Cell death regulation but not abscisic acid signaling is required for enhanced immunity to Botrytis in Arabidopsis cuticle-permeable mutants

Abstract

Prevailing evidence indicates that abscisic acid (ABA) negatively influences immunity to the fungal pathogen Botrytis cinerea in most but not all cases. ABA is required for cuticle biosynthesis, and cuticle permeability enhances immunity to Botrytis via unknown mechanisms. This complex web of responses obscures the role of ABA in Botrytis immunity. Here, we addressed the relationships between ABA sensitivity, cuticle permeability, and Botrytis immunity in the Arabidopsis thaliana ABA-hypersensitive mutants protein phosphatase2c quadruple mutant (pp2c-q) and enhanced response to aba1 (era1-2). Neither pp2c-q nor era1-2 exhibited phenotypes predicted by the known roles of ABA; conversely, era1-2 had a permeable cuticle and was Botrytis resistant. We employed RNA-seq analysis in cuticle-permeable mutants of differing ABA sensitivities and identified a core set of constitutively activated genes involved in Botrytis immunity and susceptibility to biotrophs, independent of ABA signaling. Furthermore, botrytis susceptible1 (bos1), a mutant with deregulated cell death and enhanced ABA sensitivity, suppressed the Botrytis immunity of cuticle permeable mutants, and this effect was linearly correlated with the extent of spread of wound-induced cell death in bos1. Overall, our data demonstrate that Botrytis immunity conferred by cuticle permeability can be genetically uncoupled from PP2C-regulated ABA sensitivity, but requires negative regulation of a parallel ABA-dependent cell-death pathway.

Keywords: Botrytis cinerea; BOS1; ERA1; RNA sequencing; cell death; cuticle permeable; farnesyl transferase; immunity.

Fig. 1.

Enhanced Botrytis immunity in the ABA hypersensitive era1-2 mutant. (A) Botrytis infection of Col-0, pp2c-quad, and era1-2. Droplets of Botrytis conidia suspensions (3 μl, 2×10⁶ spores ml^–1) were applied to 24-day-old fully expanded leaves. Photographs were taken at 3 days post infection (dpi). Scale bar=5 mm. (B) Quantitative data of lesion sizes in (A). The lesion diameters were measured with ImageJ. Combined results of four experiments (n=10 in each independent biological repeat) were analyzed in a linear mixed model with single-step P-value adjustment. Error bars represent the SE of means. Different letters above the bars indicate significant differences (P<0.05). (C) DNA of Botrytis on leaves at 3 dpi was quantified with real-time qPCR using the Arabidopsis actin gene as a control. Different letters indicate significant differences (two-tailed t-test, P<0.05). (D–G) Leaves were stained at 36 hours post infection (hpi) for cell death with trypan blue (D, F), and at 16 hpi for H₂O₂ accumulation with 3,3′-diaminobenzidine (E, G). The percentage stained area was used for comparisons between Col-0 and era1-2. Two biological repeats with seven leaves in each repeat were analyzed with a linear model. Different letters above the bars indicate significant differences (P<0.05). Scale bar=2 mm.

Fig. 2.

Enhanced cuticle permeability in the ABA hypersensitive era1-2 mutant. (A, B) The cuticle of era1-2 was more permeable than other mutants or the wild type Col-0 under toluidine blue staining. Leaves of 24-day-old plants were stained with 5 μl droplets of 0.05% toluidine blue solution for 2 h. The ABA-insensitive snrk2.236 was used as a positive control. (C) Quantification of the toluidine blue-stained areas. Combined results of three experiments (n=12 in each independent biological repeat) were analyzed in a linear mixed model with single-step P-value adjustment. Error bars represent the SE of means. Different letters above the bars indicate significant differences (P<0.05). (D) Leaves immersed in toluidine blue solution for 20 min. Bar=5 mm.

Fig. 3.

Analysis of the differentially expressed genes (DEGs) in cuticle-defective mutants (CDMs) compared with Col-0 in mock and Botrytis treatments. Plants at 24 days old were sprayed with 2×10⁶ ml^–1Botrytis spores. Samples were collected at 20 and 44 hours post infection (hpi) for RNA-seq analysis. (A) DEGs in the CDMs under mock treatment. DEGs were identified in comparison between each CDM and wild type (fold change ≥2 and P≤0.05; see also Supplementary Table S2). There were 64 genes commonly up-regulated (left) and seven genes down-regulated (right) in the CDMs (genes are listed in Supplementary Table S2A, B). Mutant names are abbreviated: era1-2 to era1, lacs2.3 to lacs2, snrk2.236 to srk2. (B) DEGs under Botrytis treatment in the CDMs. There were 53 genes commonly up-regulated (left) and 25 genes down-regulated (right) in the CDMs (genes are listed in Supplementary Table S2C, D). The DEGs at 44 hpi are presented in Supplementary Fig. S3B and Supplementary Table S2E, F. (C, D) GO enrichment analysis of the core genes in (B). The 53 up-regulated genes were enriched in defense-related terms (C). The 25 down-regulated genes were enriched exclusively in photosynthesis-related terms (D). The GO enrichment analysis of the core genes of mock-treated CDMs at 20 hpi is presented in Supplementary Fig. S3A.

Fig. 4.

Identification of Botrytis-induced differentially expressed genes (DEGs). DEGs were identified in comparisons between Botrytis-treated CDMs and Col-0 to their corresponding genotype under mock treatment (fold change ≥2 and P≤0.05; see also Supplementary Table S3). (A) At 20 h post infection (hpi), there were 11 genes commonly up-regulated (left) and no common genes down-regulated (right) in the Botrytis-treated genotypes. The genes are listed in Supplementary Table S3E. Mutant names are abbreviated: era1-2 to era1, lacs2.3 to lacs2, snrk2.236 to srk2. (B) At 44 hpi, there were 61 genes commonly up-regulated (left) and no common genes down-regulated (right) in the Botrytis treated genotypes. The genes are listed in Supplementary Table S3J.

Fig. 5.

Relative expression of genes known to regulate Botrytis immunity. The gene list was obtained from https://www.arabidopsis.org via the gene searching tool with the keywords ‘Botrytis’ or ‘B. cinerea’. Genes related to the jasmonic acid, ethylene, and PAD3 pathways were excluded, as they are not required for Botrytis immunity conferred by cuticle permeability (Chassot et al., 2007). The expression values (log₂ of the number of transcripts per million) for each gene were used to build a heatmap. The genes are listed in alphabetical order. The treatments were mock and Botrytis spray infection at the indicated time points and are grouped according to genotypes.

Fig. 6.

The Botrytis immunity of era1-2 was fully suppressed by bos1. (A) Typical lesion symptoms of era1-2 and bos1 era1-2. The arrows indicate Botrytis-infected lesions. Droplets of Botrytis conidia suspensions (3 μl, 2×10⁶ spores ml^–1) were applied to 24-day-old fully expanded leaves. Photographs were taken at 3 dpi. Scale bar=1 cm. (B) Quantitative lesion size data. The lesion diameters were measured with ImageJ. Combined results of three biological experiments (n=36 in total) were analyzed in a linear mixed model with single-step P-value adjustment. Error bars represent the SE of means. Different letters above the bars indicate significant differences (P<0.05). (C) Representative wound-induced cell death symptoms stained with trypan blue. Needle-punctured leaves were stained with trypan blue at 6 days post wounding to detect cell death. L, Length of cell death spread; P, puncture site. Scale bar=1 mm. (B) Quantitative spreading cell death data. The length of spread of cell death was measured as indicated in (C) around each wound four times in four directions (up, down, left, and right), and the mean value was used. Data of three repeats were analyzed in a linear mixed model with single-step P-value adjustment. Error bars represent the SE of means. Different letters above the bars indicate significant differences (P<0.05). (E) Spreading cell-death symptoms were enhanced in bos1 era1-2. Four-week-old in vitro-grown plants are shown. Once buds started opening, cell death initiated in the buds and then spread along the shoots, eventually causing the death of the whole plant. Right panel, close-up of the area indicated by the dashed box in the left panel. Arrows indicate dead buds. Scale bar=1 cm.

Fig. 7.

Botrytis immunity in lacs2.3 was attenuated in the bos1 lacs2.3 double mutant. (A) Typical lesion symptoms of lacs2.3 and bos1 lacs2.3. Droplets of Botrytis conidia suspensions (3 μl, 2×10⁶ spores ml^–1) were applied to 24-day-old fully expanded leaves. Photographs were taken at 3 dpi. Scale bar=1 cm. (B) Quantitative lesion size data. Data of three biological repeats (n=36 in total) were analyzed in a linear mixed model with single-step P-value adjustment. Error bars represent the SE of means. Different letters above the bars indicate significant differences (P<0.05). (C) Representative wound-induced cell death symptoms stained with trypan blue. Needle-punctured leaves were stained at 6 days post wounding to detect cell death. L, Length of cell death spread; P, puncture site. Scale bar=1 mm. (D) Wound-induced cell death of bos1 was reduced in the bos1 lacs2.3 double mutant. Quantitative spreading cell death data. The length of spread of cell death around each wound was measured four times in four directions (up, down, left, and right), and the mean value was used. Data of three repeats were analyzed in a linear mixed model with single-step P-value adjustment. Error bars represent the SE of means. Different letters above the bars indicate significant differences (P<0.05).

Fig. 8.

ABA sensitivity is uncoupled from Botrytis resistance in cuticle-deficient plants. The relationship between Botrytis resistance and cuticle permeability of the indicated genotypes was examined. The red line was calculated with all single-mutant genotypes (not in the bos1 background), which showed a linear correlation between cuticle permeability and lesion size (R²=0.96, P≤0.001). The blue line was calculated with genotypes of the bos1 background (R²=0.52, P=0.49). The genotypes in black were examined in this study and the genotypes in grey are from previously published data (Cui et al., 2016). All experiments were repeated three to six times. All the data were normalized, pooled, and analyzed with a linear model.

Fig. 9.

Botrytis susceptibility of bos1 double mutants was positively correlated with the extent of wound-induced cell death. (A) Suppressors of spreading cell death in bos1 (aba3-1 and abi1-1) also suppressed the Botrytis susceptibility of bos1. Lesion sizes from four independent experiments were combined and analyzed in a linear mixed model with a single step P-value adjustment. Error bars represent the SE of means (n=48 in total). Different letters above the bars indicate significant differences (P<0.05). (B) Representative symptoms of plants sprayed with Botrytis spore suspensions at 3 days post infection. Scale bar=1 cm. (C) Botrytis sensitivity correlated with the extent of wound-induced cell death in bos1 double mutants (R²=0.78, P=0.047). The blue dotted line shows a linear correlation between the extent of wound-induced cell death and the Botrytis-induced lesion sizes. The genotypes indicated with blue squares were examined in this study. The spread of cell death of the genotypes indicated with grey squares are from previously published data (Cui et al., 2013), which were calculated with normalization to the reference values of the wild type and bos1. All experiments were repeated three to six times.

Fig. 10.

Model of layered defense responses to Botrytis in cuticle-deficient plants. Cuticle permeability itself leads to increased expression of defense genes related to pathogen perception and salicylic acid signaling. In addition, a permeable cuticle enhances the production of reactive oxygen species (ROS), which act as signaling molecules and regulators of cell death (see also L’Haridon et al., 2011). BOS1 is required to maintain cell viability and contain Botrytis-induced lesion expansion. The left panel represents the initial resistance and the middle panel represents a later stage of infection. The right panel illustrates signaling relationships under normal conditions, with an intact cuticle and the absence of pathogens, in which ERA1 attenuates ABA signaling and independently promotes cuticle formation via an unknown mechanism.