Increased resistance to biotrophic pathogens in the Arabidopsis constitutive induced resistance 1 mutant is EDS1 and PAD4-dependent and modulated by environmental temperature

Abstract

The Arabidopsis constitutive induced resistance 1 (cir1) mutant displays salicylic acid (SA)-dependent constitutive expression of defence genes and enhanced resistance to biotrophic pathogens. To further characterise the role of CIR1 in plant immunity we conducted epistasis analyses with two key components of the SA-signalling branch of the defence network, ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We demonstrate that the constitutive defence phenotypes of cir1 require both EDS1 and PAD4, indicating that CIR1 lies upstream of the EDS1-PAD4 regulatory node in the immune signalling network. In light of this finding we examined EDS1 expression in cir1 and observed increased protein, but not mRNA levels in this mutant, suggesting that CIR1 might act as a negative regulator of EDS1 via a post-transcriptional mechanism. Finally, as environmental temperature is known to influence the outcome of plant-pathogen interactions, we analysed cir1 plants grown at 18, 22 or 25°C. We found that susceptibility to Pseudomonas syringae pv. tomato (Pst) DC3000 is modulated by temperature in cir1. Greatest resistance to this pathogen (relative to PR-1:LUC control plants) was observed at 18°C, while at 25°C no difference in susceptibility between cir1 and control plants was apparent. The increase in resistance to Pst DC3000 at 18°C correlated with a stunted growth phenotype, suggesting that activation of defence responses may be enhanced at lower temperatures in the cir1 mutant.

Figure 1. EDS1 and PAD4 are required for cir1-mediated resistance to Pst DC3000.

Four-week old plants grown at 22°C were pressure inoculated with Pst DC3000 (10⁶ cfu mL⁻¹) and bacterial titres determined at 48 hpi. Data shown are mean values ± SEM (n = 3) from one experiment representative of three independent experiments. Mean bacterial titres (cfu cm⁻²) with different letters are significantly different (p<0.05).

Figure 2. EDS1 and PAD4 are required for cir1-mediated resistance to H. arabidopsidis Noco2.

Four-week old plants grown at 22°C were infected with H. arabidopsidis (10⁴ conidiospores mL⁻¹) and condiospore load determined at 7 dpi. ANOVA revealed a significant effect of host genotype (p<0.001) on conidiospore load. Mean conidiospore counts (spores g⁻¹ fresh weight) with different letters are significantly different (p<0.05). Data shown are mean values ± SEM (n = 4) from one experiment representative of three independent experiments.

Figure 3. EDS1 and PAD4 are required for cir1-mediated constitutive defence gene expression.

Relative expression values for At2g14160 (PR-1) and At2g31880 (suppressor of BIR1) were determined in four-week-old plants grown at 22°C using qPCR, with normalisation to Actin2 expression. Col-0 + Pst plants were inoculated with Pst DC3000 (10⁶ cfu mL⁻¹) and tissue harvested after 24 h. Values shown are the means of two independent biological repeats + SD.

Figure 4. EDS1 protein but not mRNA levels are constitutively higher in the cir1 mutant.

(A) Total protein from 4-week-old plants grown at 22°C was separated by SDS–PAGE, transferred to nitrocellulose membrane and probed with an EDS1 antibody. Equal loading of the gel was verified by Ponceau staining of the membrane after protein transfer. This experiment was repeated twice with the same results. (B) Relative EDS1 expression in 4-week-old cir1 and PR-1:LUC plants was determined using qPCR, with normalization to Actin2 expression levels. Each value is the mean of three independent biological repeats ± SEM. This experiment was repeated three times with the same results.

Figure 5. The cir1 mutant displays a temperature-sensitive growth phenotype.

Representative cir1 and PR-1:LUC plants grown for four weeks under a 16 h light/8 h dark cycle at 18, 22 or 25°C are shown. Scale bar indicates a distance of 10 mm.

Figure 6. Susceptibility to Pst DC3000 is modulated by temperature in cir1.

Four-week-old cir1 and PR-1:LUC plants grown at 18, 22 or 25°C were pressure inoculated with Pst DC3000 (10⁶ cfu mL-1) and bacterial titres determined at 48 hpi. Data shown are mean values ± SEM (n = 8–10). ANOVA revealed a significant effect of host genotype (p<0.001) and temperature (p<0.001) on bacterial titres at 48 hpi, A significant interaction between these two variables (p = 0.015) indicates that they combine non-additively to influence bacterial growth. Mean bacterial titres (cfu cm⁻²) with different letters are significantly different (p<0.05).

Figure 7. SNC1 transcript levels are not elevated in cir1.

Relative SNC1 expression in 4-week-old cir1 and PR-1:LUC plants grown at 18 or 22°C was determined using qPCR, with normalization to Actin2 expression levels. Each value is the mean of three independent biological repeats ± SEM.