Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae

Abstract

Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification.

Figure 1.

PME activity was induced after pathogen treatment. A, Total PME activity was induced upon challenge with A. brassicicola. Wild-type Col-0 plants were inoculated with A. brassicicola (Alternaria) or mock. Leaf tissue was harvested immediately (0 h) and after 24, 48, 72, 96, and 120 h. Total protein was extracted and PME activity determined using a gel diffusion assay. B, Total PME activity was induced upon challenge with Pma ES4326. Wild-type Col-0 plants were inoculated with Pma ES4326 (OD₆₀₀ = 0.002) or mock. Experiments were performed as in A. Bars represent means and ses of data from four independent experiments, each with three technical replicates analyzed together using a mixed linear model. Asterisks indicate PME activity significantly higher than in mock-treated-plants at the same time point (q < 0.01). Photographs show representative leaves infected with A. brassicicola (A) or Pma ES4326 (B) at the indicated time points. [See online article for color version of this figure.]

Figure 2.

Methylesterification of cell wall pectins was reduced after pathogen challenge. A and B, Nonmethylesterified pectin was enriched (A) and methylesterified pectin was reduced (B) after challenge with A. brassicicola. Wild-type Col-0 plants were inoculated with A. brassicicola (1 × 10⁶ spores mL⁻¹) or mock. Leaf tissue was harvested immediately (0 h) and after 48, 72, and 96 h. Pectins were extracted and samples were diluted to a final concentration equivalent to 1 nmol µL⁻¹ GalA (undiluted). Samples were serially diluted (1:3 to 1:81) and 1 µL each was applied to a nitrocellulose membrane. Membranes were probed with LM19 (A) or LM20 (B) antibodies. C and D, nonmethylesterified pectin was enriched (C) and methylesterified pectin was reduced (D) after challenge with Pma ES4326. Experiments were performed as in A and B, but sample concentration was equivalent to 2 nmol µL⁻¹ GalA for the undiluted sample. Two biological replicates were performed and yielded similar results.

Figure 3.

Induction of PME activity by A. brassicicola required JA signaling and was promoted by ERF1. A, A. brassicicola-induced PME activity required DDE2. Wild-type Col-0 (Col) and the indicated mutants were inoculated with A. brassicicola (1 × 10⁶ spores mL⁻¹). PME activity was measured in tissue harvested immediately (0 h) and 24, 48, 72, and 96 h after inoculation. B, A. brassicicola-induced PME activity was unaltered in MYC2 mutants (jin1-1, jin1-7), but reduced in both a JA biosynthesis (dde2-2) and a JA coreceptor mutant (coi1-1). Experiments were performed as in A but samples harvested only after 0 and 72 h (0 h Ab and 72 h Ab). C, A. brassicicola-induced PME activity was unaltered in myc2 myc3 myc4 mutants (myc234a or b), but increased in two ERF1 overexpression lines (ERF1-1 and ERF1-2). Experiments were performed as in B. Bars represent means and ses of data from three (A and C) or four (B) independent biological replicates each with three technical replicates combined using a mixed linear model. Asterisks indicate significant differences from Col-0 at the indicated time point (q < 0.01).

Figure 4.

Pma ES4326-induced PME activity involved both the ERF1- and the MYC2-dependent branch of JA signaling. A, Pma ES4326-induced PME activity was strongly reduced in coi1. Wild-type Col-0 (Col) plants and mutants impaired in JA perception (coi1-1), JA biosynthesis (dde2-2), ET signaling (ein2-1), PAD4-dependent signaling (pad4-1), and SA biosynthesis (sid2-2) were inoculated with Pma ES4326 (OD₆₀₀ = 0.002). Leaves were harvested immediately (0 h) and after 72 h (72 h) and total PME activity was determined. B, Pma ES4326-induced PME activity was increased in the ERF1 overexpression line ERF1-1. Experiments were performed as in A. C, Pma ES4326-induced PME activity was unaltered in MYC2 single mutants jin1-1 and jin1-7 but reduced in two myc2 myc3 myc4 triple mutants. Experiments were performed as in A. Bars represent means and ses from two (A and C) or four (B) independent biological replicates with three technical replicates per sample, combined using a mixed linear model analysis. Asterisks indicate significant differences from Col-0 at each time point (q < 0.01).

Figure 5.

Pma ES4326-dependent induction in PME activity was not reduced but increased in some pme mutants. A, Pma ES4326-dependent induction of PME activity was enhanced in pme3 mutants. Wild-type Col-0 (Col) and pme3 plants were inoculated with Pma ES4326 (OD₆₀₀ = 0.002). Leaves were harvested immediately (0 h Pma ES4326) and after 72 h (72 h Pma ES4326) and total PME activity was determined. B, Pma ES4326-dependent induction of PME activity was enhanced in pme12 mutants, but unaltered in pme17 mutants and a At1g11580 mutant. Wild-type Col-0 and pme mutant plants as specified were inoculated with Pma ES4326. Leaves were harvested immediately and after 72 h and total PME activity was determined. C, Pma ES4326-dependent induction of PME activity was enhanced in pme3 pme25 and pme3 pme44 mutants, but unaltered in ppme1 pme44, pme12 pme41, pme25 pme44, and pme35 pme39 mutants. Experiment was performed as in B. Bars represent means and ses from two independent biological replicates with three technical replicates per sample, combined using a mixed linear model. Arrows represent the change in PME activity between the 0 and the 72-h time point. Asterisks indicate significant differences to wild-type Col-0 (q < 0.01).

Figure 6.

Many pme mutants allowed enhanced growth of Pma ES4326. A, Single pme mutants and multiple mutants of the PME group D are more susceptible to Pma ES4326. Plants of the indicated genotypes were inoculated with Pma ES4326 (OD₆₀₀ = 0.0002). For multiply mutant lines, numbers indicate PME gene and allele numbers; for example, 12-1 22-1 35-1 indicates pme12-1 pme22-1 pme35-1 triple mutant. Bacterial titers were determined immediately (0 dpi) and 3 d later (3 dpi). B, Mutants with defects in PME genes that show Pma ES4326-dependent expression and mutants with multiple mutations combined according to common cell wall alterations or expression patterns were more susceptible to Pma ES4326. Plants of the indicated genotypes were inoculated with Pma ES4326 by syringe infiltration. Bacterial titers were determined immediately (0 dpi) and 3 d later (3 dpi). Each bar represents the mean and se of three independent experiments, each with 4 or 12 biological replicates at 0 and 3 dpi, respectively, combined using a mixed linear model. Asterisks indicate significant differences from Col wild-type (q < 0.01). Susceptible pad4 plants were included as a positive control. Mutants have been plotted in numerical order. Col, Col-0; dpi, days post inoculation.