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 investigated. Development of colonies of the race 2 strain 85-10 and transconjugants expressing the cloned avirulence gene avrBs3 on plasmids pL3XV1-6 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 (HR). Immunocytochemical staining allowed examination of the contribution of extracellular polysaccharide (EPS) to colony development and differentiation between material of bacterial and plant origin in the intercellular space. Early growth of colonies was identical in resistant and susceptible leaves. Bacteria were not specifically attached to mesophyll cells in resistant leaves but during compatible and incompatible interactions rapidly became coated by a thick layer of EPS within 4 hr 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. By contrast, with MAb B3, which has specificity for the pyruvylated terminal mannose of the xanthan side chain, label was primarily intracellular at the poles of bacterial cells. Labeling with B3 was greatly reduced in bacteria within tissue which had undergone the HR. The AvrBs3 protein was located by immunocytochemistry within the cytoplasm of cells of X. c. pv. vesicatoria overexpressing the avrBs3 gene in vitro and 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 collapse of pepper cells during the HR followed plasma membrane damage and vesiculation of the cytoplasm initially close to the bacterial colony. The histological studies have defined the structural and temporal framework within which recognition and response occur in pepper and indicate the changing conditions to which cells of X. c. pv. vesicatoria are exposed during colonization of the intercellular space.