TOR inhibition primes immunity and pathogen resistance in tomato in a salicylic acid-dependent manner

Abstract

All organisms need to sense and process information about the availability of nutrients, energy status, and environmental cues to determine the best time for growth and development. The conserved target of rapamycin (TOR) protein kinase has a central role in sensing and perceiving nutritional information. TOR connects environmental information about nutrient availability to developmental and metabolic processes to maintain cellular homeostasis. Under favourable energy conditions, TOR is activated and promotes anabolic processes such as cell division, while suppressing catabolic processes. Conversely, when nutrients are limited or environmental stresses are present, TOR is inactivated, and catabolic processes are promoted. Given the central role of TOR in regulating metabolism, several previous works have examined whether TOR is wired to plant defence. To date, the mechanisms by which TOR influences plant defence are not entirely clear. Here, we addressed this question by testing the effect of inhibiting TOR on immunity and pathogen resistance in tomato. Examining which hormonal defence pathways are influenced by TOR, we show that tomato immune responses and disease resistance to several pathogens increase on TOR inhibition, and that TOR inhibition-mediated resistance probably requires a functional salicylic acid, but not jasmonic acid, pathway. Our results support the notion that TOR is a master regulator of the development-defence switch in plants.

Keywords: Botrytis; Xanthomonas; TOR; immunity; pathogenesis; tobacco mosaic virus; tomato.

FIGURE 1

TOR inhibition promotes Botrytis cinerea disease resistance. Tomato cultivar M82 plants were mock treated (with 1:5000 dimethyl sulphoxide [DMSO] in double‐distilled water), treated with 2 µM Torin2 (a and b), or TOR‐silenced using the virus‐induced gene silencing (VIGS) system (c and d). Plants were challenged with B. cinerea mycelia from a 72 h culture, 24 h after Torin2 treatment, or 4 weeks after VIGS on leaflets derived from leaves five to six. (b) The experiment was repeated four independent times, n = 30. Asterisks denote a statistically significant reduction in disease on TOR inhibition in a two‐tailed t test with Welch's correction, p < 0.0001. (d) The experiment was repeated three independent times, n = 14. The asterisk denotes a statistically significant reduction in disease on TOR silencing when compared with empty vector silencing (EV) in a two‐tailed t test with Welch's correction, p = 0.038

FIGURE 2

TOR inhibition increases immune responses. Tomato cultivar M82 plants were treated with 1:5000 dimethyl sulphoxide (DMSO) in double‐distilled water (mock) or treated with 2 µM Torin2. Plants were challenged with the immunity elicitors EIX (1 µg/ml) (a and b) or flg22 (1 µM) (c and d) 24 h after Torin2 treatment. (a) Ethylene induction was measured using gas chromatography. (b) Conductivity of samples immersed in water for 40 h was measured. Average conductivity of the mock treatment was defined as 100%. (c and d) Reactive oxygen species (ROS) production was measured immediately after flg22 application every 4 min, using the horseradish peroxidase‐luminol method, and expressed as relative luminescent units (RLU). For total RLU (d), average ROS production of the mock treatment was defined as 100%. Bars represent mean ± SEM, with all points shown. Experiments were repeated three or four independent times. (a) Different letters indicate statistically significant differences between samples in Welch's analysis of variance (ANOVA) with a Dunnett post hoc test, n = 9, p = 0.0037. (b) Different letters indicate statistically significant differences between samples in one‐way ANOVA with a Tukey post hoc test, n = 15, p = 0.038. (c) Asterisks indicate a statistically significant increase from mock treatment in multiple t tests with Holm–Sidak correction. The experiment was repeated four times on at least five plants per experiment per treatment, n = 40, ****p < 0.0001, **p < 0.01. (d) Asterisks indicate a statistically significant increase from mock treatment in a t test with Welch's correction, n = 40, ***p < 0.001

FIGURE 3

TOR inhibition‐mediated disease resistance is salicylic acid (SA)‐dependent. Tomato plants of the indicated genotypes (cultivar M82 and its mutant jai‐1, cultivar Moneymaker [MM] and its transgenic line NahG) were treated with 2 µM Torin2 (a and b) or TOR‐silenced using the virus‐induced gene silencing (VIGS) system. Plants were challenged with Botrytis cinerea (Bc) mycelia from a 72 h culture, 24 h after Torin2 treatment, or 4 weeks after VIGS on leaflets derived from leaves five to six. (a and c) Normalized Bc necrotic lesion size. (b and d) Percentage of disease reduction following TOR inhibition in the different genotypes. Bars represent mean ± SEM. Experiments were repeated three or four independent times. (a) Asterisks indicate statistically significant disease reduction with Torin2 treatment and letters indicate statistically significant differences among samples in a one‐way analysis of variance (ANOVA) with a Bonferroni post hoc test, n = 9, p = 0.021 (****p < 0.0001, ***p < 0.001, **p < 0.01; ns, not significant). (b) Different letters indicate statistically significant differences between samples in Welch's ANOVA with a Dunnett post hoc test, n = 9, p < 0.006. (c) Asterisks indicate statistically significant disease reduction on TOR silencing when compared with empty vector (EV) silencing in Welch's ANOVA with a Dunnett post hoc test, n = 2 4, p = 0.03 (***p < 0.001, *p < 0.05; ns, not significant). (d) Different letters indicate statistically significant differences between samples in Welch's ANOVA with a Dunnett post hoc test, n = 24, p = 0.012

FIGURE 4

TOR inhibition‐mediated increased immunity is salicylic acid (SA)‐dependent. Tomato plants of the indicated genotypes. The SA‐deficient line NahG and its wild‐type (WT) background Moneymaker (Mm), and the jasmonic acid (JA)‐insensitive mutant jai‐1 and its WT background M82 were treated with 1:5000 dimethyl sulphoxide (DMSO) in double‐distilled water (mock) or treated with 2 µM Torin2. Plants were challenged with the immunity elicitors EIX (1 µg/ml) (a and b) or flg22 (1 µM) (c–f) 24 h after Torin2 treatment. (a) Ethylene induction was measured using gas chromatography. (b) The conductivity of samples immersed in water for 40 h was measured. Average conductivity of the mock treatment was defined as 100%. (c–f) Reactive oxygen species (ROS) production was measured immediately after flg22 application every 4 min, using the horseradish peroxidase‐luminol method, and expressed as relative luminescent units (RLU). For total RLU (e and f), average ROS production of the mock treatment was defined as 100%. Bars represent mean ± SEM. Experiments were repeated three independent times on at least five plants per experiment per treatment, (c) n = 40, (d) n = 24. (a) Different letters indicate statistically significant differences between samples in Welch's analysis of variance (ANOVA) with a Dunnett post hoc test, n = 9, p = 0.042. (b) Different letters indicate statistically significant differences between samples in one‐way ANOVA with a Tukey post hoc test, n = 9, p = 0.04. (e) Different letters indicate statistically significant differences between samples in Welch's ANOVA with a Dunnett post hoc test, n = 40, p = 0.044. (f) Different letters indicate statistically significant differences between samples in Welch'’s ANOVA with a Dunnett post hoc test, n = 24, p = 0.0037

FIGURE 5

TOR inhibition and the salicylic acid (SA) analogue acibenzolar‐S‐methyl (ASM) generate a nonadditive increase in immunity. Tomato cultivar M82 plants were treated with 1:5000 dimethyl sulphoxide (DMSO, mock) or treated with 0.001% ASM, with or without the addition of 2 µM Torin2. Plants were challenged with flg22 (1 µM) 24 h after ASM and/or Torin2 treatment. Reactive oxygen species (ROS) production was measured immediately after flg22 application every 3 min, using the horseradish peroxidase‐luminol method, and expressed as relative luminescent units (RLU, a). For total RLU (b), average ROS production of the mock treatement was defined as 100%. Bars represent mean ± SEM, with all points shown. Experiments were repeated four independent times. (b) Different letters indicate statistically significant differences between samples in a Kruskal–Wallis test with Dunn's post hoc test, n = 48, p = 0.047

FIGURE 6

Defence genes are induced by TOR inhibition. Gene expression analysis of the indicated defence genes in indicated samples. Mock (1:5000 dimethyl sulphoxide [DMSO] in double‐distilled water), Torin2 (2 µM) treatment, Botrytis cinerea (Bc) infection, and Bc infection combined with Torin2 treatment were measured by reverse transcription‐quantitative PCR. Relative expression was calculated using the mean between the gene copy number obtained for three reference genes, RPL8 (Solyc10g006580), EXP (Solyc07g025390), and CYP (Solyc01g111170), and normalized to the mock treatment. Analysis was conducted on four or five individual plants. Bars represent mean ± SEM with all points shown. Different letters indicate statistically significant differences between samples in Welch's t test comparing each gene, p = 0.034

FIGURE 7

TOR inhibition promotes Alternaria alternata and Xanthomonas euvesicatoria disease resistance. Tomato cultivar M82 plants (a, b, and d) or Nicotiana benthamiana plants (c) were TOR‐silenced using the virus‐induced gene silencing (VIGS) system. Plants were naturally infected with A. alternata (a and b) or challenged with X. euvesicatoria (Xcv) (c and d) 4 weeks after VIGS. Bars represent mean ± SEM, with all points shown. At least seven individual plants were analysed. Asterisks denote statistical significance in a two‐tailed t test, (a) **p < 0.01, (b) *p < 0.05

FIGURE 8

TOR inhibition promotes tobacco mosaic virus (TMV) disease resistance. Nicotiana benthamiana plants were TOR‐silenced using the virus‐induced gene silencing (VIGS) system. Plants were infected with TMV‐GFP at a single injection site (indicated) on leaf four, 4 weeks after VIGS. Virus movement was assessed by measuring green fluorescent protein (GFP) radiance in all leaves above the injection site, 7 days postinoculation (a and b). In (b), L1 denotes the leaf immediately above the injection site, with leaves numbered successively upward until the youngest leaf (L5). (c) Average TMV distribution in the youngest system leaf (L5). Asterisks indicate a significant reduction in TMV observed in L5 in the Mann–Whitney U test, n = 26, ***p < 0.001. (d) Average total TMV distribution in all systemic leaves (L1–L5). Asterisks indicate a significant reduction in TMV observed in L1–L5 in a two‐tailed t test with Welch's correction, n = 75, ****p < 0.0001. (c and d) Boxplots represent minimum to maximum values with inner quartile ranges (box), outer quartile ranges (whiskers), median (line in box), all points shown. The experiment was repeated three times