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The molecular basis of pathogenesis by Xanthomonas oryzae pv. oryzae has been partly elucidated by the identification of a gene, hrpXo, required for bacterial blight on rice. A mutation in hrpXo results in the loss of pathogenicity on rice and the loss of hypersensitivity on nonhosts such as Datura stramonium and radishes. Pathogenicity and its ability to cause Xanthomonas oryzae pv. oryzae (30), a yellow-pigmented gram-negative member of the family Pseudomonadaceae, causes bacterial blight on rice, a disease of considerable importance in the lowland rice-growing regions of the world (21). X. oryzae pv. oryzae infects through natural openings by first multiplying at that site and then invading primarily the vascular tissues by propagating in the xylem (22, 31). Severe necrosis along the interveinal regions, known as leaf blight, occurs as a result of pathogen ingression and multiplication.X. campestris pv. campestris causes the black rot disease of cruciferous plants. Like X. oryzae pv. oryzae, X. campestnis pv. campestris enters through natural openings, mainly hydathodes located along the leaf edge. This organism propagates in the xylem, which results in the necrosis of interveinal tissues and blackening along the veins.For both bacteria, the mechanism of pathogenesis is poorly understood. this gene, designated hrpXo (for hypersensitive reaction pathogenicity), has been examined by protein sequence analysis. Unlike the so-called avirulence (avr) genes, whose products catalyze the formation of metabolites that differentially elicit hypersensitivity responses on incompatible cultivars of a particular host (recently reviewed in references 14, 17a, 27, and 38), the hrpX gene product prevents a vascular hypersensitivity response (VHR), which is a rapid tissue necrosis in the vascular system, in the respective compatible host (17).The clustered hrp genes that are likely involved in secretion (7) and that are unrelated to hrpX have been studied with X. campestris pv. campestris (1)

We have devised a rapid four-step procedure for the generation of directed and unmarked chromosomal deletions in bacteria, based on the use of a novel cloning vector containing the Bacillus subtilis sacB gene that encodes levansucrase and confers sucrose sensitivity, which can be used for counter-selection. Using this technique, we describe the construction of a 6.5 kb directed and unmarked deletion in a phytopathogenicity region of the chromosome in Xanthomonas campestris. This procedure allows rapid and easy transfer of a wide variety of mutant allelic DNA to the bacterial chromosome, and should be adaptable to various bacteria besides Xanthomonas spp.

Cosmid cloning and mutagenesis were used to identify genes involved in the production of phaseolotoxin, the chlorosis-inducing phytotoxin of Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of bean (Phaseolus vulgaris L.). Eight stable clones were isolated from a genomic cosmid library by en masse mating to 10 ethyl methanesulfonate (EMS)-induced Tox-mutants. In cross-matings, each suppressed all 10 mutants as well as an additional 70 EMS-induced Tox-mutants (and one UV-induced Tox-mutant). On the basis of restriction endonuclease analysis and hybridization studies, the clones were grouped into three classes. Clones in a particular class shared common fragments, whereas clones in different classes did not. Clones from class I (but not classes H and III) also suppressed TnS-induced Tox-mutants. Interposon mutagenesis and marker exchange of a representative clone from class III into the wild-type genome did not alter its Tox+ phenotype, indicating that this clone does not harbor structural or regulatory genes involved in phaseolotoxin production. We suggest that the genome of P. syringae pv. phaseolicola contains a "hot spot" in one of the functions involved in toxin production which is affected by EMS and UV and that heterologous clones are able to suppress the Tox-phenotype because their inserts encode products that are able to substitute for the product of the mutated gene. Alternatively, the inserts may contain sequences which titrate a repressor protein. In either case, the data suggest that suppression of EMS-and UV-induced mutants occurs when heterologous clones are present in multiple copies.Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of bean (Phaseolus vulgaris L.), produces a nonspecific, extracellular phytotoxin known as phaseolotoxin, which induces chlorosis, ornithine accumulation, and growth inhibition (6,21,28,29) in infected bean cultivars. Phaseolotoxin inhibits the enzyme ornithine carbamoyltransferase (OCT), which catalyzes the conversion of ornithine to citrulline in arginine biosynthesis (23). In planta its effects can be reversed by the addition of L-citrulline or L-arginine (24). In infected plants the dipeptide alanylhomoarginine is cleaved from phaseolotoxin (19) to produce octicidin ( Fig. 1) (20), which acts as the functional toxin (19). Octicidin is -20-fold more active against bean OCT than is phaseolotoxin (14). Phaseolotoxin also inhibits Escherichia coli OCT, and this inhibition was used to develop a quantitative microbiological assay for phaseolotoxin (33).Using the flanking regions of a TnS insertion fragment from a toxin-deficient (Tox-) mutant of P. syringae pv. phaseolicola as a probe, Peet et al. (25)