Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana

Abstract

The plant-signaling molecules salicylic acid (SA) and jasmonic acid (JA) play an important role in induced disease resistance pathways. Cross-talk between SA- and JA-dependent pathways can result in inhibition of JA-mediated defense responses. We investigated possible antagonistic interactions between the SA-dependent systemic acquired resistance (SAR) pathway, which is induced upon pathogen infection, and the JA-dependent induced systemic resistance (ISR) pathway, which is triggered by nonpathogenic Pseudomonas rhizobacteria. In Arabidopsis thaliana, SAR and ISR are effective against a broad spectrum of pathogens, including the foliar pathogen Pseudomonas syringae pv. tomato (Pst). Simultaneous activation of SAR and ISR resulted in an additive effect on the level of induced protection against Pst. In Arabidopsis genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, induction of ISR did not affect the expression of the SAR marker gene PR-1 in plants expressing SAR. Together, these observations demonstrate that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these pathways. SAR and ISR both require the key regulatory protein NPR1. Plants expressing both types of induced resistance did not show elevated Npr1 transcript levels, indicating that the constitutive level of NPR1 is sufficient to facilitate simultaneous expression of SAR and ISR. These results suggest that the enhanced level of protection is established through parallel activation of complementary, NPR1-dependent defense responses that are both active against Pst. Therefore, combining SAR and ISR provides an attractive tool for the improvement of disease control.

Figure 1

Induced protection against Pst in Arabidopsis plants expressing ISR, SAR, or both types of induced resistance. ISR was induced by growing the plants in soil containing WCS417r (417r) at 5 × 10⁷ cfu/g. SAR was induced in wild-type Col-0 plants by preinfecting three leaves per plant with avirulent Pst(avrRpt2) (avrPst) at 10⁷ cfu/ml (A), or by exogenous application of 1 mM SA (B) 3 days before challenge inoculation. Mutant cpr1 constitutively expressed SAR (C). The disease index is the mean ± SE (n = 20 plants) of the proportion of leaves with symptoms per plant relative to that of control-treated (Ctrl) Col-0 plants (set at 100%), 4 days after challenge with virulent Pst. The absolute proportions of diseased leaves of the control-treated Col-0 plants depicted in A, B, and C were 55%, 52, and 71%, respectively. Within each frame, different letters indicate statistically significant differences between treatments (Fischer's Least Significant Differences test; α = 0.05). Corresponding bacterial growth data are given in Table 1. The data presented are from representative experiments that were repeated at least twice with similar results. In D, growth curves of Pst in Col-0 plants expressing ISR and in cpr1 plants expressing either SAR or both SAR and ISR. Values presented are average numbers (±SE) of cfu/g fresh weight, each from five whole shoots harvested 0, 1, 2, or 3 days after challenge with Pst. For experimental details see text and Table 1 legend. The additive effect on inhibition of pathogen growth in the combination treatment was statistically significant at all time points tested (Fisher's Least Significant Differences test; α = 0.05). Circles, Col-0 plants; triangles, cpr1 plants; solid lines with closed symbols, control treatment; and dotted lines with open symbols, WCS417r treatment.

Figure 2

Quantification of protection against Pst in Arabidopsis genotypes jar1, etr1, NahG, and npr1 after treatment with the ISR inducer WCS417r, the SAR inducer Pst(avrRpt2), or a combination of both inducers. For experimental details see the text and Fig. 1 legend. The absolute proportions of diseased leaves of control-treated jar1, etr1, NahG, and npr1 plants were 82%, 75%, 89%, and 70%, respectively. Within each frame, different letters indicate statistically significant differences between treatments (Fischer's Least Significant Differences test; α = 0.05). The data presented are from a representative experiment that was repeated twice with similar results.

Figure 3

RNA blot analysis of the expression of the SAR response gene PR-1 in Arabidopsis plants expressing ISR, SAR, or both. ISR was induced by growing the plants in soil containing WCS417r (417r). SAR was induced in wild-type Col-0 plants either by preinfecting three leaves per plant with Pst(avrRpt2) (avrPst) or by dipping the plants in 1 mM SA 3 days before harvest, or was constitutively expressed in mutant cpr1. Of the Pst(avrRpt2)-induced plants, the systemic, noninoculated tissue was collected. A PR-1 gene-specific probe was used to detect PR-1 transcripts. To check for equal loading, the blots were stripped and hybridized with a gene-specific probe for β-tubulin (Tub).

Figure 4

RNA blot analysis of the expression of the SAR and ISR regulatory gene Npr1 in Arabidopsis Col-0 plants expressing ISR, SAR, or both types of induced resistance. ISR was induced by growing the plants in soil containing WCS417r (417r). SAR was induced 3 days before harvest by preinfecting three leaves per plant with Pst(avrRpt2) (avrPst). Of the Pst(avrRpt2)-induced plants, the systemic, noninoculated tissue was collected. A Npr1 gene-specific probe was used to detect Npr1 transcripts. To check for equal loading, the blots were stripped and hybridized with a gene-specific probe for β-tubulin (Tub).

Figure 5

Model for the enhanced level of induced protection against Pst in Arabidopsis plants simultaneously expressing SAR and ISR. Pathogen-induced SAR is dependent on SA, requires NPR1, and is associated with PR gene expression (25, 54). WCS417r-mediated ISR requires responsiveness to JA and ethylene, also is dependent on NPR1 but is not associated with PR gene expression (35), indicating that downstream of NPR1 the pathways diverge. PR proteins that accumulate in plants expressing SAR are unlikely to contribute to induced resistance against P. syringae pathogens (53). Cross-talk between the SAR and the ISR pathway is absent. Simultaneous activation of SAR and ISR results in an additive effect on the level of protection against Pst. This is not accompanied by an increase in the expression of the Npr1 gene. Therefore, the enhanced level of induced protection against Pst must be accomplished through the parallel activation of so far unidentified defense responses that are all effective against Pst. The complementary effects on the level of protection could be achieved through the production of distinct (model I) or more of the same antibacterial gene products (model II). In both cases, simultaneous induction of SAR and ISR leads to enhanced levels of defensive components that are active against Pst. Question marks indicate unidentified defensive components.