Costs and Benefits of Transgenerational Induced Resistance in Arabidopsis

Abstract

Recent evidence suggests that stressed plants employ epigenetic mechanisms to transmit acquired resistance traits to their progeny. However, the evolutionary and ecological significance of transgenerational induced resistance (t-IR) is poorly understood because a clear understanding of how parents interpret environmental cues in relation to the effectiveness, stability, and anticipated ecological costs of t-IR is lacking. Here, we have used a full factorial design to study the specificity, costs, and transgenerational stability of t-IR following exposure of Arabidopsis thaliana to increasing stress intensities by a biotrophic pathogen, a necrotrophic pathogen, and salinity. We show that t-IR in response to infection by biotrophic or necrotrophic pathogens is effective against pathogens of the same lifestyle. This pathogen-mediated t-IR is associated with ecological costs, since progeny from biotroph-infected parents were more susceptible to both necrotrophic pathogens and salt stress, whereas progeny from necrotroph-infected parents were more susceptible to biotrophic pathogens. Hence, pathogen-mediated t-IR provides benefits when parents and progeny are in matched environments but is associated with costs that become apparent in mismatched environments. By contrast, soil salinity failed to mediate t-IR against salt stress in matched environments but caused non-specific t-IR against both biotrophic and necrotrophic pathogens in mismatched environments. However, the ecological relevance of this non-specific t-IR response remains questionable as its induction was offset by major reproductive costs arising from dramatically reduced seed production and viability. Finally, we show that the costs and transgenerational stability of pathogen-mediated t-IR are proportional to disease pressure experienced by the parents, suggesting that plants use disease severity as an environmental proxy to adjust investment in t-IR.

Keywords: Arabidopsis; costs and benefits; induced resistance; transgenerational effects; transgenerational phenotypic plasticity.

FIGURE 1

Full factorial experimental design of the study. Arabidopsis thaliana plants (accession Col-0) from a common ancestor were exposed to increasing stress intensities (Mock, Low, Medium, and High) by the (hemi)biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (green), the necrotrophic fungal pathogen Plectosphaerella cucumerina (blue), or soil salinity (NaCl; orange). Plants in this parental generation (P) were evaluated for impacts on fitness parameters. Four plants per stress level were selected to generate F1 populations, which were analyzed for transgenerational changes in resistance against all three stresses, in order to determine the specificity of transgenerational induced resistance (t-IR), potential costs arising from increased susceptibility, and dose-dependency of t-IR intensity on parental stress. For each parental treatment, four individual plants from three independent F1 populations were randomly selected to set seed in the absence of stress. The resulting F2 populations were analyzed for resistance against the parental stress to examine dose-dependent effects on t-IR stability. Circles indicate individual plants; small (thin-lined) boxes indicate F1/F2 populations derived from a common ancestor in the previous generation; big (bold-lined) boxes indicate pooled F1/F2 populations from a common ancestor two generations earlier.

FIGURE 2

Differential impacts of three (a)biotic stresses on parental fitness parameters. Plants in the parental generation (4.5 weeks old) were exposed to varying stress intensities by P. syringae pv tomato (Mock, Pst-I, Pst-II, and Pst-III), P. cucumerina (Mock, Pc-I, Pc-II, and Pc-III), or soil salinity (Mock, S-I, S-II, and S-III) over a 3-week period before transferring to long-day conditions to trigger flowering and set seed. Boxplots show the interquartile range (IQR; box) ± 1.5xIQR (whiskers), including median (horizontal line) and replication units (single dots). (A) Impacts on relative growth rate (RGR) during the period of stress exposure. Data represent RGR values of single plants (n = 5–6) normalized to the average RGR of Mock-treated plants (100%). Different letters indicate statistically significant differences (ANOVA + Tukey’s post hoc test, α = 0.05). (B) Impacts on seed production. Data represent seed numbers per plant (n = 5–6) normalized to average value of Mock-treated plants (100%). Different letters indicate statistically significant differences (Pst: Welch ANOVA + Games-Howell post hoc test, α = 0.05; Pc and salt: ANOVA + Tukey’s post hoc test, α = 0.05). (C) Impacts on seed viability. Seed viability was determined 5 days after planting of surface-sterilized and stratified seeds onto 0.2× Murashige and Skoog (MS) agar plates. Data represent mean germination percentages per plate (25 seeds/plate) of seed batches from four similarly treated parents (n = 15–60). Different letters indicate statistically significant differences (Welch ANOVA + Games-Howell post hoc test; α = 0.05). Viability data for seed batches from individual plants are presented in Supplementary Figures 1A–C.

FIGURE 3

Intensity and transgenerational stability of Pseudomonas syringae pv. tomato-mediated t-IR in matched environments. Parental plants had been exposed to different disease severities by the biotrophic bacterium Pseudomonas syringae pv. tomato (Pst; Mock, Pst-I, Pst-II, and Pst-III). F1 and F2 plants were analyzed for resistance against the same pathogen (Pst) and/or the biotrophic Oomycete Hyaloperonospora arabidopsidis (Hpa). (A) t-IR against Pst in F1 progeny at 3 dpi. Boxplots show the interquartile range (IQR; box) ± 1.5xIQR (whiskers), including median (horizontal line) and replication units (dots). Data represent ¹⁰log-transformed bacterial titers (log cfu/cm²) in leaves of single plants within F1 populations from similarly treated parents (n = 42). Different letters indicate statistically significant differences (Welch ANOVA + Games-Howell test, α = 0.05). Data for individual F1 populations are shown in Supplementary Figure 2A. (B) t-IR against Hpa in F1 progeny. Hpa colonization was quantified at 6 dpi by assigning trypan-blue stained leaves to four Hpa resistance classes (I, healthy; II, hyphal colonization only; III, hyphal colonization with conidiospores; and IV, hyphal colonization with conidiospores and oospores). Stacked bars show leaf frequency distributions within F1 populations from similarly treated parental plants (n = 600–1,000). Different letters indicate statistically significant differences (pairwise Fisher’s exact tests + Bonferroni FDR, α = 0.05). Data for individual F1 populations are shown in Supplementary Figure 2B. (C) t-IR against Hpa in F2 progeny at 6 dpi after one stress-free F1 generation. Stacked bars show leaf frequency distributions across Hpa resistance classes within F2 populations that share a common parental ancestor (n = 300–350). Different letters indicate statistically significant differences (pairwise Fisher’s exact tests + Bonferroni FDR; α = 0.05). Data for individual F2 populations are shown in Supplementary Figure 2C.

FIGURE 4

Intensity and transgenerational stability of Plectosphaerella cucumerina-mediated t-IR in matched environments. Parental plants had been exposed to different disease severities by necrotrophic Plectosphaerella cucumerina (Pc; Mock, Pc-I, Pc-II, and Pc-III). F1 and F2 plants were analyzed for resistance against the same pathogen. Lesion diameters were determined in four leaves/plant at 15 dpi and the average lesion diameter per plant was used as statistical unit of replication. Boxplots show the interquartile range (IQR; box) ± 1.5xIQR (whiskers), including median (horizontal line) and replication units (dots). (A) t-IR against Pc in F1 progeny. Data represent lesion diameters (mm) of plants within F1 populations from similarly treated parents (n = 40). Different letters indicate statistically significant differences (Welch ANOVA + Games-Howell test, α = 0.05). Data for individual F1 populations are shown in Supplementary Figure 3A. (B) t-IR against Pc in F2 progeny after a stress-free F1 generation. Data represent lesion diameters of plants within F2 populations that share a common parental ancestor (n = 20). Different letters indicate statistically significant differences (ANOVA + Tukey’s post hoc test; α = 0.05). Data for individual F2 populations are shown in Supplementary Figure 3B.

FIGURE 5

Lack of salt-mediated t-IR in matched environments. Parental plants had been exposed to different stress intensities by soil salinity (NaCl; Mock, S-I, S-II, and S-III). Salt tolerance of F1 and F2 plants was quantified by root growth reduction (%) over a 5-day period on NaCl-containing agar medium relative to the average root growth on agar medium without NaCl. Boxplots show the interquartile range (IQR; box) ± 1.5xIQR (whiskers), including median (horizontal line) and replication units (dots). (A) Unaltered tolerance of F1 plants to 50 and 100 mM NaCl. Data represent growth reduction percentages of single plants within F1 populations from similarly treated parents (n = 60). ns, no statistically significant differences (ANOVA; α = 0.05). Root growth data for individual F1 populations are shown in Supplementary Figure 4A; root tolerance data for individual F1 populations are shown in Supplementary Figure 4C. (B) Unaltered tolerance of F2 plants to 50 and 100 mM NaCl after one stress-free F1 generation. Data represent growth reduction percentages of single plants within F2 populations that share a common parental ancestor (n = 18–20). ns, no statistically significant differences (ANOVA; α = 0.05). Root growth data for individual F2 populations are shown in Supplementary Figure 4B; root tolerance data for individual F2 populations are shown in Supplementary Figure 4D.

FIGURE 6

Costs and benefits of t-IR in mismatched environments. Parental plants had been exposed to different stress severities by Pst (green), Pc (blue), or soil salinity (orange). F1 plants were tested for resistance against different stresses than the parental stress. (A) Increased Pc susceptibility in F1 progeny from Pst-exposed parents. Box plots show lesion diameters (mm) of plants within F1 populations from similarly treated parental plants (n = 76–80). See the legend of Figure 4 for details. Different letters indicate statistically significant differences between parental treatments (ANOVA + Tukey’s post hoc test; α = 0.05). Data for individual F1 populations are shown in Supplementary Figure 5A. (B) Reduced salt tolerance in F1 progeny from Pst-exposed parents. Box plots show root growth reduction percentages by 50 mM NaCl of plants within F1 populations from similarly treated parental plants (n = 40). See the legend of Figure 5 for details. Different letters indicate statistically significant differences (ANOVA + Tukey’s post hoc test; α = 0.05). Root growth data for individual F1 populations at 0, 50, and 100 mM NaCl are shown in Supplementary Figure 5B; tolerance data for individual F1 populations to 50 and 100 mM NaCl are shown in Supplementary Figure 5C. (C) Increased Hpa susceptibility in F1 progeny from Pc-exposed parents. Stacked bars show leaf frequency distributions across Hpa resistance classes within F1 populations from similarly treated parents (n = 400–500). See the legend of Figure 3B for details. Different letters indicate statistically significant differences (pairwise Fisher’s exact tests + Bonferroni FDR, α = 0.05). Data for individual F1 populations are shown in Supplementary Figure 6A. (D) Unaltered salt tolerance in F1 progeny from Pc-exposed parents. Box plots show root growth reduction percentages by 50 mM NaCl of plants within F1 populations from similarly treated parental plants (n = 47). See the legend of Figure 5 for details. ns, no statistically significant differences (ANOVA; α = 0.05). Root growth data for individual F1 populations at 0, 50, and 100 mM NaCl are shown in Supplementary Figure 6B; tolerance data for individual F1 populations to 50 and 100 mM NaCl are shown in Supplementary Figure 6C. (E) Non-specific t-IR against Hpa in F1 progeny from NaCl-exposed parents. Stacked bars show leaf frequency distributions across Hpa resistance classes within F1 populations from similarly treated parents (n = 350–800). See the legend of Figure 3B for details. Different letters indicate statistically significant differences (pairwise Fisher’s exact tests + Bonferroni FDR, α = 0.05). Data for individual F1 populations are shown in Supplementary Figure 7A. (F) Non-specific t-IR against Pc in F1 progeny from NaCl-treated parents. Box plots show lesion diameters (mm) of plants within F1 populations from similarly treated parental plants (n = 30–60). See the legend of Figure 4 for details. Different letters indicate statistically significant differences between parental treatments (ANOVA + Tukey’s post hoc test; α = 0.05). Data for individual F1 populations are shown in Supplementary Figure 7B.