Regulation of tradeoffs between plant defenses against pathogens with different lifestyles

Abstract

Plants activate distinct defense responses depending on the lifestyle of the attacker encountered. In these responses, salicylic acid (SA) and jasmonic acid (JA) play important signaling roles. SA induces defense against biotrophic pathogens that feed and reproduce on live host cells, whereas JA activates defense against necrotrophic pathogens that kill host cells for nutrition and reproduction. Cross-talk between these defense signaling pathways has been shown to optimize the response against a single attacker. However, its role in defense against multiple pathogens with distinct lifestyles is unknown. Here we show that infection with biotrophic Pseudomonas syringae, which induces SA-mediated defense, rendered plants more susceptible to the necrotrophic pathogen Alternaria brassicicola by suppression of the JA signaling pathway. This process was partly dependent on the cross-talk modulator NPR1. Surprisingly, this tradeoff was restricted to tissues adjacent to the site of initial infection; A. brassicicola infection in systemic tissue was not affected. Even more surprisingly, tradeoff occurred only with the virulent Pseudomonas strain. Avirulent strains that induced programmed cell death (PCD), an effective plant-resistance mechanism against biotrophs, did not cause suppression of JA-dependent defense. This result might be advantageous to the plant by preventing necrotrophic pathogen growth in tissues undergoing PCD. Our findings show that plants tightly control cross-talk between SA- and JA-dependent defenses in a previously unrecognized spatial and pathogen type-specific fashion. This process allows them to prevent unfavorable signal interactions and maximize their ability to concomitantly fend off multiple pathogens.

Fig. 1.

Effect of SA on JA-mediated defenses against the necrotroph A. brassicicola. (A) In planta-formed spores per lesion 4 dpi of mutant pad3 plants with A. brassicicola. Before inoculation, plants were treated with a solution of 10 mM MgSO₄ containing either 1 mM SA, 50 μM methyl-JA, or a combination of both. Asterisks indicate statistically significant differences compared with control treatment (Tukey–Kramer ANOVA test; α = 0.05, n = 3). (B and C) In planta-formed spores per lesion (B) and lesion size 4 dpi of WT Col-0 plants with A. brassicicola (C). Before inoculation plants were treated with a solution of 10 mM MgSO₄ supplemented with or without 1 mM SA. The absolute percentage of diseased leaves after SA treatment was 77.0% in this experiment and was always >50.0% in three replicate experiments. (D) SA-responsive PR-1 and JA-responsive PDF1.2 gene expression in untreated and SA-treated Col-0 plants at different dpi of A. brassicicola. Note that day 0 is shown only once because it is identical for untreated and SA-treated plants. To check for equal loading, blots were stripped and hybridized with a gene-specific probe for ubiquitin (UBQ). (E and F) Lesion size on right leaf halves of Col-0 plants 5 days after challenge inoculation with A. brassicicola. Two days before challenge inoculation, left halves of leaves were pressure-infiltrated with 10 mM MgSO₄ or biotrophic virulent Pst DC3000 (10⁷ cfu/ml). The absolute percentage of leaves diseased with A. brassicicola was 80.0%. Error bars in graphs represent SE. An asterisk indicates statistically significant differences compared with the control (Student's t test; α = 0.05).

Fig. 2.

Biotroph infection locally suppresses JA-mediated defenses against necrotrophic A. brassicicola through SA and NPR1. (A) Percentage of spreading A. brassicicola lesions on WT, sid2, and npr1 plants. Left halves of leaves were pressure-infiltrated with 10 mM MgSO₄ alone or with biotrophic virulent Pst DC3000 (10⁷ cfu/ml). After 2 days, the right halves of these leaves were challenge-inoculated with A. brassicicola. Error bars indicate SE. An asterisk indicates statistically significant differences compared with the control (Student's t test; α = 0.05, n = 30). (B) RNA gel blot analysis of SA-responsive PR-1 and JA/ethylene-responsive PDF1.2, HEL, CHI-B, and LOX2 gene expression in pad3 plants at different dpi with A. brassicicola. At −2 dpi, plants were infected with Pst DC3000 and, at 0 dpi, challenge inoculated with A. brassicicola. Only A. brassicicola-inoculated leaf halves were collected for RNA extraction. To check for equal loading, blots were stripped and hybridized for constitutively expressed ubiquitin (UBQ). (C) RNA gel blot analysis of JA/ethylene-responsive PDF1.2, HEL, and CHI-B gene expression in pad3 and pad3 npr1 plants at different dpi with A. brassicicola. To check for equal loading, rRNA was stained with ethidium bromide.

Fig. 3.

Systemic SA signaling and R protein-mediated resistance to biotrophs do not suppress JA-dependent defense against necrotrophic A. brassicicola. (A) In planta-formed spores per lesion 5 days after A. brassicicola challenge-inoculation of control (MgSO₄) or Pst DC3000-infected mutant pad3 plants. Two days before challenge inoculation, three lower leaves per plant were pressure-infiltrated with 10 mM MgSO₄ alone or with biotrophic virulent Pst DC3000 (10⁷ cfu/ml). Next, three systemic intact upper leaves were challenge-inoculated with A. brassicicola. Error bars indicate SE. (B) In planta-formed spores per lesion 4 days after A. brassicicola challenge-inoculation of pad3 plants that were previously treated with 10 mM MgSO₄, virulent Pst DC3000 (vir.) or Pst DC3000 carrying the avirulence genes avrRpt2 or avrRpm1. Plants received half-leaf pathogen inoculations as described in the legend of Fig. 2. Error bars indicate SE. Asterisk indicates statistically significant differences compared with the control (Tukey–Kramer ANOVA test; α = 0.05, n = 3). (C) RNA gel blot analysis of systemic expression of SA-responsive PR-1 and JA-responsive PDF1.2 genes from plants described in A. To check for equal loading, blots were stripped and hybridized for constitutively expressed ubiquitin (UBQ). (D) RNA gel blot analysis of SA-responsive PR-1 and JA-responsive PDF1.2 gene expression from plants described in B. To check for equal loading, rRNA was stained with ethidium bromide.

Fig. 4.

Models for tradeoffs between plant defenses against biotrophic and necrotrophic pathogens. (A) Proposed model for the spatial regulation of tradeoff between defenses against virulent (vir) biotrophs and necrotrophs. Virulent biotroph infection induces strong SA signaling in local and adjacent tissues, which tapers off with increasing distance from the site of infection. As a result of cross-talk, this gradient of SA signaling from local to systemic tissues is inverse-correlated to JA-mediated defenses launched against necrotrophs. In systemic tissues, SA signaling is relatively low, compared with JA signaling, and biological tradeoffs are diminished. (B) Proposed model for interaction between R protein-mediated resistance to biotrophs and JA-mediated resistance to necrotrophs. R protein-mediated resistance to avirulent (avr) biotrophs also induces a gradient of SA signaling from local to systemic tissues. Because of an unidentified factor, this SA signaling is not sufficient to suppress JA-mediated defenses against necrotrophs.