Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker

Abstract

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.

FIG. 1

Symptoms expressed on citrus leaves 2 weeks after inoculation with X. campestris pv. citri XW47 (A), XT906 (B), or XT906(pUW906XAp) (C).

FIG. 2

Growth in planta of X. campestris pv. citri XW47, XT906, and XT906(pUW906XAp). Data are means ± standard errors of the means (SEM) of values from three independent experiments, each performed in duplicate.

FIG. 3

Structural organization of the pgi (A) and hrpX (B) loci of X. campestris pv. citri and of plasmids derived therefrom. (A) Restriction map of the genomic region containing pgi showing the orientation of the gene (horizontal arrow) and the site of transposon insertion in the mutant XT906. The plasmids pUW906XH, pUW906XAp, and pUW906P were used for complementation studies (the results of which are summarized), and the plasmid pUW906PGUS (containing a pgi::gus fusion gene) was used for the analysis of regulation of pgi expression. (B) Restriction map of the genomic region containing hrpX showing the orientation of the gene (horizontal arrow) and the structure of the plasmid pUW10PGUS, which contains an hrpX::gus fusion gene.

FIG. 4

Expression of pgi and hrpX genes in the wild-type XW47 and mutant XT906 strains of X. campestris pv. citri grown in various culture media. Strains XW47 or XT906 were transformed with the plasmid pUW906PGUS (pgi::gus), the plasmid pUW10PGUS (hrpX::gus), or the promoterless control pUGUS (gus) and were grown for 16 h in either TSG or XVM2 medium in the absence or presence of 10, 20, or 30 mM glucose, as indicated. Cell extracts were then prepared and assayed for GUS activity. Data are means ± SEM of values from three experiments.

FIG. 5

Expression of pgi and hrpX genes in the wild-type XW47 (top) and mutant XT10 (middle) and XT906 (bottom) strains of X. campestris pv. citri grown in the presence of extracts of lemon, common bean, or tobacco plants. Bacteria were transformed with pUW906PGUS (pgi::gus), pUW10PGUS (hrpX::gus), or pUGUS (gus) and grown for 16 h in either TSG medium or plant extract. Bacterial extracts were then prepared and assayed for GUS activity. Data are means ± SEM of values from three experiments.