Downstream divergence of the ethylene signaling pathway for harpin-stimulated Arabidopsis growth and insect defense

Abstract

Ethylene (ET) signal transduction may regulate plant growth and defense, depending on which components are recruited into the pathway in response to different stimuli. We report here that the ET pathway controls both insect resistance (IR) and plant growth enhancement (PGE) in Arabidopsis (Arabidopsis thaliana) plants responding to harpin, a protein produced by a plant pathogenic bacterium. PGE may result from spraying plant tops with harpin or by soaking seeds in harpin solution; the latter especially enhances root growth. Plants treated similarly develop resistance to the green peach aphid (Myzus persicae). The salicylic acid pathway, although activated by harpin, does not lead to PGE and IR. By contrast, PGE and IR are induced in both wild-type plants and genotypes that have defects in salicylic acid signaling. In response to harpin, levels of jasmonic acid (JA) decrease, and the COI1 gene, which is indispensable for JA signal transduction, is not expressed in wild-type plants. However, PGE and IR are stimulated in the JA-resistant mutant jar1-1. In the wild type, PGE and IR develop coincidently with increases in ET levels and the expression of several genes essential for ET signaling. The ET receptor gene ETR1 is required because both phenotypes are arrested in the etr1-1 mutant. Consistently, inhibition of ET perception nullifies the induction of both PGE and IR. The signal transducer EIN2 is required for IR, and EIN5 is required for PGE because IR and PGE are impaired correspondingly in the ein2-1 and ein5-1 mutants. Therefore, harpin activates ET signaling while conscribing EIN2 and EIN5 to confer IR and PGE, respectively.

Figure 1.

Effects of harpin on the growth of Arabidopsis. A, Appearance of plants grown in pots. B, Quantification of plant growth in pots. Subsection a, Increase in plant weight with time. Subsection b, Effects of harpin dose on growth. C, Appearance of roots grown on agar medium. D, Quantification of root growth on agar medium. Subsection a, Increase of root length with time. Subsection b, Effects of harpin dose on root growth. Plants in A and B were grown in pots in a controlled-environment chamber. Harpin at 15 μg mL⁻¹, or the indicated dose, and 15 μg mL⁻¹ EVP were applied 20 d after sowing by spaying plants to runoff. The plants shown in the insets in A are representative of those grown from seeds that were soaked in 15 μg mL⁻¹ harpin or 15 μg mL⁻¹ EVP for 6 h. Plants shown in the pots in the main photo of A were grown from untreated seeds. In B, plant weight was determined at the indicated times (a) or at 25 dpt (b). In C, plants shown are representative of those that grew from seeds soaked in 15 μg mL⁻¹ harpin or 15 μg mL⁻¹ EVP for 6 h prior to sowing on agar medium. In D, root length was measured at indicated times (a) or at 15 dpt (b). Quantitative assays were done 20 (A and B) or 10 (C and D) times; each assay was done with 3 replicates (B–D); each replicate involved 5 plants. In graphs and histograms (B and D), bars refer to statistical deviation.

Figure 2.

Effects of harpin on colonization of Arabidopsis by green peach aphids. A, Appearance of plants following treatment and infestation with aphids. Subsection a, Overall damage by the insects to densely grown plants. Subsection b, Insect colonies on lower surfaces of single leaves from treated plants. Subsection c, Insects on single plants. B, Quantification of insect reproduction. Subsection a, Insect multiplication progressed with time following treatment. Subsection b, Effects of harpin concentration on insect reproduction. Twenty-day-old plants had been sprayed separately with solutions of EVP and harpin at 15 μg mL⁻¹ or the indicated concentrations. At 5 dpt, aphids were moved from nurse plants to the treated plants. Plants in A, subsection a, were photographed 30 d after colonization by approximately 200 aphids per pot. In A, b and c, and B, mature aphids were placed on the lower sides of two leaves of a plant, 10 insects per leaf for A, b and c, and 5 insects per leaf for B. Leaves and plants were photographed 7 d later. The number of nymphs per plant was counted at 5 dpt or at the indicated times. Quantitative assays were done five times. For each assay, five plants were treated and evaluated. The data are presented as means ± sd.

Figure 3.

Effects of harpin on the JA-, SA-, and ET-signaling pathways in Arabidopsis. A, Quantification of the hormones. Subsections a and b, Endogenous levels of free JA and SA. Subsections c and d, Amounts of ET released. B, The effects of harpin and known elicitors on expression of genes involved in the pathways. Subsections a to c, RT-PCR analysis of signaling regulatory genes, defense genes, and expansin genes. Subsection d, RNA gel-blot analysis to confirm RT-PCR results. In A, a solution containing 15 μg mL⁻¹ harpin (rectangles) or 15 μg mL⁻¹ EVP (triangles) was applied by spraying the seedlings (A, a–c) or by soaking seeds for 6 h before incubation on agar medium (A, d). Levels of JA, SA, and ET were quantified as described in the text based on fresh weight of tissues (A, a–c) or germinated seeds (A, d). The data points indicate means ± sd of results. In B, lower leaves of plants were sprayed separately with solutions containing the indicated compounds. Twelve hours later, or at the indicated times, RNA was extracted from untreated upper leaves. The constitutively expressed gene EF1α was used as a standard in the RT-PCR protocol. RT-PCR products of genes in B, a and b, were loaded to gel separately; products of genes in B, subsection c, were loaded in a mixture of equal amounts. In B, subsection d, harpin 12 and harpin 24 refer to leaf sampling at 12 and 24 hpt with harpin. Treatment with harpin resulted in increased expression of ETR1 and ERS1 encoding the ET receptors ERF1 and NPR1 that are required, respectively, for ET and SA signal transduction. Harpin did not induce expression of CTR1, which functions in the absence of an ET signal. Harpin also did not induce expression of COI1, which is required for JA signaling.

Figure 4.

Effects of Arabidopsis genotypes on expression of several effector genes in SA, ET, and JA signaling in response to harpin. A, Gel patterns of gene expression. B, Relative levels of gene expression. RT-PCR was conducted with RNA isolated at 3 dpt from untreated upper leaves of plants that had been sprayed on lower leaves with EVP or harpin. In gel photos, the brightest band of the 100-bp ladder is 600 bp. Gene expression levels are presented as white and black bars, respectively, for treatments with EVP (control) and harpin. For each gene, the arbitrary units were determined by verification based on defining the expression level in controls as 10. Data are given as means ± sd from three replicates.

Figure 5.

Effects of Arabidopsis genotypes on the development of PGE and IR in response to harpin. A, Root length of seedlings grown on agar medium. B, The number of aphids on leaves of potted plants. Plants were observed at the indicated times after soaking seeds (A) or spraying seedlings (B) with EVP (triangles and left insets) or harpin (rectangles and right insets). Seedlings in insets were photographed when the effects were evident by 5 dpt for the wild type, jar1-1, and NahG and 8 dpt for others. The data points indicate means ± sd of results from three replicates each containing approximately 120 seedlings in A and 15 plants in B.

Figure 6.

Effects of inhibitors and inducers of ET synthesis and perception on the induction of PGE and IR by harpin in wild-type Arabidopsis and ein mutants. A, Assays of wild-type plants in response to different treatments. Subsection a, Fresh weight of plants grown in pots. Subsection b, Numbers of aphids on leaves of plants grown in pots. Subsection c, Length of roots grown on the agar medium. B, Assays of ein2-1 plants grown on the medium. Subsection a, ET levels from roots. Subsection b, Root length. C, Assays of ein5-1 plants grown in pots. Subsection a, ET levels from leaves. Subsection b, Numbers of aphids on leaves. Solutions that contained the indicated compounds were applied separately by spraying 20-d-old potted plants or soaking seeds for 6 h before germination. Soaked seeds were placed on the agar medium for root growth. AOA and AgNO₃ (Ag) were applied together with harpin and labeled as H + AOA and H + Ag, respectively. Fresh weight of potted plants was determined at 20 dpt and is shown as weight per plant. Nymphs present on each plant were counted at 7 dpt and are shown as the number of nymphs per plant. Lengths of roots grown on medium were measured at 10 dpt. At 12 hpt, ET was quantified with fresh weight (FW) of leaves. Bars in the histograms represent sd.