heat stress at 30°C (C), H₂O₂ (2 mM), SDS (0.2 mg/ml), Congo red (CR, 0.8 mg/ml), NaCl (1.2 M), and stored spores for 60 d at 26°C (D). *, P < 0.05, **, P < 0.01.

FIG 3

BbCmr1 affects cell wall properties of conidia. (A) Representative TEM images of conidia. (B) Fluorescence images of lecting binding profiles of aerial conidia. Fluorescent lectins WGA, PNA and ConA were specific for β-GluNAc and sialic acid residues, β-galactose, and α-Glc/α-GlcNA respectively (scale bar, 5 μm). (C) chitin content, (D) hydrophobicity, and (E) intracellular trehalose accumulation from 15-day-old aerial conidia incubated on 1/4SDAY. *, P < 0.05, **, P < 0.01.

FIG 4

Transcriptional analyses of genes influenced by Bbcmr1. (A) Cluster analysis of differentially expressed genes (DEGs) in the transcriptomes from the 10 d-old PDA cultures of ΔBbcmr1 and WT strains. Distributions of log₂ ratios (≥1 or≤ −1) and adjusted P values (P_adjust <0.05) for 1061 DEGs identified from ΔBbcmr1. Upregulated (B) and downregulated (C) genes in ΔBbcmr1 were categorized according to putative functions gathered from Fungal Genome Database (FunCat).

FIG 5

(A) Relative transcript levels of the conidial wall component genes in ΔBbcmr1 strain culture grown for 10 d or 15 d on PDA under 26°C. (B) Subcellular localization of BbRds1::mCherry fusion proteins expressed in Bbcmr1 deletion and WT strains. Fungal strains were cultured on the solid medium PDA for 12–360 h. Red fluorescent signals in different cell types were observed. (Scale bar, 2 μm).

FIG 6

(A) BbwetA expressed in the conidia of fungi. Relative transcript levels of BbwetA in ΔBbcmr1 and wide-type strains grown on 1/4SDAY for 5–20 d at 26°C. (B) Yeast one-hybrid analysis of the interaction of BbCmr1 and the BbwetA promoter. The promoter of BbwetA was divided into P1 (−1049 ∼ −1500), P2 (−548 ∼ −1048), and P3 (−1 ∼ −547).

FIG 7

The relative expressions of conidial wall component genes (A-C) and intracellular trehalose synthase genes (D) in ΔBbcmr1/BbwetA^OE and ΔBbwetA/Bbcmr1^OE grown on 1/4SDAY for 10 d.

FIG 8

Conidial germinations of the fungal strains under SDS stress. (A) Conidial suspensions (100 μl, 1×l0⁸ conidia/ml) were sprayed onto CZA plates (90 mm) containing SDS (0.2 mg/ml) and cultivated for 24 h. Germinated conidia were microscopically observed (A) and the germination rates were analyzed (B). *, P < 0.05. (scale bars, 5 μm).

FIG 9

(A) Relative transcript levels of conidiation-related genes from 10-day-old cultures of Bbcmr1 deletion mutant on PDA. (B) Relative transcript level of Bbcmr1 in ΔBbsmr1, ΔBbbrlA, and ΔBbsmr1/BbbrlA^OE (overexpression BbbrlA in ΔBbsmr1 background) strains grown on PDA for 10 d, respectively.

FIG 10

(A) EMSA of the interaction between BbBrlA and promoter of Bbcmr1. The promoter region of Bbcmr1 (642 bp to 692 bp upstream of ATG) was used as labeled probe, and unlabeled probe was added in a 200-fold excess. (B) Yeast one-hybrid analysis of BrlA binding the promoter of Bbcmr1. (C) EMSA of the binding activity of BbCmr1 with the promoter of BbbrlA (586 bp to 637 bp upstream of ATG). Each lane contained 10 ng labeled probe, purified protein (0.5 2 μg) or purified protein (2 μg) and unlabeled probe (200 800–fold excess) were added in reactions. (D) Yeast one-hybrid analysis for the interaction between BbCmr1 and the promoter of BbbrlA. (E) A4GA3 was the possible binding site of BbCmr1.

FIG 11

Regulatory networks of conidium maturation. BbCmr1 was responsible for trehalose accumulation and cell wall integrity via BbWetA. BbWetA and BbVosA were both required for conidium maturation. Green arrows indicated positive regulation, and red lines indicated negative regulation, dotted lines represented the uncertain control relationship.