Date of Award

1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Abstract

Pseudomonas syringae pv. tomato is the causal agent of bacterial speck on tomato. An essential feature of this organism, in the pathogenic process, is its ability to enter the intercellular spaces of its host and use the available plant substrates for growth. Tomato leaves contain quantities of carbon energy in the form of carboxylates. A Tn5 mutant, DC3481, cannot utilize carboxylates as a source of carbon energy for growth (CA{dollar}\sp-{dollar}) and is nonpathogenic (Path{dollar}\sp-{dollar}) on tomato. The focus of this thesis was to utilize DC3481 to isolate and characterize P. syringae pv. tomato DNA sequences involved with growth and pathogenicity on tomato.;Initial studies produced a specific DNA probe, pGB25, to screen a wild-type (DC3000) gene library. The probe contained P. syringae pv. tomato DNA sequences, from DC3481, which flank the transposon Tn5. Four members of the DC3000 library hybridized to pG825. Two members, pDJ208 and pDJ400, both complemented DC3481 and restored the Path{dollar}\sp+{dollar} CA{dollar}\sp+{dollar} phenotype. Southern blot analysis of pDJ208 showed that two EcoR1 fragments, 3.8- and 2.5-kb in size, hybridized to pGB25. Further analysis revealed that the transposon had inserted into the 3.8-kb EcoR1 fragment. Complementation studies, with DC3481, showed that only the 3.8-kb EcoR1 fragment restored the CA{dollar}\sp+{dollar} Path{dollar}\sp+{dollar} phenotype.;Sequencing studies, on the 3.8-kb EcoR1 fragment, identified three potential open reading frames (ORFs) which could be directly affected by transposon Tn5 insertion. It appears that these sequences are not involved with dicarboxylic acid transport. Instead, these sequences appear to be associated with utilization of Entner-Doudoroff and Tricarboxylic Acid Cycle (TCA) substrates. Computer analysis revealed that one ORF, ORF1, shared amino acid residue homology to both phosphoglycerate mutase and enzymes forming part of the glyceraldehyde 3-phosphate operon.;Results of this study support the involvement of carboxylate assimilation for growth of P. syringae pv. tomato and subsequent disease expression. The mutation in DC3481 may provide further insight into nutrient assimilation beyond transport of these substrates.

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