The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis

Abstract

The molecular and cellular mechanisms involved in plant resistance to the necrotrophic fungal pathogen Botrytis cinerea and their genetic control are poorly understood. Botrytis causes severe disease in a wide range of plant species, both in the field and in postharvest situations, resulting in significant economic losses. We have isolated the BOS1 (BOTRYTIS-SUSCEPTIBLE1) gene of Arabidopsis based on a T-DNA insertion allele that resulted in increased susceptibility to Botrytis infection. The BOS1 gene is required to restrict the spread of another necrotrophic pathogen, Alternaria brassicicola, suggesting a common host response strategy against these pathogens. In the case of the biotrophic pathogens Pseudomonas syringae pv tomato and the oomycete parasite Peronospora parasitica, bos1 exhibits enhanced disease symptoms, but pathogen growth is similar in bos1 and wild-type plants. Strikingly, bos1 plants have impaired tolerance to water deficit, increased salinity, and oxidative stress. Botrytis infection induces the expression of the BOS1 gene. This increased expression is severely impaired in the coi1 mutant, suggesting an interaction of BOS1 with the jasmonate signaling pathway. BOS1 encodes an R2R3MYB transcription factor protein, and our results suggest that it mediates responses to signals, possibly mediated by reactive oxygen intermediates from both biotic and abiotic stress agents.

Figure 1.

Progress of Disease Development in bos1 Plants Inoculated with Necrotrophic Pathogens. (A) and (B) Wild-type (left) and bos1 (right) homozygous plants at 5 (A) and 10 (B) days after inoculation with Botrytis. Plants were inoculated by spraying spore suspension at a density of 10⁵ spores/mL and kept under high humidity. (C) Leaves from wild-type (Wt) and bos1 homozygous plants at 5 days after inoculation with a 4-μL droplet of A. brassicicola spores (10⁵ spores/mL). (D) Lesion size was measured at 3 days after inoculation. Data points represent average lesion size ± se of measurements from a minimum of 40 lesions. This experiment was repeated three times with similar results. WT, wild type. (E) Accumulation of the Botrytis (B.c) β-tubulin mRNA in inoculated plants. Twenty micrograms of total RNA extracted from inoculated plants was loaded per lane. dpi, days after inoculation.

Figure 2.

bos1 Plants Show Wild-Type Levels of Resistance but Increased Symptom Development in Response to Bacterial Pathogens. (A) and (B) P. syringae pv tomato avirulent strain DC3000 carrying avrRpm1 (A) and virulent strain DC3000 (B). Representative leaves are shown at 4 days after inoculation. Wt, wild type. (C) and (D) Bacterial growth for DC3000 carrying avrRpm1 (C) and DC3000 (D). Four-week-old plants were infiltrated with bacterial suspension (OD₆₀₀ = 0.001), and the number of bacteria was determined per area of leaf plotted for wild-type (green bars) and bos1 (red bars) plants. CFU, colony-forming units.

Figure 3.

Impaired Abiotic Stress Tolerance in bos1. (A) Sensitivity to NaCl. WT, wild type. (B) Percentage plant survival after drought stress. (C) Percentage germination of seeds in the presence of polyethylene glycol 8000 (PEG). Wt, wild type. Data points represent average values ± se.

Figure 4.

bos1 Plants Have Increased Sensitivity to Compounds That Generate Oxidative Stress. (A) Five-day-old seedlings germinated on MS medium were transferred to MS medium containing rose bengal. Photographs were taken 2 weeks after transfer to the medium containing rose bengal. Wt, wild type. (B) Response to paraquat was assayed by depositing a single 2-μL droplet of methyl viologen dissolved in water on individual leaves. Control leaves were treated with water alone. Photographs were taken 4 days after treatment.

Figure 5.

Generation of ROI in Botrytis-Infected Tissues. (A) to (D) Production of O₂⁻ in wild-type ([A] and [B]) and bos1 ([C] and [D]) plants at 2 days after inoculation after treatment with buffer ([A] and [C]) or Botrytis spore suspension ([B] and [D]). (E) to (H) Production of H₂O₂ in wild-type ([E] and [F]) and bos1 ([G] and [H]) plants treated with buffer ([E] and [G]) or Botrytis ([F] and [H]). ROI production was assayed using nitroblue tetrazolium for O₂⁻ and 3,3′-diaminobenzidine for H₂O₂. O₂⁻ is indicated by the blue spots, and the reddish-brown coloration indicates the polymerization of 3,3′-diaminobenzidine at the site of H₂O₂ production.

Figure 6.

Genomic Structure of the bos1 Mutant Allele, Expression of the BOS1 Gene, and Complementation of the bos1 Phenotype. (A) Structure of the BOS1 gene and position of the T-DNA insertion in the bos1 mutant allele. Exons are shown as hatched boxes. The arrow shows the predicted direction of transcription. LB, left border of the T-DNA; RB, right border of the T-DNA. (B) BOS1 expression in homozygous wild-type (BOS1/BOS1), heterozygous mutant (BOS1/bos1), and homozygous mutant (bos1/bos1) plants. mRNA was extracted from leaf tissue from 3-week-old plants and separated on a formaldehyde agarose gel. An RNA gel blot from the gel was hybridized to a probe representing the 3′ part of the BOS1 gene. (C) and (D) The genomic clone containing the BOS1 gene rescues Botrytis susceptibility (C) and oxidative stress sensitivity (D) of bos1 plants. Transgenic plants were generated by transforming bos1 plants with constructs containing the BOS1 genomic clone, including the 1.5-kb promoter region. The Botrytis assay was performed by drop inoculation (see Methods). The oxidative stress assay was performed using rose bengal. Plants were photographed 3 weeks after transfer to medium containing rose bengal. Wt, wild type.

Figure 7.

Sequence Comparison between BOS1 and Other Plant MYB Proteins. (A) Alignment showing the amino acid sequences of the R2 and R3 repeats and flanking regions. The repeats are indicated with asterisks. The conserved region after the R3 repeat is shown with dots. (B) Sequence alignment showing C-terminal amino acid sequences. Sequences shown are from Arabidopsis (AtBOS1, AtMYB78, AtMYB112, AtMYB62, AtMYB116, AtMYB2, AtMYB57, and AtMYB24), Craterostigma plantagineum (CPM10, CPM7, and CPM5), Oryza sativa (JaMYB and OsCPM7), and Gossypium hirsutum (GhMYB5). Numbers above the alignments correspond to amino acid positions in BOS1. Black shading indicates amino acids conserved in all entries, and gray shading indicates amino acids identical to the BOS1 sequence.

Figure 8.

Expression of BOS1 and Defense Response Marker Genes. RNA gel blot analysis of BOS1 (A) and PDF1-2 and PR-1 (B) expression in Botrytis-infected tissues. Wild-type (Wt), ein2, coi1, and bos1 plants were inoculated by spraying Botrytis at 10⁵ spores/mL or buffer, and tissue was frozen for RNA extraction. Twenty micrograms of total RNA was loaded per lane. Numbers indicate hours after inoculation. The bottom gels show total RNA as a loading control. The experiment was repeated two times with similar results.