Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.

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Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of 83-1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of 8-1,3-glucanase was observed after inoculation of leaves with heat-or nfampicin-killed X. c. vitians, Escherichia coii, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major ,-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pi] -8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pi 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2.gens. All plant lysozymes so far described have been shown to act as endochitinases (3). (3-1,3-Glucanase and chitinase are coordinately induced in a number of plant tissues by pathogen attack and elicitors (25).We are interested in the mechanisms of resistance of Brassica spp., in particular turnip (Brassica campestris) to incompatible pathovars of the Gram-negative phytopathogen Xanthomonas campestris. X. campestris pv campestris (hereafter referred to as X. c. campestris) is an important worldwide pathogen of almost all cultivated brassicas and noncrop crucifers (27) whereas X. c. vitians, a pathogen of lettuce (Lactuca sativa) is incompatible with brassica and triggers HR characterized by tissue collapse (6). As part of our study on defence mechanisms in Brassica, we have investigated the induction of CHL and (3-1,3-glucanase in response to X. c. campestris, X. c. vitians, and Escherichia coli (a nonpathogen). Lysozyme activity in turnip was originally described by Fleming (10) and the enzymes from turnip root (1) and cauliflower (9) have been partially characterized. In this paper we report on the characterization of the (-1,3-glucanase and of isoforms of CHL that show differential regulation in response to the different bacteria.The induction of the hydrolytic enzymes (3-1,3-glucanase and chitinase has been studied in a number of plants in response to infection, heat-killed pathogens, pathogen cell walls, or wounding (2,3,11). Some of the PR2 proteins induced in several different plants in response to infection have now been identified as (3-1,3-glucanases and chitinases (12,13,18). These hydrolases are of interest as plant defence products because of their...