SummaryImmunogold labelling was used to probe the responses of mesophyll cells in French bean (Phaseolus vulgaris L.) to an hrpA mutant of Xanthomonas campestris pv. vesicatoria and a saprophytic strain of X.c. The non-pathogenic strains both caused localized alterations to the plant cell wall and formation of large papillae in adjacent cells. Immunocytochemistry showed the co-localization, in the cell wall and paramural deposits, of an M r 42 000 proline-rich glycoprotein with chitin-binding activity (CBPRP) and the enzyme responsible for its immobilization, an M r 46 000 peroxidase. The CBPRP appeared to lose antigenicity after cross-linking, and, unlike the peroxidase, was not detected consistently in the extracellular matrix that encapsulated bacteria onto the plant cell wall. The peroxidase may have a dual function in both the generation and utilization of H 2 O 2 for crosslinking of proteins and phenolics during the construction of papillae. A burst of H 2 O 2 was detected 1-5 h after inoculation at reaction sites by histochemical staining with cerium chloride. Progressive expansion of papillae and cell-wall alterations was, however, not associated with the maintenance of high levels of H 2 O 2 . Co-localization of callose and an M r 65 000 polypeptide component of callose synthase was also demonstrated. Synthesis of callose appeared so rapid that the enzyme became embedded in the polysaccharide so that both were detected as integral to the developing papilla. Localized alterations to the cell wall and deposition of papillae were found to involve co-ordinated synthetic and oxidative activities at microsites within responding cells, without activation of the hypersensitive reaction.
A characteristic of the defence response is the immobilisation of wall proteins possibly through the formation of covalent cross-links and the subsequent barrier formation against pathogens. A requirement for this is the generation of active oxygen species, particularly hydrogen peroxide. In the present work, we examine in depth the requirement for H2O2 and the specificity of the immobilisation with respect to particular wall proteins. Salt-extractable wall proteins were analysed for hydroxyproline content and the subset of proteins with this post-translational modification was found to be small. About 50 proteins were found to be easily salt-extractable and in response to elicitor treatment about 5 were found to be specifically immobilised. Immobilisation was very rapid and completed within 15 min after elicitation, and dependent upon the type of elicitor and the intensity of the production of active oxygen species. N-terminal sequencing and amino acid analysis revealed that, apart from one polypeptide, all immobilised proteins were (hydroxy)proline-containing glycoproteins with O-linked oligosaccharide side chains. In contrast, N-linked glycoproteins were not immobilised. N-terminal protein sequencing revealed the immobilised HRGPs to be novel, but both extensin and PRP-like. Implications of these findings for both pathogenic and symbiotic processes are also discussed.
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