Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in arabidopsis

Abstract

Disease resistance in Arabidopsis is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) function as key signaling molecules. Epistasis analyses were performed between mutants that disrupt these pathways (npr1, eds5, ein2, and jar1) and mutants that constitutively activate these pathways (cpr1, cpr5, and cpr6), allowing exploration of the relationship between the SA- and JA/ET-mediated resistance responses. Two important findings were made. First, the constitutive disease resistance exhibited by cpr1, cpr5, and cpr6 is completely suppressed by the SA-deficient eds5 mutant but is only partially affected by the SA-insensitive npr1 mutant. Moreover, eds5 suppresses the SA-accumulating phenotype of the cpr mutants, whereas npr1 enhances it. These data indicate the existence of an SA-mediated, NPR1-independent resistance response. Second, the ET-insensitive mutation ein2 and the JA-insensitive mutation jar1 suppress the NPR1-independent resistance response exhibited by cpr5 and cpr6. Furthermore, ein2 potentiates SA accumulation in cpr5 and cpr5 npr1 while dampening SA accumulation in cpr6 and cpr6 npr1. These latter results indicate that cpr5 and cpr6 regulate resistance through distinct pathways and that SA-mediated, NPR1-independent resistance works in combination with components of the JA/ET-mediated response pathways.

Figure 1.

Effects of eds5 on PR-1, PR-2, PR-5, and PDF1.2 Gene Expression in the cpr Mutants. PR-1, PR-2, PR-5, and PDF1.2 gene-specific probes were used for RNA gel blot analysis of the indicated genotypes. The UBQ5 transcript was used as a loading standard. RNA was extracted from 3-week-old soil-grown plants. RNA gel blot analysis was performed at both Duke and Massachusetts General Hospital with similar results. c1e5, cpr1 eds5; c5e5, cpr5 eds5; c6e5, cpr6 eds5; WT, wild-type BGL2-GUS transgenic line.

Figure 2.

Effects of eds5 and npr1 on the Growth of P. s. maculicola ES4326 in the cpr Mutants. (A) Growth of P. s. maculicola ES4326 on the cpr eds5 double mutants compared with that on the cpr mutants. (B) Growth of P. s. maculicola ES4326 on the cpr npr1 double mutants compared with that on the cpr mutants. Plants were infected by infiltrating a suspension of P. s. maculicola ES4326 in 10 mM MgCl₂, corresponding to an OD₆₀₀ of 0.0001. Leaf discs were collected immediately after infection (day 0) and 3 days later. Four samples from each genotype were collected on day 0; six samples from each genotype were collected on day 3. The cpr1 npr1 double mutant was not tested because of its prohibitively small size. The results obtained in this experiment are different from those reported previously (Bowling et al., 1997; Clarke et al., 1998) because we used 10-fold less bacterial inoculum in the experiments reported here. Although this low bacterial inoculum produced consistent results in distinguishing the resistant mutants from the susceptible ones, the growth of the pathogen varied significantly in wild-type plants. Error bars represent 95% confidence limits of log-transformed data (Sokal and Rohlf, 1981). Growth analysis of P. s. maculicola ES4326 in the cpr eds5 mutants was performed at both Duke and Massachusetts General Hospital with similar results. cfu, colony-forming unit; c1e5, cpr1 eds5; c5e5, cpr5 eds5; c5n1, cpr5 npr1; c6e5, cpr6 eds5; c6n1, cpr6 npr1; WT, wild-type BGL2-GUS transgenic line.

Figure 3.

Effects of eds5 and npr1 on Resistance to P. parasitica Noco2 in the cpr Mutants. (A) Growth of P. parasitica Noco2 on the cpr eds5 double mutants compared with that on the cpr mutants. (B) Growth of P. parasitica Noco2 on the cpr npr1 double mutants compared with that on the cpr mutants. P. parasitica Noco2 infection was accomplished by spraying a conidiospore suspension (3 × 10⁴ spores mL⁻¹) onto 2-week-old plants and assaying for pathogen growth 7 days later. The infection was quantified using a hemacytometer to count the number of spores in a 10-μL aliquot of spores harvested from 25 leaves in 1 mL of water. Two independent counts from each sample were averaged. The averages from three independent samples were used to compute the number of spores per 25 leaves per milliliter (±sd). c1e5, cpr1 eds5; c1n1, cpr1 npr1; c5e5, cpr5 eds5; c5n1, cpr5 npr1; c6e5, cpr6 eds5; c6n1, cpr6 npr1; WT, wild-type BGL2-GUS transgenic line.

Figure 4.

Effects of eds5 and npr1 on SA Concentrations in the cpr Mutants. (A) Free SA in the cpr eds5 double mutants in comparison with that in the cpr mutants. (B) Free SA in the cpr npr1 double mutants in comparison with that in the cpr mutants. Leaves from 4-week-old soil-grown plants were collected and analyzed by HPLC for free SA. The values are an average of three replicates ±sd. The cpr1 npr1 double mutant was not tested because of its prohibitively small size. +avr, plants infected with P. s. maculicola ES4326/avrRpt2 3 days before tissue harvest; c1e5, cpr1 eds5; c5e5, cpr5 eds5; c5n1, cpr5 npr1; c6e5, cpr6 eds5; c6n1, cpr6 npr1; FW, fresh weight; WT, wild-type BGL2-GUS transgenic line.

Figure 5.

Effects of npr1, eds5, and nahG on HR-Mediated Resistance to P. s. maculicola ES4326/avrRpt2. (A) Symptoms observed after infection with P. s. maculicola ES4326/ avrRpt2 in wild type, npr1, eds5, npr1 eds5 (n1e5), and nahG. (B) Quantification of P. s. maculicola ES4326/avrRpt2 growth in wild type, npr1, eds5, n1e5, and nahG. cfu, colony-forming unit; n1e5, npr1 eds5. Plants were infected with a suspension of P. s. maculicola ES4326/avrRpt2 corresponding to an OD₆₀₀ of 0.001. Pictures were taken 3 days after infection. Each leaf in (A) is a representative sample from a population of 10 leaves. Leaf discs were collected immediately after infection (day 0) and 3 days later. Four samples from each genotype were collected on day 0; six samples from each genotype were collected on day 3. Error bars represent 95% confidence limits of log-transformed data (Sokal and Rohlf, 1981). The growth of P. s. maculicola ES4326/avrRpt2 on eds5 plants was similar to that observed by Nawrath and Métraux (1999) but different from that reported by Rogers and Ausubel (1997), who used a less concentrated bacterial inoculum. nahG, transgenic line expressing salicylate hydroxylase; WT, wild-type BGL2-GUS transgenic line.

Figure 6.

Effects of jar1 and ein2 on PR-1 and PDF1.2 Gene Expression in cpr5 and cpr6. PR-1 and PDF1.2 gene-specific probes were used for RNA gel blot analysis of the indicated genotypes. The UBQ5 transcript was used as a loading standard. RNA was extracted from 4-week-old soil-grown plants. c5n1, cpr5 npr1; c5e2, cpr5 ein2; c5j1, cpr5 jar1; c5n1e2, cpr5 npr1 ein2; c5n1j1, cpr5 npr1 jar1; c6n1, cpr6 npr1; c6e2, cpr6 ein2; c6j1, cpr6 jar1; c6n1e2, cpr6 npr1 ein2; c6n1j1, cpr6 npr1 jar1; WT, wild-type BGL2-GUS transgenic line.

Figure 7.

Effects of jar1 and ein2 on Resistance in cpr5 and cpr6. (A) Growth of P. s. maculicola ES4326 on wild type, cpr5, c5n1, c5n1e2, cpr6, c6n1, and c6n1e2. Error bars represent 95% confidence limits of log-transformed data. (B) Growth of P. parasitica Noco2 on wild type, cpr5, c5n1, c5e2, c5j1, c5n1e2, c5n1j1, cpr6, c6n1, c6e2, c6j1, c6n1e2, and c6n1j1. Error bars indicate se. Plants were infected and resistance was determined as described in Figures 2 and 3. cfu, colony-forming unit; c5n1, cpr5 npr1; c5e2, cpr5 ein2; c5j1, cpr5 jar1; c5n1e2, cpr5 npr1 ein2; c5n1j1, cpr5 npr1 jar1; c6n1, cpr6 npr1; c6e2, cpr6 ein2; c6j1, cpr6 jar1; c6n1e2, cpr6 npr1 ein2; c6n1j1, cpr6 npr1 jar1; WT, wild-type BGL2-GUS transgenic line.

Figure 8.

Effects of ein2 on SA Concentrations in the cpr ein2 and cpr npr1 ein2 Mutants. Leaves from 4-week-old, soil-grown plants were collected and analyzed by HPLC for free SA. The SA value in c5n1e2 (which is off the scale of the graph) is printed next to the bar to allow a better comparison between samples. The values are an average of two replicates ± range. +avr, plants infected with P. s. maculicola ES4326/avrRpt2 at 3 days before tissue harvest; c5n1, cpr5 npr1; c5e2, cpr5 ein2; c5n1e2, cpr5 npr1 ein2; c6n1, cpr6 npr1; c6e2, cpr6 ein2; c6n1e2, cpr6 npr1 ein2; FW, fresh weight; WT, wild-type BGL2-GUS transgenic line.

Figure 9.

Effects of jar1, ein2, and npr1 on HR-Mediated Resistance to P. s. maculicola ES4326/avrRpt2. (A) Symptoms observed after infection with P. s. maculicola ES4326/avrRpt2 in wild type, npr1, ein2, jar1, e2j1, n1e2, n1j1, and n1e2j1. (B) Quantification of P. s. maculicola ES4326/avrRpt2 growth in wild type, npr1, ein2, jar1, e2j1, n1e2, n1j1, and n1e2j1. Plants were infected and resistance was determined as described in Figure 5. Error bars represent 95% confidence limits of log-transformed data (Sokal and Rohlf, 1981). cfu, colony-forming unit; e2j1, ein2 jar1; n1e2, npr1 ein2; n1j1, npr1 jar1; n1e2j1, npr1 ein2 jar1; WT, wild-type BGL2-GUS transgenic line.

Figure 10.

Model of Interacting Defense Response Pathways. (A) Model showing the requirements for cpr-mediated resistance. Our data indicate that the cpr mutants activate both NPR1-dependent and NPR1-independent resistance to P. s. maculicola ES4326 and P. parasitica Noco2. Both pathways are mediated by SA and are blocked by the eds5 mutation. The NPR1-independent pathway also requires sensitivity to JA/ET and is blocked by the jar1 and ein2 mutations. It is not clear at this time whether the resistance to P. parasitica Noco2 in cpr6, which is suppressed by the ein2 and jar1 mutations, is NPR1 dependent or NPR1 independent. (B) Model overlaying NPR1-dependent and NPR1-independent resistance with HR-mediated local and systemic resistance. Our data indicate that NPR1-independent resistance in the cpr mutants resembles the local resistance response triggered during the HR. Avirulent pathogen resistance and NPR1-independent resistance both require SA- and JA/ET–mediated signaling pathways. JA/ET or sensitivity to JA/ET may be required for perception of the signal (elicitor) necessary to bypass the npr1 mutation or for expression of downstream antimicrobial proteins. Therefore, HR-mediated local resistance is shown as a function of overlapping components from both SA- and JA/ET–mediated pathways. Cell death is shown initiating this response, and elements from both the SA and JA/ET pathways are shown to be enhancing the formation of lesions. This feedback loop accounts for SA-dependent lesion mimic mutants as well as the reduction of the lesion-forming phenotype of cpr5 by eds5, ein2, and jar1. NPR1 is shown as being required only for systemic resistance. A second function for NPR1 in feedback regulation of SA accumulation is indicated by a blocked arrow. The cpr mutants are not included in this model because their locations in the signal transduction pathways are not clear. They could function either in the local pathway, inducing NPR1-dependent and NPR1-independent resistance simultaneously, or in the systemic pathway, triggering NPR1-independent resistance only when combined with npr1.