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. 2021 May 29;7(6):431.
doi: 10.3390/jof7060431.

Botrytis cinerea G Protein β Subunit Bcgb1 Controls Growth, Development and Virulence by Regulating cAMP Signaling and MAPK Signaling

Jiejing Tang  1 , Mingde Wu  1 , Jing Zhang  1 , Guoqing Li  1 , Long Yang  1
Affiliations

Botrytis cinerea G Protein β Subunit Bcgb1 Controls Growth, Development and Virulence by Regulating cAMP Signaling and MAPK Signaling

Jiejing Tang et al. J Fungi (Basel). .

Abstract

Botrytis cinerea is a necrotrophic phytopathogenic fungus that causes gray mold disease in many crops. To better understand the role of G protein signaling in the development and virulence of this fungus, the G protein β subunit gene Bcgb1 was knocked out in this study. The ΔBcgb1 mutants showed reduced mycelial growth rate, but increased aerial hyphae and mycelial biomass, lack of conidiation, failed to form sclerotia, increased resistance to cell wall and oxidative stresses, delayed formation of infection cushions, and decreased virulence. Deletion of Bcgb1 resulted in a significant reduction in the expression of several genes involved in cAMP signaling, and caused a notable increase in intracellular cAMP levels, suggesting that G protein β subunit Bcgb1 plays an important role in cAMP signaling. Furthermore, phosphorylation levels of MAP kinases (Bmp1 and Bmp3) were increased in the ΔBcgb1 mutants. Yeast two-hybrid assays showed that Bcgb1 interacts with MAPK (Bmp1 and Bmp3) cascade proteins (BcSte11, BcBck1, BcMkk1, and BcSte50), and the Bmp1-regulated gene Bcgas2 was up-regulated in the ΔBcgb1 mutant. These results indicated that Gβ protein Bcgb1 is involved in the MAPK signaling pathway in B. cinerea. In summary, our results revealed that Gβ protein Bcgb1 controls development and virulence through both the cAMP and MAPK (Bmp1 and Bmp3) signaling pathways in B. cinerea.

Keywords: Botrytis cinerea; Gβ subunit; MAPK signaling pathway; cAMP signaling pathway.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Sequence analysis of Bcgb1 in B. cinerea. (A) Amino sequence alignment of Bcgb1 orthologues. All conversed residues are shown in black and similar residues in grey. The positions of the seven WD repeats are labeled and indicated by arrows. ** means the conserved WD residues in WD repeats. (B) The crystal model of Bcgb1. The Gβ protein of R. norvegicus Gβ (PDB: 7cfm.1.B) was used as the template for the Bcgb1 model at the SWISS-MODEL website. The seven β-propeller blades were numbered. (C) A neighbor-joining tree based on amino acid sequences of Gβ protein in fungi. The following protein sequences were used: XP_018249805 (fgb1), ABE67098 (GBB1), XP_028497849 (VGB), BAC01165 (mgb1), XP_009851210 (gnb-1), XP_024550185 (Bcgb1), XP_001595393 (SS1G_03482), XP_024345016 (SfaD), XP_657685 (sfaD-1), AAO25585 (cgb1), AAD03596 (GPB1), XP_011386498 (bpp1), 5TDH_B (R. norvegicus Gβ protein), NP_014855 (STE4), AAC37501 (gpb1), XP_024548939 (Bcg1), XP_024552854 (Bcg2), and XP_024553380 (Bcg3). Bootstrap values (%) from 1000 replicates of the data are indicated above the nodes. The red dot represents the Gβ protein Bcgb1 of B. cinerea in this study.
Figure 2
Figure 2
Bcgb1 is required for mycelial growth, conidiation, and sclerotia formation. (A) Colony morphology (3 d and 15 d) and mycelium tips (48 h) of the indicated strains cultured on PDA at 20 ℃. (B) Aerial hyphae growth is increased in the ΔBcgb1 mutants after incubation on PDA for 7 days at 20 °C. (C) Mycelial growth rate of the indicated strains cultured on PDA at 20 °C *** p < 0.001. (D) Mycelial biomass of the indicated strains cultured in PDB at 20 °C for 2 d. * p < 0.05.
Figure 3
Figure 3
Bcgb1 is involved in responses to cell-wall and oxidative stresses. (A) Sensitivity test of strains to salt stress (NaCl or KCl), osmotic stress (sucrose or sorbitol), cell-wall stress (SDS, CR, or CFW), and oxidative stress (H2O2). Strains were incubated on PDA supplemented with 1 M NaCl, 1 M KCl, 1 M sucrose, 1 M sorbitol, 0.1 mg/mL SDS, 0.3 mg/mL CR, 0.2 mg/mL CFW, and 5 mM H2O2 at 20 °C for 72 h. (B) The relative mycelial growth rate of the indicated strains in the presence of various stresses. * p < 0.05.
Figure 4
Figure 4
Bcgb1 is important for virulence in B. cinerea. (A) Pathogenicity test of the indicated strains on unwounded and wounded tobacco leaves. Disease symptoms were photographed at 72 h post inoculation (20 °C). (B) Lesion size caused by the indicated strains in A. (C) Infection cushion formation by mycelium plugs of the indicated strains on onion epidermis at 12 h or 24 h post inoculation (20 °C). IC: infection cushion, IH: infectious hyphae. (D) Quantitative analysis of infection cushions of the indicated strains in C. *** p < 0.001.
Figure 5
Figure 5
Bcgb1 is involved in the regulation of intracellular cAMP levels. (A) Quantitative determination of intracellular cAMP levels in mycelia of the indicated strains cultured in PDB for 2 days. Two biological repetitions with three replicates were assayed. The error bars represent the SD of three replicates. (B) Transcript level of Bac, BcPde1, and BcPde2 in the WT and the ΔBcgb1 mutants of B. cinerea. (C) Transcript level of BcPka1, BcPka2, and BcPkaR in the WT and the ΔBcgb1 mutants of B. cinerea. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
Bcgb1 negatively regulates the Bmp1 and Bmp3 MAPK pathway in B. cinerea. (A) Phosphorylation level of MAPK (Bmp1 and Bmp3) in the ΔBcgb1 mutants. Bmp1 and Bmp3 and their phosphorylated proteins were detected using the ERK1/2 and phospho-p44/42 MAPK antibodies, respectively. The intensity of the phosphorylated Bmp1 and Bmp3 band for each strain is relative to that of the Bmp1 and Bmp3 band, respectively. (B) Yeast two-hybrid assay between Bcgb1 and BcSte11/BcSte7/Bmp1 and BcBck1/BcMkk1/Bmp3 cassette. The pGBKT7-53 and pGADT7-T pair of plasmids served as the positive control. The pGBKT7-Lam and pGADT7-T pair of plasmids served as the negative control. Yeast cells were drop-plated on SD-Trp/-Leu/-His with x-α-gal. (C) Transcript level of the Bmp1 MAPK-regulated gene Bcgas2 in the WT and the ΔBcgb1 mutants of B. cinerea. *** p < 0.001.
Figure 7
Figure 7
Transcript level of the sclerotia formation-related genes in the WT and the ΔBcgb1 mutants of B. cinerea. ** p < 0.01, *** p < 0.001.
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