ULTRASTRUCTURE OF INTERACTIONS BETWEEN XANTHOMONAS-CAMPESTRIS PV VESICATORIA AND PEPPER, INCLUDING IMMUNOCYTOCHEMICAL LOCALIZATION OF EXTRACELLULAR POLYSACCHARIDES AND THE AVRBS3 PROTEIN

The ultrastructure of interactions between the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria and its host, pepper, was inve stigated. Development of colonies of the race 2 strain 85-10 and trans conjugants expressing the cloned avirulence gene avrBs3 on plasmids pL 3XV1-6 and pD36 was examined in pepper cultivars Early Cal Wonder (ECW ) and ECW-30R; these cultivars are isogenic apart from the absence or presence, respectively, of the Bs3 allele for resistance to X. c. pv. vesicatoria. Resistance is expressed by the hypersensitive reaction (H R). Immunocytochemical staining allowed examination of the contributio n of extracellular polysaccharide (EPS) to colony development and diff erentiation between material of bacterial and plant origin in the inte rcellular space. Early growth of colonies was identical in resistant a nd susceptible leaves. Bacteria were not specifically attached to meso phyll cells in resistant leaves but during compatible and incompatible interactions rapidly became coated by a thick layer of EPS within 4 h r after inoculation. Labeling with monoclonal antibodies (MAbs) A6 and D1 with specificity for the xanthan side chain was most dense around the bacterial cell wall; more extracellular label was found with A6. B y contrast, with MAb B3, which has specificity for the pyruvylated ter minal mannose of the xanthan side chain, label was primarily intracell ular at the poles of bacterial cells. Labeling with B3 was greatly red uced in bacteria within tissue which had undergone the HR. The AvrBs3 protein was located by immunocytochemistry within the cytoplasm of cel ls of X. c. pv. vesicatoria overexpressing the avrBs3 gene in vitro an d in the plant. Exchange of signals between bacteria and plant during both the compatible and incompatible interactions was indicated by the localized deposition of paramural papillae in mesophyll cells. The co llapse of pepper cells during the HR followed plasma membrane damage a nd vesiculation of the cytoplasm initially close to the bacterial colo ny. The histological studies have defined the structural and temporal framework within which recognition and response occur in pepper and in dicate the changing conditions to which cells of X. c. pv. vesicatoria are exposed during colonization of the intercellular space.