Phosphorylation status of a conserved residue in the adenylate cyclase of Botrytis cinerea is involved in regulating photomorphogenesis, circadian rhythm, and pathogenicity

Abstract

Adenylate cyclase (AC) regulates growth, reproduction, and pathogenicity in many fungi by synthesizing cyclic adenosine monophosphate (cAMP) and activating downstream protein kinase A (PKA). Botrytis cinerea is a typical necrotrophic plant-pathogenic fungus. It shows a typical photomorphogenic phenotype of conidiation under light and sclerotia formation under dark; both are important reproduction structures for the dispersal and stress resistance of the fungus. The report of B. cinerea adenylate cyclase (BAC) mutation showed it affects the production of conidia and sclerotia. However, the regulatory mechanisms of the cAMP signaling pathways in photomorphogenesis have not been clarified. In this study, the S1407 site was proven to be an important conserved residue in the PP2C domain which poses a remarkable impact on the phosphorylation levels and enzyme activity of the BAC and the overall phosphorylation status of total proteins. The point mutation bac^S1407P , complementation bac^P1407S , phosphomimetic mutation bac^S1407D , and phosphodeficient mutation bac^S1407A strains were used for comparison with the light receptor white-collar mutant Δbcwcl1 to elucidate the relationship between the cAMP signaling pathway and the light response. The comparison of photomorphogenesis and pathogenicity phenotype, evaluation of circadian clock components, and expression analysis of light response transcription factor genes Bcltf1, Bcltf2, and Bcltf3 showed that the cAMP signaling pathway could stabilize the circadian rhythm that is associated with pathogenicity, conidiation, and sclerotium production. Collectively, this reveals that the conserved S1407 residue of BAC is a vital phosphorylation site to regulate the cAMP signaling pathway and affects the photomorphogenesis, circadian rhythm, and pathogenicity of B. cinerea.

Keywords: Botrytis cinerea; adenylate cyclase; cAMP; circadian clock; pathogenicity; photomorphogenesis.

Figure 1

(A) Schematic representation of the in situ point mutation strategy. (B) PCR amplification of target sequences (bac^S1407D and bac^S1407A) by different primer sets (right), and electrophoretic identification of the target sequences (left). (C) Intracellular levels of cAMP in the wild type (B05.10), mutant strain (bac^S1407P, bac^S1407A, bac^S1407D, and bac^P1407S) observed after incubation on CM for 3 days under conditions involving light. The different letters on the columns indicate significant differences at p < 0.05. The bars present mean values ± SD (n = 3).

Figure 2

(A) Botrytis cinerea adenylate cyclase (BAC) domains analyzed using InterPro and SMART. (B) Conservation analysis of the S1407 locus of BAC. (C) The phosphorylation of BAC-His₆ and BAC^S1407P-His₆ was detected by a mobility shift on a Phos-tag SDS–PAGE gel. Red arrows represent different positions of migration (migration direction from bottom to top). (D) Concentration curve of cAMP synthesized by BAC-His₆ PP2C and AC domains and BAC^S1407P-His₆ PP2C and AC domains under different concentrations of ATP.

Figure 3

Differences in phosphorylation levels of total proteins of mycelia of wild-type (B05.10) and mutation strains (bac^S1407P, bac^S1407A, and bac^S1407D) growing for 3 days on CM under light (A) and dark (B) conditions.

Figure 4

Growth and pathogenic phenotype analysis of different strains. (A) Wild-type (B05.10) and mutant strains (bac^S1407P, bac^S1407A, bac^S1407D, and bac^P1407S) were incubated onto solid CM medium for 3 or 10 days under continuous white light (LL) and dark (DD) conditions. (B–D) Statistical analyses of the colony diameters, conidia, and sclerotia of (A). (E) Wild-type (B05.10) and mutant strains (bac^S1407P, bac^S1407A, bac^S1407D, and bac^P1407S) were incubated onto grape (72 h), tomato leaf (48 h), and apple surfaces (96 h). (F) Statistical analysis of the lesion area of (E). Different letters indicate significant differences at p < 0.01. The bars present mean values ± SD (n = 3).

Figure 5

(A) Colony appearance of different strains under blue light (BL), red light (RL), continuous dark (DD), and continuous light (LL) conditions. Wild-type (B05.10) and mutant strains (bac^S1407P, bac^S1407A, bac^S1407D, and bac^P1407S) were incubated onto the solid CM medium for 3 days under BL, RL, DD, and LL conditions. The white bar represents 10 mm. (B) The colony growth rate of test strains under the same conditions as those depicted in (A). Different letters indicate significant differences at p < 0.05. The bars present mean values ± SDs (n = 3).

Figure 6

Comparison of lesion areas at dawn and dusk during the interaction of B. cinerea and A. thaliana leaves (A) and B. cinerea and tomato fruits (D). The conidia were cultured under LD conditions. PDB was used to suspend the conidia to 1 × 10⁶ conidia/μl, the suspension was then placed in the dark for 24 h after inoculation. The white bar represents 5 mm. (B,E) Statistical analysis of contrast of gray values of the lesions recorded at dusk and dawn in (A,D). (C,F) Statistical analysis of lesion diameter in (A,D). Different letters indicate significant differences at p < 0.05. The bars present mean values ± SDs (n = 3).

Figure 7

(A) The banding pattern of the mycelial colony produced by the wild type (B05.10) and mutant (bac^S1407P, bac^S1407A, bac^S1407D, bac^P1407S, Δbcwcl1, and Δbcwcl1-com) strains on SDS-containing CM medium for 7 days under LD conditions. SDS-containing CM medium was used to reduce mycelial growth to approximately 50% of ordinary radial growth to obtain a clearer pattern. The white bar represents 10 mm. The expression of circadian clock components Bcfrq1 in wild-type B05.10, bac^S1407P (B) and Δbcwcl1 (E), and Bcwcl1 in the wild type (C,D). The expression of B05.10 Bcfrq1 at 3 h (B,E) and B05.10 Bcwcl1 at 3 h (C,D) was used as a ruler to compare the expression changes of genes at different time points under LD conditions for 2 days. Error bars are standard deviations (n = 3).

Figure 8

(A) Race tube assay for the same strains grown on CM media with or without IBMX at 9 days under LD conditions along with the circadian rhythms after incubation. (B) Comparison of amino acid sequences in phosphorylation sites between N. crassa WC1 and B. cinerea BcWCL. (C) Yeast two-hybrid analysis of BcPKAR, BcFRQ1, and BcWCL1. In this assay, yeast cells were used at various densities (10⁻¹ to 10⁻³ conidia/μl dilution). AD and BD represent empty plasmids.

Figure 9

The conidia and sclerotia reproduction-related genes, Bcltf1, Bcltf2, and Bcltf3 in the wild type (B05.10) (A,D,G), bac^S1407P (B,E,H), and Δbcwcl1 (C,F,I) at different time points under LD conditions. The expression of B05.10 Bcltf1 (A–C), B05.10 Bcltf2 (D–F), and B05.10 Bcltf3 (G–I) recorded after 3 h was used as a ruler to compare the changes in the expression of genes at different time points under LD conditions for 2 days. Error bars represent standard deviations (n = 3).

Figure 10

(A) WCC binding site analysis of the Bcfrq1, Bcltf1, Bcltf2, and Bcltf3 promoter regions. The red and black regions represent the gene 5’UTR and the genome region before the 5’UTR, respectively. (B) Promoter binding evaluation of BcWCL1 and Bcfrq1, Bcltf1, Bcltf2, and Bcltf3 by Yeast one-hybrid analysis.

Figure 11

The conidiation and sclerotia formation in B. cinerea are regulated by the cAMP signaling pathway.