BcRPD3-Mediated Histone Deacetylation Is Involved in Growth and Pathogenicity of Botrytis cinerea

Abstract

Histone deacetylase activity plays an important role in transcriptional repression. Botrytis cinerea is an important necrotrophic fungal pathogen distributed worldwide and parasites a wide range of hosts. However, the molecular mechanisms of how B. cinerea regulates growth and host infection remain largely unknown. Here, the function of BcRPD3, a histone deacetylase of B. cinerea, was investigated. Overexpression of the BcRPD3 gene resulted in significantly decreased acetylation levels of histone H3 and H4. The BcRPD3 overexpression strains showed slightly delayed vegetative growth, dramatically impaired infection structure formation, oxidative stress response, and virulence. RNA-Seq analysis revealed that enzymatic activity related genes, including 9 genes reported to function as virulence factors, were downregulated in BcRPD3 overexpression strain. Chromatin immunoprecipitation followed by qPCR confirmed the enrichment of BcRPD3 and H3Kac at the promoter regions of these nine genes. These observations indicated that BcRPD3 regulated the transcription of enzymatic activity related genes by controlling the acetylation level of histones, thereby affecting the vegetative growth, infection structure formation, oxidative stress response, and virulence of B. cinerea.

Keywords: Botrytis cinerea; RNA-seq; deacetylase; histone acetylation; pathogenicity.

FIGURE 1

Classification and localization of BcRPD3. (A) Phylogenetic tree of BcRPD3 based on a neighbor-joining analysis using MEGA-X. The orthologs are from V. dahliae RPD3 (Ensembl: PNH36023), C. incanum RPD3 (Ensembl: KZL80576), F. graminearum RPD3 (Ensembl: ESU06006), M. oryzae RPD3 (Ensembl: MGG_05857T0), B. graminis Hda1 (Ensembl: BLGH_01902), B. zeicola HDA1 (Ensembl: EUC28153), A. fumigatus RPDA (Ensembl: OXN03576), C. jadinii Hda1 (Ensembl: ODV71565), K. marxianus RPD3 (Ensembl: BAO39086), S. cerevisiae RPD3 (Ensembl: KZV08295), Z. bailii RPD3 (Ensembl: SJM84961), M. larici-populina Hda1 (Ensembl: EGG09787), S. reilianum Hda1 (Ensembl: CBQ72606), and U. maydis HDA1 (Ensembl: KIS69526). (B) Schematic representation of different RPD3 orthologs including their catalytic domains. HDA, histone deacetylase domain. (C) Mycelia images for subcellular localization of the GFP-tagged BcRPD3 and BcRPD3-VRPP. Bars = 10 μm. (D) Western blotting for the expression levels of histone H4, H3 and acetylation levels of H4, H3 and its Kac sites in wild type B05.10, VRPP-3, and BcRPD3-1 strains.

FIGURE 2

Effects of BcRPD3 overexpression on mycelial growth, sporulation and conidial germination. The bars represent standard errors from three replicates, and statistical tests were conducted using Tukey’s test for multiple comparisons. Values on the bars followed by different letters are significantly different at p < 0.05. (A) Mycelial growth of B05.10, BcRPD3-1 and VRPP-3 strains on PDA plates after 3 days of cultivation. (B) Quantification of colony diameter of the indicated strains grown on PDA plates for 3 days. (C) Conidiation of different strains on PDA after 10 days of cultivation. (D) Quantification of conidia produced by the indicated strains on PDA plates (diameter, 4 cm). (E) Quantification of the conidial germination of the indicated strains during a time course (6 h) of germination in PDB on glass slides.

FIGURE 3

Impact of BcRPD3 overexpression on virulence, infection cushion formation and oxidative stress response. The bars represent standard errors from five replicates, and statistical tests were conducted using Tukey’s test for multiple comparisons. Values on the bars followed by different letters are significantly different at p < 0.05. (A) Pathogenicity on tomato leaves after 3 days of incubation. (B) Quantification of disease lesions on tomato leaves 3 days after inoculation. (C) Infection cushion formation of the indicated strains after 24 and 48 h of incubation on glass slides. Bars = 20 μm. (D) Quantification of infection cushion (IC) numbers produced by the indicated strains over a time course. (E) Sensitivity of the indicated strains after cultivation on PDA plates supplemented with 0.2% H₂O₂ for 3 days. (F) Relative growth reduction of the indicated strains which represented the reduction of colony diameters between normal and stress conditions normalized to colony diameters in normal condition.

FIGURE 4

RNA-Seq Analysis of BcRPD3 overexpression strain. (A) Numbers of up- and down regulated genes (P-value < 0.05, fold change > 2) in BcRPD3-1 strain compared with wild type B05.10. (B) Distribution of functional classification of downregulated genes in BcRPD3-1. Histograms indicate P-values of the enriched functional categories. (C) RNA-seq analysis of 9 downregulated genes involved in enzymatic activity. Differential expression in three biological replicates is illustrated using a heat map with colored squares indicating the range of expression levels referred to log2 FPKM value. (D) qRT-PCR validation of downregulated genes. Expression levels were normalized to β-tubulin gene. The bars represent standard errors from three replicates, and statistical tests were conducted using Tukey’s test for multiple comparisons. Values on the bars followed by different letters are significantly different at p < 0.05. (E) RNA-seq analysis of BcBoa and BcBot family genes. Differential expression in three biological replicates is illustrated using a heat map with colored squares indicating the range of expression levels referred to log2 FPKM value. (F) KEGG pathway enrichment of differentially expressed genes in BcRPD3-1 compared with wild type.

FIGURE 5

Relative enrichment levels of immunoprecipitated promoter regions of enzymatic activity related genes. The fold enrichment was normalized to input and internal control gene (β-tubulin-p). Data are means ± SD (n = 3). (A) Relative levels of the immunoprecipitated chromatin using anti-GFP antibody from the BcRPD3-1 strain and wild type containing GFP alone. (B) Relative levels of the immunoprecipitated DNA using anti-H3Kac from wild type containing GFP alone and BcRPD3-1 strain.