Next-generation systemic acquired resistance

Abstract

Systemic acquired resistance (SAR) is a plant immune response to pathogen attack. Recent evidence suggests that plant immunity involves regulation by chromatin remodeling and DNA methylation. We investigated whether SAR can be inherited epigenetically following disease pressure by Pseudomonas syringae pv tomato DC3000 (PstDC3000). Compared to progeny from control-treated Arabidopsis (Arabidopsis thaliana; C(1)), progeny from PstDC3000-inoculated Arabidopsis (P(1)) were primed to activate salicylic acid (SA)-inducible defense genes and were more resistant to the (hemi)biotrophic pathogens Hyaloperonospora arabidopsidis and PstDC3000. This transgenerational SAR was sustained over one stress-free generation, indicating an epigenetic basis of the phenomenon. Furthermore, P(1) progeny displayed reduced responsiveness of jasmonic acid (JA)-inducible genes and enhanced susceptibility to the necrotrophic fungus Alternaria brassicicola. This shift in SA- and JA-dependent gene responsiveness was not associated with changes in corresponding hormone levels. Instead, chromatin immunoprecipitation analyses revealed that SA-inducible promoters of PATHOGENESIS-RELATED GENE1, WRKY6, and WRKY53 in P(1) plants are enriched with acetylated histone H3 at lysine 9, a chromatin mark associated with a permissive state of transcription. Conversely, the JA-inducible promoter of PLANT DEFENSIN1.2 showed increased H3 triple methylation at lysine 27, a mark related to repressed gene transcription. P(1) progeny from the defense regulatory mutant non expressor of PR1 (npr1)-1 failed to develop transgenerational defense phenotypes, demonstrating a critical role for NPR1 in expression of transgenerational SAR. Furthermore, the drm1drm2cmt3 mutant that is affected in non-CpG DNA methylation mimicked the transgenerational SAR phenotype. Since PstDC3000 induces DNA hypomethylation in Arabidopsis, our results suggest that transgenerational SAR is transmitted by hypomethylated genes that direct priming of SA-dependent defenses in the following generations.

Figure 1.

Transgenerational SAR in progeny from healthy and diseased Arabidopsis. A, Experimental design for the generation of progeny lines. Plants were inoculated five times at intervals of 3 to 4 d by dipping the leaves in a control solution (C₀) or a solution containing PstDC3000 (P₀), after which plants were allowed to set seed to provide and P₁ progenies, respectively. Insets show representative growth phenotypes of C₀ and P₀ after mock and PstDC3000 inoculations. C₁ and P₁ plants were allowed to set seed under stress-free conditions, providing C₁C₂ and P₁C₂ progeny, respectively. A separate batch of P₁ plants was exposed to similar PstDC3000 disease pressure as P₀ plants to provide P₁P₂ progeny. B, Seed production by mock- and PstDC3000-inoculated parental plants. Data represent mean values (±se; n = 4–6) of grams of seed weight per plant. Asterisks indicate statistically significant differences compared to mock-inoculated C₀ or C₁ plants (Student’s t test; α = 0.05) C, Basal resistance against H. arabidopsidis WACO9 in C₁ and P₁ progenies of wild-type plants (Col-0; Experiment 1), C₁ and P₁ progenies of Col-0 and npr1-1 (Experiment 2), and C₁C₂, C₁P₂, and P₁P₂ progenies of Col-0 (Experiment 3). At 6 d after conidiospore inoculation, stained leaves were microscopically examined and assigned to different classes. Asterisks indicate statistically significant differences in class distributions in comparison to C₁ or C₁C₂ plants (χ² test; α = 0.05).

Figure 2.

Transgenerational priming of SA-induced defense gene expression. A, Quantitative reverse transcription PCR (qRT-PCR) analysis of SA-induced PR-1 transcription in C₁ and P₁ progenies (left), C₁C₂ and C₁P₂ progenies (center), and C₁C₂ and P₁P₂ progenies (right) at 4, 8, and 24 h after treatment with 0.5 mm SA. B, qRT-PCR analysis of SA-induced transcription of WRKY6, WRKY53, and WRKY70 in C₁ and P₁ progenies at 2, 4, and 8 h after treatment with 0.5 mm SA. Gene expression analyses were performed in 2-week-old plants. Data represent average fold induction values (±se; n = 3), relative to transcription levels before hormone treatment in C₁ plants (2^ΔCt_PR1 = 0.00042; 2^ΔCt_WRKY6 = 0.0076; 2^ΔCt_WRKY53 = 0.00092; 2^ΔCt_WRKY70 = 0.0078) or in C₁C₂ plants (2^ΔC_PR1 = 0.00030). Asterisks indicate statistically significant differences in gene induction values (Student’s t test; α = 0.05).

Figure 3.

Transgenerational cross-effects on JA-dependent resistance. A, Resistance against the necrotrophic fungus A. brassicicola in 5-week-old C₁ and P₁ plants. Left: Average lesion diameters (±se; n = 15) at 5 d after spore inoculation. Asterisk indicates a statistically significant difference in lesion diameter between lines (Student’s t test; α = 0.05). Right: Representative photographs of fungal colonization, visualized by trypan blue staining at 4 d after inoculation. Bars = 100 μm. Red arrows indicate germinated spores. B, qRT-PCR analysis of JA-induced transcription of PDF1.2 and VSP2 in C₁ and P₁ progenies at 4, 8, and 24 h after treatment of the leaves with 0.1 mm JA. Gene expression analyses were based on 2-week-old plants from C₁ and P₁ progenies. Data represent average fold induction values (±se; n = 3) relative to transcription levels before hormone treatment in C₁ plants (2^ΔCt_PDF1-2 = 0.0011; 2^ΔCt_VSP2 = 0.0060). Asterisks indicate statistically significant differences in gene induction (Student’s t test; α = 0.05). C, UPLC-MS/MS quantification of JA, JA-Ile, OPDA, SA, and SAG in mature leaves from 5-week-old plants. Shown are average values in nanograms per gram of dry weight (DW; ±se; n = 3). No statistically significant differences were detected between C₁ and P₁ plants (Student’s t test; α = 0.05).

Figure 4.

Transgenerational modifications of histone H3 at defense gene promoters in Col-0 (A) but not in npr1-1 (B). After ChIP, promoter DNA of SA-inducible PR-1, WRKY6, WRKY53, and JA-inducible PDF1.2 was quantified by qPCR relative to DNA amounts in chromatin extracts before immunoprecipitation (input) using antibodies against H3K9ac or H3K27me3. Data represent average fold change values (±se; n = 3) in P₁ plants compared to C₁ plants. P values indicate statistical differences between C₁ and P₁ progenies (Student’s t test).

Figure 5.

Role of non-CpG DNA methylation in transgenerational SAR. A, Level of resistance against H. arabidopsidis WACO9 in C₁ and P₁ progeny from Col-0 and the dmr1dmr2ctm3 (ddc) mutant. At 6 d after conidiospore inoculation, stained leaves were microscopically examined and assigned to different severity classes. P values indicate statistical differences in class distributions between P₁ and C₁ progeny of each genotype (χ² test). B, qRT-PCR analysis of SA-induced PR-1 transcription at 4, 8, and 24 h after treatment of C₁ and P₁ progeny of Col-0 and ddc plants with 0.5 mm SA. Data represent average fold induction values (±se; n = 3) relative to transcription levels before hormone treatment in C₁ Col-0 plants (2^ΔCt_PR1 = 0.0020).