Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae

Abstract

Vascular wilts caused by Verticillium spp. are very difficult to control and, as a result, are the cause of severe yield losses in a wide range of economically important crops. The responses of Arabidopsis thaliana mutant plants impaired in known pathogen response pathways were used to explore the components in defence against Verticillium dahliae. Analysis of the mutant responses revealed enhanced resistance in etr1-1[ethylene (ET) receptor mutant] plants, but not in salicylic acid-, jasmonic acid- or other ET-deficient mutants, indicating a crucial role of ETR1 in defence against this pathogen. Quantitative polymerase chain reaction analysis revealed that the decrease in symptom severity shown in etr1-1 plants was associated with significant reductions in the growth of the pathogen in the vascular tissues of the plants, suggesting that impaired perception of ET via ETR1 results in increased disease resistance. Furthermore, the activation and increased accumulation of the PR-1, PR-2, PR-5, GSTF12, GSTU16, CHI-1, CHI-2 and Myb75 genes, observed in etr1-1 plants after V. dahliae inoculation, indicate that the outcome of the induced defence response of etr1-1 plants seems to be dependent on a set of defence genes activated on pathogen attack.

Figure 1

(A) Percentage disease severity of Arabidopsis thaliana plants after inoculation by Verticillium dahliae. Disease severity at each observation was calculated from the number of leaves that showed wilting as a percentage of the total number of leaves of each plant. Each genotype consisted of 15 plants and the experiment was repeated three times with similar results. The columns represent the means of 15 plants and the vertical bars indicate the standard errors. (B) Disease ratings were plotted over time to generate disease progress curves; subsequently, the area under the disease progress curve (AUDPC) was calculated by the trapezoidal integration method (Campbell and Madden, 1990), and disease was expressed as a percentage of the maximum possible area for the whole period of the experiment, which is referred to as the relative AUDPC (Korolev et al., 2001). Columns with different letters are statistically significantly different according to Tukey's multiple range test at P≤ 0.05.

Figure 2

Typical symptoms caused by Verticillium dahliae on Arabidopsis salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) mutants. The mutants etr1‐1, ein2‐1, ein3‐1, ein4, ein5‐1, jar1‐1, pad3‐1, pad4‐1, sid2, npr1‐1, NahG, eds5/sid1 and the corresponding wild‐type Col‐0 were inoculated with V. dahliae or mock inoculated. V. dahliae‐inoculated etr1‐1 mutants show reduced symptom development, including less severe stunting, wilting and tissue necrosis, when compared with Col‐0 and the other mutant plants. Photographs were taken at 20 days post‐inoculation.

Figure 3

Quantification of the Verticillium dahliae DNA levels in the ethylene (ET) mutants etr1‐1, ein2‐1, ein3‐1, ein4, ein5‐1 and Col‐0 plants. The fungal DNA levels were estimated by quantitative polymerase chain reaction (qPCR) using total plant DNA isolated from the above‐ground parts of 10 plants per genotype and sampling at 5, 10, 15, 20 and 25 days post‐inoculation (dpi). The experiment was repeated three times. The columns represent the means of 30 plants and the vertical bars indicate the standard errors. At each sampling day, columns with different letters differ significantly according to Tukey's multiple range test at P≤ 0.05.

Figure 4

Expression of GSTF12 (A), GSTU16 (B), CHI‐1 (C), CHI‐2 (D), PR‐1 (E), PR‐2 (F), PR‐5 (G) and Myb75 (H) in Arabidopsis thaliana Col‐0, ein4 and etr1‐1 plants in response to infection with Verticillium dahliae. RNA was isolated from the above‐ground parts of 10 plants per genotype with sampling at 2, 6, 10 and 14 days post‐inoculation (dpi). Poly(A)⁺‐RNA was reverse transcribed to cDNA, and subjected to quantitative polymerase chain reaction (qPCR). Transcript levels in the different samples were normalized to those of the constitutive gene α‐tubulin. The relative mRNA level was calculated with respect to the level of the corresponding transcript in uninfected plants. Each genotype consisted of 80 plants (40 V. dahliae‐ and 40 mock‐inoculated) and the experiment was repeated twice. The columns represent the means of 20 plants and the vertical bars indicate the standard errors.