| Summary: | Pseudomanas syringae pv. tomato was used to investigate the role of cell wall alteration in early basal defences against microbial challenge in Arabidopsis thaliana. Wild - type DC3000 and the hrpA mutant, deficient in a structural component of the type three secretion system and consequently non-pathogenic, were used for a comparison of pathogenic and non-pathogenic interactions. A comprehensive population study was undertaken to pinpoint the crucial time, post inoculation, when bacterial populations grew in planta .The results indicated that key differences between mutant and wild - type populations occurred between 6 to 8 h post inoculation and that the hrpA mutant bacteria failed to maintain their rate of multiplication, forming a static population from 6 h after inoculation until up to twelve days after inoculation. Electron microscopy, including immunocytochemi~1and histochemical approaches were used to define changes in the cell wall between 2 and 8 h after inoculation and results indicated that similar swelling of the pectin matrix, cellulose and callose deposition could be identified in both the wild - type and mutant bacterial interactions 4 to 8 h after inoculation. Trans-vacuolar movement of arabinogalactan proteins was identified, followed by their subsequent deposition in the apoplast. Further studies using extracted intercellular wash fluid measured levels of reducing sugars, protein, and ascorbate 2 to 8 h after inoculation. Methods used included colorimetric assays and SOS - PAGE protein separation Importantly, ascorbate redox ratios differed between wild-type and mutant bacteria implicating H20 2 production as a key defence response early in the interaction. Finally confocal microscopy combined with a fluorescent probe was used to localize H20 2 to the mesophyll walls and chloroplasts in tissue challenged with the hrpA mutant. The restriction of hrp mutant bacteria appeared not to be due to any generation of antibacterial compounds, but the physical entrapment of bacterial cells in a proteinaceous and carbohydrate containing matrix.
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