The role of cell wall-based defences in the early restriction of non-pathogenic hrp mutant bacteria in Arabidopsis

Accumulation of reactive oxygen species was identified as the first plant response differentiating between wild-type and hrp mutant bacteria in Arabidopsis. The image shows detection of ROS in chloroplasts causing pale green fluorescence after staining with H2DCFDA 4h after inoculation with the hrpA mutant. [Display omitted] •hrp mutant bacteria are restricted within 6h after inoculation.•Secretion occurs at similar frequency next to wild type and hrp mutant bacteria.•H2O2 and peroxidase accumulate more rapidly around the hrp mutant bacteria.•Chloroplasts generate ROS after challenge with the hrp mutant.•Generation of ROS has been identified as a potential target for effector proteins. We have investigated the cause of the restricted multiplication of hrp mutant bacteria in leaves of Arabidopsis. Our focus was on early interactions leading to differentiation between virulent wild-type and non-pathogenic hrpA mutant strains of Pseudomonas syringae pv. tomato. An initial drop in recoverable bacteria detected 0–4h after inoculation with either strain was dependent on a functional FLS2 receptor and H2O2 accumulation in challenged leaves. Wild-type bacteria subsequently multiplied rapidly whereas the hrpA mutant was restricted within 6h. Despite the early restriction, the hrpA mutant was still viable several days after inoculation. Analysis of intercellular washing fluids (IWFs), showed that high levels of nutrients were readily available to bacteria in the apoplast and that no diffusible inhibitors were produced in response to bacterial challenge. Histochemical and immunocytochemical methods were used to detect changes in polysaccharides (callose, two forms of cellulose, and pectin), arabinogalactan proteins (AGPs), H2O2 and peroxidase. Quantitative analysis showed very similar changes in localisation of AGPs, cellulose epitopes and callose 2 and 4h after inoculation with either strain. However from 6 to 12h after inoculation papillae expanded only next to the hrp mutant. In contrast to the similar patterns of secretory activity recorded from mesophyll cells, accumulation of H2O2 and peroxidase was significantly greater around the hrpA mutant within the first 4h after inoculation. A striking differential accumulation of H2O2 was also found in chloroplasts in cells next to the mutant. Ascorbate levels were lower in the IWFs recovered from sites inoculated with the hrp mutant than with wild-type bacteria. The critical response, observed at the right time and pla