Arabidopsis SNI1 and RAD51D regulate both gene transcription and DNA recombination during the defense response

Abstract

The plant immune response known as systemic acquired resistance (SAR) is a general defense mechanism that confers long-lasting resistance against a broad spectrum of pathogens. SAR triggers many molecular changes including accumulation of antimicrobial pathogenesis-related (PR) proteins. Transcription of PR genes in Arabidopsis is regulated by the coactivator NPR1 and the repressor SNI1. Pathogen infection also triggers an increase in somatic DNA recombination, which results in transmission of changes to the offspring of infected plants. However, it is not known how the induction of homologous recombination during SAR is controlled. Here, we show that SNI1 and RAD51D regulate both gene expression and DNA recombination. In a genetic screen for suppressors of sni1, we discovered that RAD51D is required for NPR1-independent PR gene expression. As a result, the rad51d mutant has enhanced disease susceptibility. Besides altered PR gene expression, rad51d plants are hypersensitive to DNA-damaging agents and are impaired in homologous recombination. The dual role of RAD51D and SNI1 in PR gene transcription and DNA recombination suggests a mechanistic link between the short-term defense response and a long-term survival strategy.

Fig. 1.

A simplified model for plant defense signaling. Plant immunity is determined by the hundreds of resistance (R₁, R₂, R₃ …) genes that are present in clusters in the genome (hollow and filled tandem arrows) (27). When an R protein recognizes a specific signal produced by the pathogen, it can trigger a series of rapid physiological responses to restrict pathogen growth. These local resistance responses also result in an increase in SA levels through an unknown systemic signal(s) (2). SA is necessary and sufficient for the induction of defense-related genes and systemic resistance to a broad-spectrum of pathogens. NPR1 is required for SA signaling (7). In the npr1 mutant, SA-induced gene expression and resistance is completely abolished. SA controls the translocation of NPR1 to the nucleus where it serves as a cofactor of TGA transcription factors (TF_TGAs). NPR1 is also proposed to inactivate the transcriptional repressor SNI1, as the sni1 mutation can suppress the npr1 phenotype. In the sni1 single mutant, NPR1-dependent genes are specifically derepressed and induced-state chromatin modification (red dots) was observed at a defense gene (PR1) promoter (9). Because in the sni1 and sni1 npr1 mutants, SA is still required for full induction, there must be signaling components, including RAD51D, whose activities depend on SA but not NPR1. RAD51D activity makes the chromosome more accessible for transcription and homologous recombination. Defense-associated homologous DNA recombination may result in generation of new R genes (R₄) and new pathogen recognition capability.

Fig. 2.

The rad51d mutant is a suppressor of sni1 and is impaired in disease resistance. (A) Morphological phenotypes of 4-week-old, soil-grown WT and mutant plants. (B and C) Expression of BGL2:GUS in untreated plants (B) or plants sprayed with 0.3 mM BTH 2 days previously (C). (D) PR1 gene expression in WT and mutant plants. (E–G) Growth of Psm ES4326 in WT and mutant plants. (E) Leaf discs were collected from six plants for day 0 (gray bars) and 10–12 plants for day 3. Error bars represent 95% confidence intervals of log-transformed data. The data were analyzed by Student's t test. (F and G) Leaf discs were collected from 4–16 plants for day 0 (gray bars) and 8–16 plants for day 3 (black bars). The data were analyzed by two-way ANOVA. Results show means for five pooled experiments (n = 60 plants per genotype; ANOVA, genotype: P < 0.0001; experiment: P < 0.0001; genotype × experiment: P = 0.5255) (F) and three pooled experiments (n = 28 plants per genotype; ANOVA, genotype: P < 0.0001; experiment: P < 0.0001; genotype × experiment: P = 0.3645) (G). Letters above bars indicate statistically significant differences between genotypes (Bonferroni correction, P < 0.01).

Fig. 3.

SSN1 corresponds to At1g07745, which encodes RAD51D. (A and B) Complementation of the ssn1 mutation by RAD51D. The RAD51D gene driven by its own promoter and the RAD51D cDNA driven by the constitutive 35S promoter were transformed into sni1 rad51d plants. Complementation restored sni1 morphology (A) and BGL2:GUS expression (B). From left to right are photographs of sni1, sni1rads51d, the RAD51D gene in the sni1rad51d background, and the RAD51D cDNA in the sni1rad51d background. (C) Sequence alignment of Arabidopsis (At), human (Hs), and mouse (Mm) RAD51D proteins. Identical and conserved amino acids are highlighted in black and gray, respectively. Dashes indicate gaps in the sequence to optimize the alignment. The conserved Walker A and B motifs and the 7-bp deletion in Arabidopsis rad51d are indicated by dashed, dotted, and solid lines, respectively.

Fig. 4.

RAD51D plays a role in DNA repair. The rad51d mutant is hypersensitive to the DNA-damaging agents MMC (A) and bleomycin (B). For each data point values represent three replicate Petri plates with each replicate containing ≈100 plants. Error bars represent standard error.

Fig. 5.

Somatic recombination is affected in the sni1 and rad51d mutants. Frequency of recombination sectors per plant in WT or mutant lines containing the reporter transgene 1445 without (A) and with 50 mM INA induction (B). Error bars represent standard error; letters above bars indicate statistically significant differences (Bonferroni correction, P < 0.01; for Col n = 71, sni1 n = 107, rad51d n = 220, sni1 rad51d n = 214).