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The overall research interests of my laboratory are to determine the molecular/ biochemical interactions between plants and their pathogens. Over the years, we have employed several plant-pathogen systems,however, the model sysem that has been used most intensively has been the Pseudomonas syringae pv. phaseolicola-bean system. During the recent past our laboratory has focused its attention on the molecular genetics of production and regulation of phaseolotoxin, a virulence factor produced by Pseudomonas syringae pv. phaseolicola. We have cloned and characterized a gene cluster encoding many of the proteins involved in the biosynthesis of phaseolotoxin. The bacterium protects itself from its own toxin by producing a resistant form of its target, ornithine carbamoyltransferase (ROCT), under permissive conditions. One of the interesting aspects about phaseolotoxin regulation is that it and ROCT (encoded by argK ) are produced at low temperature (18ƒC) but not at high (28ƒC-30ƒC) temperature. At 30ƒC (but not at 18ƒC) the pathogen produces a DNA-binding protein that binds (in vitro) to sites in the upstream region of argK. We postulated that this protein negatively regulates argK and phaseolotoxin gene expression at 30ƒC. We have purified the DNA-binding protein (37 kDa) from bacteria grown at the higher temperature, by using heparin-agarose and other column chromatographic techniques and by southwestern blotting employing the upstream fragment from argK which harbors the binding sites as a probe. Based on the N'-terminal amino acid sequence of this protein a degenerate oligonucleotide probe was synthesized and used to screen a genomic library of P.s. pv. phaseolicola. This yielded a clone which harbors the gene encoding the 37 kDa protein. Sequence alignment has indicated that the regulatory protein has significant homology to a class of DNA-binding and chaperone-like proteins. The molecular/ genetic and biochemical techniques used in the laboratory are cloning , sequencing, transposon mutagenesis, HPLC, Southern, and Southwestern gel electrophoresis, SDS gel electrophoresis, DNA mobility shift assays, footprinting assays and related techniques.

SELECTED PUBLICATIONS

Kamdar HV, Rowley KB, Clements D, Patil SS. Pseudomonas syringae pv. phaseolicola genomic clones harboring heterologous DNA sequences suppress the same phaseolotoxin-deficient mutants. J Bacteriol. 1991;173:1073-9.

Borchert S, Patil SS, Marahiel MA. Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases. FEMS Microbiol Lett. 1991;92:175-80.

Rowley KB, Clements DE, Mandel M, Humphreys T, Patil SS. Multiple copies of a DNA sequence from Pseudomonas syringae pv. phaseolicola abolish thermoregulation of phaseolotoxin production. Mol Microbiol. 1993;8:625-35

Zhang Y, Rowley KB, Patil SS. Genetic organization of a cluster of genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. phaseolicola. J Bacteriol. 1993;175:6451-8.

Turgay K, Bachmann AS, Marahiel M, Patil SS. Cloning of a putative peptide synthetase gene involved in the synthesis of phaseolotoxin. In: Pseudomonas syringae Pathovars and Related pathogens. (Rudolph K, ed.) Kluwer Academic Publishers, dordrecht, The Netherlands; 1996, pp. 248-54.

Zhang YX, Patil SS. The PhtE locus in the phaseolotoxin gene cluster has ORFs with homologies to genes encoding amino acid transferases, the AraC family of transcriptional factors, and fatty acid desaturases. Mol Plant-Microb Interact. 1997;10:947-60

Xu R, Rowley KB, Mocz G, Patil SS. Purification of a putative repressor involved in the thermoregulation of the phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) of Pseudomonas syringae pv. phaseolicola. Phytopathol. 1999;89:S86.

Rowley KB, Xu R, Patil SS. Molecular analysis of thermoregulation of phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) from Pseudomonas syringae pv. phaseolicola. Mol Plant-Microbe Intearct. 2000;13:1071-80.

Patil SS, Steadman JR. Halo blight of bean. In: Encyclopedia of Plant Pathology (DC Maloy and TD Murray, eds.), John Wiley & Sons, Inc. 2000; pp. 521-2.

Shigaki T, Gabriel DW, Patil SS, Borthakur D, Choi JH, Alvarez AM. Blight-associated epitope and DNA fragment from Xanthomonas campestris pv. campestris are not required for Blight. Plant Biol. 2001; 106-112.

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