The coronatine toxin of Pseudomonas syringae is a multifunctional suppressor of Arabidopsis defense

Abstract

The phytotoxin coronatine (COR) promotes various aspects of Pseudomonas syringae virulence, including invasion through stomata, growth in the apoplast, and induction of disease symptoms. COR is a structural mimic of active jasmonic acid (JA) conjugates. Known activities of COR are mediated through its binding to the F-box-containing JA coreceptor CORONATINE INSENSITIVE1. By analyzing the interaction of P. syringae mutants with Arabidopsis thaliana mutants, we demonstrate that, in the apoplastic space of Arabidopsis, COR is a multifunctional defense suppressor. COR and the critical P. syringae type III effector HopM1 target distinct signaling steps to suppress callose deposition. In addition to its well-documented ability to suppress salicylic acid (SA) signaling, COR suppresses an SA-independent pathway contributing to callose deposition by reducing accumulation of an indole glucosinolate upstream of the activity of the PEN2 myrosinase. COR also suppresses callose deposition and promotes bacterial growth in coi1 mutant plants, indicating that COR may have multiple targets inside plant cells.

Figure 1.

COR Promotes Bacterial Virulence in SA Signaling–Deficient Plants. (A) Callose deposition in Col-0, sid2, and npr1 Arabidopsis leaves after infiltration with the indicated bacterial strains or buffer. Shown are representative fluorescence microscopy images of aniline blue–stained leaves. Bar = 0.2 mm. (B) Quantification of callose deposits following treatments as in (A). Shown are the mean and se of combined data from two independent biological replicates. Statistical analyses of log-transformed data of the indicated samples were by one-way ANOVA and Tukey HSD test with significant differences (P < 0.05) indicated by lowercase letters. (C) Effect of exogenous COR on callose deposition elicited by ΔCEL cor- in sid2 mutant plants. Shown are the mean and sd of combined data from two independent biological replicates. (D) Growth of the indicated strains 4 d after inoculation into Col-0, sid2, and npr1 Arabidopsis leaves. The dashed line indicates the starting inoculum of bacteria. Shown are the mean and se of four biological replicates. Different letter types (uppercase, lowercase, and lowercase’) indicate significant differences (P < 0.05) by one-way ANOVA and Tukey HSD test of comparisons between the different bacterial strains on individual plant genotypes.

Figure 2.

HopM1 Promotes Bacterial Virulence in SA Signaling–Deficient Plants. (A) Quantification of callose deposits following infiltration of the indicated strains into Col-0 and sid2 leaves. Shown are the mean and se of combined data from two independent biological replicates. (B) Growth of the indicated strains 4 d after inoculation into Col-0 and sid2 plants. The dashed line indicates the starting inoculum of bacteria. Shown are the mean and se of four biological replicates. Different letter types indicate significant differences (P < 0.05) by one-way ANOVA and Tukey HSD test of comparisons between the indicated bacterial strains on individual plant genotypes.

Figure 3.

COR Promotes Bacterial Virulence by Targeting COI1. Quantification of callose deposits following infiltration of the indicated strains into Col-0 and coi1 leaves. Shown are the mean and se of combined data from two independent biological replicates. Statistical analyses of log-transformed data of indicated samples were by one-way ANOVA and Tukey HSD test (P < 0.05).

Figure 4.

COR Promotes Bacterial Virulence in SA Signaling–Deficient Plants Independent of Targeting COI1. (A) Quantification of callose deposits following infiltration of the indicated strains into coi1, sid2 coi1, and npr1 coi1. Shown are the mean and se of combined data from two independent biological replicates. Statistical analyses of log transformed data of indicated samples were by one-way ANOVA and Tukey HSD test (P < 0.05). See also Supplemental Figure 4 online. (B) Effect of exogenous COR on callose deposition elicited by PtoΔCEL cor- in sid2 and sid2 coi1 mutant plants. Shown are the mean and sd of combined data from two independent biological replicates. (C) Growth of the indicated strains 4 d after inoculation into Col-0, coi1, sid2 coi1, and npr1 coi1 plants. The dashed line indicates the starting inoculum of bacteria. Shown are the mean and se of four biological replicates. Different letter types indicate significant differences (P < 0.05) by one-way ANOVA and Tukey HSD test of comparisons between the indicated plant genotypes with individual bacterial strains. Asterisks indicate significant differences between indicated samples as determined by two-tailed t test with * P < 0.05. See also Supplemental Figure 4 online.

Figure 5.

COR Inhibits Indole Glucosinolate Accumulation Upstream of PEN2. (A) Quantification of callose deposits following infiltration of PtoΔCEL or PtoΔCEL cor- into Col-0, sid2, pen2-1, and sid2 pen2-4 leaves. Shown are the mean and se of combined data from two independent biological replicates. Statistical analyses of log-transformed data were by one-way ANOVA and Tukey HSD test (P < 0.05). (B) Accumulation of 4MI3G after infiltration of buffer, flg22, or the indicated bacterial strains, with or without 3 μM COR, into Col-0, sid2, pen2-1, and sid2 pen2-4 leaves. Quantities of 4MI3G were calculated relative to sinigrin (spiked into each sample) and normalized with the amount elicited by PtoΔCEL in pen2-1 set to 1. Shown are the means and se from three biological replicates. Different letter types indicate significant differences (P < 0.05) by one-way ANOVA and Tukey HSD test of comparisons between the indicated bacterial strains on individual plant genotypes. See also Supplemental Figure 6 online. (C) Growth of the indicated strains 4 d after inoculation into Col-0, sid2, pen2-1, pen2-4, and sid2 pen2-4 plants. The dashed line indicates the starting inoculum of bacteria. Shown are the mean and se of five biological replicates, except pen2-4 data, which are from two biological replicates. Different letter types indicate significant differences (P < 0.05) by one-way ANOVA and Tukey HSD test of comparisons between plant genotypes with individual bacterial strains.

Figure 6.

Bacterially Produced COR Variably Affects Expression of Genes Involved in Indole Glucosinolate Metabolism. qRT-PCR analysis of MYB51, CYP79B2, CYP79B3, CYP83B1, and CYP81F2 expression 6 h after infiltration of Col-0 leaves with buffer, PtoΔCEL, or PtoΔCEL cor-. Shown are the average and se of normalized data from of four (MYB51) or three (CYP79B2, CYP79B3, CYP83B1, and CYP81F2) biological repeats with the level of each transcript induced by PtoΔCEL set to 1. Asterisks indicate significant differences between the indicated samples and ΔCEL sample as determined by two-tailed t test with *P < 0.005.

Figure 7.

Model: Suppression of the Arabidopsis Immune Response by COR. Unknown elicitors from Pto (PAMPs and/or T3Es) activate SA signaling and indole glucosinolate metabolism. SA signaling and JA signaling are antagonistic, with COR activating COI1 to suppress SA accumulation. COR also suppresses indole glucosinolate metabolism upstream of 4MI3G accumulation. SA signaling promotes indole glucosinolate metabolism, perhaps by suppressing JA accumulation, and also promotes callose deposition independent of COI1 through an unknown pathway. COR suppresses defense in a COI1-independent manner through an unknown pathway.