Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis

Abstract

The deposition of lignin during plant-pathogen interactions is thought to play a role in plant defence. However, the function of lignification genes in plant disease resistance is poorly understood. In this article, we provide genetic evidence that the primary genes involved in lignin biosynthesis in Arabidopsis, CAD-C and CAD-D, act as essential components of defence to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato, possibly through the salicylic acid defence pathway. Thus, in contrast with cellulose synthesis, whose alteration leads to an increase in disease resistance, alteration of the cell wall lignin content leads directly or indirectly to defects in some defence components.

Figure 1

Expression pattern of cinnamyl alcohol dehydrogenase (CAD) family members in response to pathogen inoculation. Gene expression level [determined by quantitative real‐time polymerase chain reaction (Q‐RT‐PCR) analysis] in leaves of 4‐week‐old Arabidopsis plants inoculated with water (blue), Pseudomonas syringae pv. tomato (Pst) DC3000 (pink) and Pst DC3000/avrRpt2 (green). The expression values of each gene were normalized using the expression level of β‐tubulin4 as an internal standard. The mean values and standard errors were calculated from two independent experiments. AU, arbitrary units.

Figure 2

Phenotypes of cad‐C, cad‐D, cad‐G, cad‐B2, cad‐1, cad‐B1, eds‐1, pad‐4 single mutants, cad‐C/cad‐D double mutant and the wild‐type ecotypes Ws and La‐er, after inoculation with Pseudomonas syringae pv. tomato (Pst). Growth of Pst DC3000 (A) and Pst DC3000/avrPphB (B) in wild‐type and mutant plants, determined at 0 days (white bars) and 3 days (black bars) post‐inoculation with a bacterial suspension of 10⁵ cfu/mL. The mean bacterial densities were calculated from 9–12 replicates and are representative of two independent experiments. According to analysis of variance (anova) test, means of colony‐forming units (cfu) do not differ significantly if they are indicated by the same lowercase letter. (C) Representative mutant and wild‐type leaves 3 days after inoculation with a suspension of the bacterial strain indicated at a density of 10⁷ cfu/mL.

Figure 3

Defence gene expression (A) and salicylic acid (SA) production (B) in the double mutant cad‐C/cad‐D (triangles) and wild‐type (squares) after inoculation with water (blue), Pseudomonas syringae pv. tomato (Pst) DC3000 (pink) and Pst DC3000/avrRpt2 (green). (A) The expression values of each gene were normalized using the expression level of β‐tubulin4 as an internal standard. The mean values and standard errors were calculated from two independent experiments. AU, arbitrary units. (B) For SA measurement, the mean values and standard errors were calculated from two independent experiments.

Figure 4

Effects of CAD‐C and CAD‐D depletion on the expression of the other members of the cinnamyl alcohol dehydrogenase (CAD) gene family and of other lignin biosynthesis‐related genes in response to Pseudomonas syringae pv. tomato (Pst). Gene expression in the wild‐type (white bars) and double mutant (gray bars), 0 h (T0) and 24 h after inoculation with water, Pst DC3000 (C) and Pst DC3000/avrRpt2 (I). The expression values of each gene were normalized using the expression level of β‐tubulin4 as an internal standard. Mean expression values and standard errors were calculated from three replicates of a representative experiment.