The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia

Abstract

The fungal necrotrophic pathogen Rhizoctonia solani is a significant constraint to a range of crops as diverse as cereals, canola, and legumes. Despite wide-ranging germplasm screens in many of these crops, no strong genetic resistance has been identified, suggesting that alternative strategies to improve resistance are required. In this study, we characterize moderate resistance to R. solani anastomosis group 8 identified in Medicago truncatula. The activity of the ethylene- and jasmonate-responsive GCC box promoter element was associated with moderate resistance, as was the induction of the B-3 subgroup of ethylene response transcription factors (ERFs). Genes of the B-1 subgroup showed no significant response to R. solani infection. Overexpression of a B-3 ERF, MtERF1-1, in Medicago roots increased resistance to R. solani as well as an oomycete root pathogen, Phytophthora medicaginis, but not root knot nematode. These results indicate that targeting specific regulators of ethylene defense may enhance resistance to an important subset of root pathogens. We also demonstrate that overexpression of MtERF1-1 enhances disease resistance without apparent impact on nodulation in the A17 background, while overexpression in sickle reduced the hypernodulation phenotype. This suggests that under normal regulation of nodulation, enhanced resistance to root diseases can be uncoupled from symbiotic plant-microbe interactions in the same tissue and that ethylene/ERF regulation of nodule number is distinct from the defenses regulated by B-3 ERFs. Furthermore, unlike the stunted phenotype previously described for Arabidopsis (Arabidopsis thaliana) ubiquitously overexpressing B-3 ERFs, overexpression of MtERF1-1 in M. truncatula roots did not show adverse effects on plant development.

Figure 1.

Effect of exogenous ethylene treatment of A17 on susceptibility to R. solani. A, The average number of survivors (out of four seedlings planted per pot) 1 week after inoculation. B, The number of healthy leaves at 3 weeks after inoculation. C and D, Shoot dry weight (C) and root dry weight (D) 3 weeks after inoculation. Columns present group means, and the vertical lines represent their respective se values. For each graph, columns joined by the same letter are not significantly different according to the Tukey-Kramer HSD (P < 0.05). The experiment consisted of four complete blocks with four plants per pot, making a total of 16 plants per treatment.

Figure 2.

The response of a GCC box promoter element tetramer (ethylene and jasmonate responsive) to inoculation with R. solani AG8. Transgenic hairy roots containing a tetramer of the GCC box fused to the luciferase reporter gene were generated for A17 and skl and challenged with R. solani. Relative luminescence is the light emitted from the roots relative to that at time zero before inoculation. Averages and se of four replicate plants are shown.

Figure 3.

Expression of defense-related genes in response to inoculation with R. solani AG8 at 12 and 24 h post inoculation. A, Salicylate-associated gene PR-1. B and C, Salicylate- and ethylene-responsive genes PR10 (B) and BGL (C). D and E, Ethylene-responsive genes ACC oxidase (D) and Hel1 (E). F, Jasmonate-responsive gene VSP. The expression data are shown relative to Mock-A17-12 h. Columns represent group means, and the vertical lines represent their respective se values. Comparison of means was done using the Tukey-Kramer HSD test; columns connected with the same letter are not significantly different (P < 0.05). Results of two-way ANOVA are presented in Supplemental Table S1.

Figure 4.

Phylogenetic tree of selected Arabidopsis and Medicago ERF genes of subgroups B-1 and B-3.

Figure 5.

Expression of selected Medicago ERF genes in response to R. solani inoculation. B-3 subgroup ERF genes (A–F) and subgroup B-1 ERF genes (G–I) are represented at 12 and 24 h after inoculation with R. solani AG8. The expression data are shown relative to Mock-A17-12 h. All statistical analyses were done using the log-transformed values of the relative expression data. P values indicate significance levels of the linear statistical model used to evaluate the effect of the treatments (inoculation, genotype, and time; Supplemental Table S1). Columns represent group means, and the vertical lines represent their respective se values. Comparison of means was done exclusively in cases where the linear model significantly fitted the data (P < 0.05; A–F) using the Tukey-Kramer HSD test; means connected with the same letter are not significantly different (P < 0.05).

Figure 6.

Overexpression of MtERF1-1 in roots of composite plants inoculated with R. solani AG8. A, The number of healthy leaves per plant. B, Length of root lesion per plant. C and D, Shoot (C) and root (D) weights per plant. E, Relative fungal biomass as the relative abundance of P. medicaginis DNA to plant DNA measured by quantitative PCR. F, Relative expression of MtERF1-1 in roots. Ox-MtERF1-1, Composite plants with transgenic roots overexpressing MtERF1-1; Ox-GFP, composite plants with transgenic roots overexpressing GFP as a control. White bars indicate noninoculated controls, and gray bars indicate plants inoculated with R. solani AG8. In each graph, columns not connected by the same letter are significantly different according to the Tukey-Kramer HSD test for multiple comparisons (A, C, and D) or a t test for pair comparisons (B, E, and F). n ≥ 6.

Figure 7.

Performance of composite plants inoculated with other root pathogens. A, The number of healthy leaves on composite plants with and without inoculation with the oomycete P. medicaginis (Pm; n = 7). Columns not connected with the same letter are significantly different according the Tukey-Kramer HSD test (P < 0.05). B, Relative P. medicaginis biomass in planta as the relative abundance of P. medicaginis DNA to plant DNA measured by quantitative PCR. The asterisk indicates that the relative amount of pathogen DNA within Ox-GFP roots is significantly greater than that in Ox-MtERF1-1 roots (P < 0.05, as indicated by t test). C, The number of galls caused by the root knot nematode (n ≥ 12).

Figure 8.

Performance of A17 composite plants overexpressing MtERF1-1 and GFP. A, Composite plants 14 weeks after transformation. B, Number of leaves (healthy and dead) on composite plants 13 weeks after transformation. Means show no significant difference (P > 0.05) between Ox-GFP and Ox-MtERF1-1 according to t test (n = 16). C, Fluorescence microscopy of transgenic roots inoculated with GFP expressing S. meliloti. M. truncatula overexpression constructs contain DsRed as a visible marker. The hash mark indicates nontransgenic root, asterisks indicate transgenic roots, and arrows indicate the location of root nodules containing GFP expressing S. meliloti. D, Number of nodules on transgenic roots of composite plants. A minimum of 19 composite plants were scored.