Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development

Abstract

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces.

Figure 1

att1-1 specifically enhances type III gene expression. (A) CCD image of avrPto-luc expression in wild-type Col-gl (WT) and att1-1 leaves. (B) Activity of avrPto-luc and trp-luc in wild-type Col-gl (WT) and att1-1 leaves. Plants were inoculated with P. syringae pv. phaseolicola carrying avrPto-luc or trp-luc, and leaves were detached at 0 or 12 h for luciferase assay. Error bars indicate standard errors. The experiments were repeated three times with similar results.

Figure 2

Kinetics of type III gene expression in wild-type Col-gl (WT) and att1-1 leaves. Plants were inoculated with P. syringae pv. phaseolicola carrying avrPto-luc (A) or hrpL-luc (B), and leaves were detached at the indicated times for luciferase assay. Error bars indicate standard errors. The experiments were repeated three times with similar results.

Figure 3

att1 displays enhanced disease severity. (A) Disease symptoms. (B) Bacterial growth assay. Wild-type Col-gl (WT) and att1-1 plants were dip-inoculated with DC3000 at 1 × 10⁸ CFU/ml. Disease symptoms were photographed 7 days after inoculation. Leaf bacterial number was determined at 0 and 4 days after inoculation. The experiment was repeated twice with similar results.

Figure 4

Map-based cloning of ATT1. (A) Physical mapping of ATT1. ATT1 was mapped to the upper arm of chromosome IV and positioned on the BAC clone F5I10. (B) T-DNA insertion in At4g00360 results in att1 phenotype. Col-gl (WT), att1-1, and the T-DNA line (Salk-005826; att1-2) were infiltrated with P. syringae pv. phaseolicola carrying the avrPto-luc reporter. Relative LUC activity was measured 12 h after inoculation. Error bars represent standard error. (C) At4g00360 complements the att1-1 mutation. An 8 kb PvuII fragment containing At4g00360 was mobilized from the BAC clone F5I10 to pBI121 (by replacing the 35S-GUS fragment) and transformed into att1-1 mutant plants. Four of the five putative primary transgenic plants tested contained the transgene. These plants (FX179-1, -3, -4, -5) were tested for avrPto-luc activation and compared with wild-type and untransformed att1-1 plants. Error bars indicate standard error.

Figure 5

Bacteria repress ATT1 expression. Wild-type plants were infiltrated with water (mock) or the indicated bacterial strains at a concentration of 2 × 10⁶ CFU/ml, and tissues were harvested at the indicated times for RNA isolation. RNA blots were hybridized with a radiolabeled ATT1 probe. The rRNA gel pictures indicate equal loading of RNA in lanes. The Northern analysis was repeated three times with similar results.

Figure 6

Sequence alignment of ATT1 with other CYP86A subfamily members. Sequences underlined with a solid line, a dashed line, and a double line indicate oxygen binding and activation motif, ERR triad, and heme binding motif, respectively.

Figure 7

cyp86a2-1 displays altered cuticle membrane ultrastructure. Ultrastructure of the wild-type Col-gl (A, C) and the cyp86a2-1 mutant (B, D) cuticle membrane from an epidermal cell (A, B) and substomatal chamber mesophyll cell (C, D). The cuticle (arrowhead) and cell wall (CW) are shown. Note the lower electron density of the cyp86a2-1 cuticle membrane, which was found in all samples examined, compared with the dense, compact cuticle in the wild-type plant. Bar equals 200 nm.

Figure 8

cyp86a2-1 has a higher transpiration rate. Whole flowering plant in-pot transpiration rates were measured for consecutive 16 h light and 8 h dark photoperiods. Error bars indicate standard deviation.

Figure 9

wax2 mutant shows normal induction of avrPto-luc. Col-0 (WT), cyp86a2-2, and wax2 plants were infiltrated with P. syringae pv. phaseolicola (avrPto-luc), and the luciferase activity was measured at 0, 6, and 12 h after inoculation. Bars indicate standard errors.

Figure 10

Induction of avrPto-gfp in the intercellular spaces. avrPto-gfp expression in the intercellular spaces (A, B) or at the leaf surface (C, D) of the wild-type (A, C) and cyp86a2-2 (B, D) plants. Leaves were inoculated with P. syringae pv. phaseolicola (avrPto-gfp) for 6 h before being examined under a confocal microscope. The images are representative of multiple plants in two experiments.