Salicylic acid activates DNA damage responses to potentiate plant immunity

Abstract

DNA damage is normally detrimental to living organisms. Here we show that it can also serve as a signal to promote immune responses in plants. We found that the plant immune hormone salicylic acid (SA) can trigger DNA damage in the absence of a genotoxic agent. The DNA damage sensor proteins RAD17 and ATR are required for effective immune responses. These sensor proteins are negatively regulated by a key immune regulator, SNI1 (suppressor of npr1-1, inducible 1), which we found is a subunit of the structural maintenance of chromosome (SMC) 5/6 complex required for controlling DNA damage. Elevated DNA damage caused by the sni1 mutation or treatment with a DNA-damaging agent markedly enhances SA-mediated defense gene expression. Our study suggests that activation of DNA damage responses is an intrinsic component of the plant immune responses.

Figure 1. SNI1 is a Subunit of the SMC5/6 Complex

(A) SNI1 forms a complex in plants. Total protein from the SNI1-TAP transgenic line was separated on a Blue-Native PAGE gel and detected with an anti-TAP antibody. The major SNI1-TAP band (arrowed) was ∼350 kDa. The untransformed wild-type (WT) plant (left lane) was used as a negative control. (B) Proteins identified in the SNI1 complex. MP, the number of matched peptides; CV, the percentage of sequence coverage; MW, molecular weight. (C) In vitro pull-down assays. GST and GST-SNI1 were expressed in E. coli and purified. SMC5-MYC, SMC6B-MYC and ASAP1-MYC were in vitro translated. The blots were detected with an anti-MYC antibody. (D) Split luciferase assays. The proteins were fused to either the C- or N-terminal half of luciferase (cLUC or nLUC) and transiently expressed in N. benthamiana. The luciferase activities were monitored by a CCD camera. (E and F) The 3D structures of SNI1, NSE6, ASAP1 and NSE5 predicted by the I-TASSER server. SNI1 and NSE6 were similar to 1B3UA; ASAP1 and NSE5 were similar to 1JDHA in the PDB database. (G) The short-root phenotype observed in sni1, asap1, smc6a (6a), smc6b (6b), and smc6a/SMC6A smc6b (smc6) seedlings in comparison to wild-type (WT). (H and I) The expression of PR1 (H) and PR2 (I) measured by qRT-PCR. Plants were grown on medium with 10 μM INA (Low INA) for 9 days. The expression level was normalized to ubiquitin 5 (UBQ5). The data are presented as mean ± SD (n = 3). See also Figure S1, Tables S1 and S3.

Figure 2. Mutation in SNI1 or SA Treatment Induces DNA Damage

(A and B) The sni1 mutant shows more DNA damage than WT in a comet assay. (A) Representative pictures of the comet assay. The scale bar is 100 μm. (B) Quantification of the percentage of DNA in the comet tails. The data are presented as mean ± SEM (n > 200). (C) Spontaneous cell death in sni1 seedlings indicated by trypan blue staining. (D) Relative expression of DDR-related genes in sni1 compared to WT. The results are shown as mean ± SD (n = 3). (E–H) SA treatment induces DNA damage in both WT (E and F) and the npr1 mutant (G and H). Comet assay was performed 4 h after plants were treated with water or 1 mM SA. The scale bar is 100 μm. The data are presented as mean ± SEM (n > 200). ***, P < 0.001 (Student’s t-test, two-tailed). Two-week-old plants were used in all the experiments in this figure.

Figure 3. The SMC5/6 Complex Negatively Regulates RAD17 and ATR

(A and B) The ssn4 mutant suppresses the sni1 morphology (A) and basal defense gene expression (B). In (B), the blue color indicates expression of the defense gene reporter PR2:GUS. (C) Map-based cloning of SSN4. The number of recombinants between the markers and SSN4 are shown below each marker. (D) The gene structure of RAD17. The boxes represent exons and the lines represent introns. Start codon (ATG) and stop codon (TGA) are shown. The deletion mutation in ssn4 is from nucleotide 2345 to nucleotide 3150. (E) The T-DNA insertion mutant rad17-2 suppresses the sni1 morphology. (F) Transforming the RAD17 gene into the sni1 ssn4 double mutant can suppress the ssn4 phenotype and restore the sni1 morphology. (G) The mutation in ATR suppresses the sni1 morphology. (H and I) Comet assays. (H) Representative pictures of the comet assay. The scale bar is 100 μm. (I) Quantification of the percentage of DNA in the comet tails. The data are presented as mean ± SEM (n > 200). ***, P < 0.001 (one-way ANOVA followed by Dunnett's multiple comparison test). (J) RAD17 interacts with SNI1 in the Y2H assay. The yeast growth on media lacking Trp, Leu, Ade and His indicates interaction. AD, activation domain; BD, DNA-binding domain. (K) RAD17-MYC can be pulled down by HisMBP-SNI1. HisMBP-SNI1 and HisMBP-GFP were expressed in E. coli and purified. RAD17-MYC was in vitro translated. The blots were detected with an anti-MYC antibody. (L) RAD17 interacts with SNI1 in the split luciferase assay. See also Figure S2.

Figure 4. DNA Damage Responses Potentiate Plant Immunity

(A) The binding of RAD51 to the PR1 promoter depends on RAD17 and ATR. ChIP assays were performed in WT, rad17 and atr treated with water or 1 mM SA for 16 h. The ChIP samples were subjected for qPCR analysis for the promoter or the coding region of PR1 (PR1-PRO or PR1-CDS) and the promoter region of ACTIN7 (ACT7-PRO). The fold enrichment between SA-treated and H₂O-treated samples is shown. The error bars represent SEM (n = 3). (B–D) DNA-damaging agent bleomycin (BLM) and low dose of the immune inducer INA synergistically induce defense gene expression. Plants were grown on medium with 4 μg/ml BLM and/or low INA (10 μM) for 9 days. (B) PR2:GUS expression. WT+BLM, WT plants treated with BLM. (C and D) Whole-genome microarray analysis. (C) Venn diagram analysis of induced genes (Fold change > 2, P < 0.05). (D) Simulation-based analysis of the 131 synergistically induced genes. The green line represents the sum of the BLM effect and the INA effect (additive), and the red line represents the effect of BLM+INA co-treatment (synergistic). The numbers on the X-axis represent genes listed in Table S4. (E) The rad17 and atr mutants are more susceptible to the bacterial pathogen Psm ES4326 than WT. cfu, colony forming unit. Error bars represent 95% confidence intervals (n = 7). ***, P < 0.001 (Student’s t-test, two-tailed). (F) A working model that shows how DDR facilitates defense gene expression independent of NPR1. TF, transcription factor. HR, homologous recombination. NHEJ, non-homologous end joining. See also Figure S3, Tables S4 and S5.