Pseudomonas syringae pv. phaseolicola genomic clones harboring heterologous DNA sequences suppress the same phaseolotoxin-deficient mutants

Abstract

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)

Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv. phaseolicola

“…Three TnS mutants, TNM5, TNM7, and TNM11, previously obtained by random Tn5 mutagenesis of the chromosome of the wild-type strain (13), were complemented by pHK120, implying that the Tn5 insertions were located within the chromosomal region represented by the insert in pHK120. Southern blots of the genomic DNA from the three TnS mutants were probed with 32P-labeled pHK120 DNA, and a comparison of the size of the hybridizing genomic fragments from the three mutants with that of pHK120 revealed that the insertions were located within the pHK120 region at the positions denoted by the symbol # (Fig.…”

“…Unlike pDC938, pHK120 (a class I clone) complements five chromosomal Tn5 mutants in addition to the UV-and ethyl methanesulfonate-induced mutants (13). The complementation of all five TnS mutants by pHK120 suggested that its insert harbored a cluster of genes involved in phaseolotoxin production, because the Tn5 insertions in the mutants are located in different chromosomal EcoRI fragments.…”

“…Kamdar et al (13) reported the isolation of three nonoverlapping classes of genomic cosmid clones from a library of P. syringae pv. phaseolicola that complements 1 UV-induced and 80 ethyl methanesulfonate-induced Tox-mutants.…”

“…Plasmid DNA was extracted by the rapid boiling method or by the alkaline lysis method (2) and purified by CsCl gradient centrifugation. Genomic DNA extraction and Southern hybridization were performed as previously described (13), except when noted otherwise. DNA fragments for subcloning were isolated from low-melting-point agarose after electrophoresis and purified with the USBioclean kit (U.S. Biochemicals, Cleveland, Ohio).…”

Molecular Biology in Plant Pathogenesis and Disease Management

“…phaseolicola (Psp). The genes involved in the production of these toxins have been cloned and characterized (Xu and Gross 1988;Willis et al 1991;Kamdar et al 1991). These genes may be located in one or more clusters.…”