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Bacterial citrus canker disease, which is caused by Xanthomonas citri subsp. citri, is one of the most devastating diseases of citrus plants. In this study, we characterized the role of the two-component regulatory system ColR/ColS in the pathogenicity of X. citri subsp. citri. colS mutants (256A10 and 421E7), colR mutants (386C6 and 417E10), and a colR colS double mutant (306DSR) all lost pathogenicity and produced no symptoms on grapefruit leaves inoculated by either pressure infiltration or the spray method. The pathogenicity defect of the colS, colR, and colR colS mutants could be complemented using the wild-type colS, colR, and colR colS genes, respectively. Mutation of colS or colR significantly reduced X. citri subsp. citri growth in planta. The ColR/ColS system also played important roles in bacterial biofilm formation in glass tubes and on leaf surfaces, lipopolysaccharide (LPS) production, catalase activity, and tolerance of environmental stress, including phenol, copper, and hydrogen peroxide. Furthermore, quantitative reverse transcription-PCR assays demonstrated that the ColR/ColS system positively regulated the expression of important virulence genes, including hrpD6, hpaF, the O-antigen LPS synthesis gene rfbC, and the catalase gene katE. Overall, our data indicate that the two-component regulatory system ColR/ColS is critical for X. citri subsp. citri virulence, growth in planta, biofilm formation, catalase activity, LPS production, and resistance to environmental stress.

Xanthomonas citri subsp. citri is the causal agent of citrus canker, which has a significant impact on citrus production. In this study, we characterized the galU gene of X. citri subsp. citri. Two galU mutants (F6 and D12) were identified in an X. citri subsp. citri EZ-Tn5 Tnp transposon library. Rescue cloning, sequence analysis, and Southern blot analysis indicated that both of these mutants had a single copy of the EZ-Tn5 transposon inserted in galU in the chromosome. Further study showed that galU was required for biosynthesis of extracellular polysaccharides (EPS; xanthan gum) and capsular polysaccharide (CPS) and biofilm formation. Mutation of galU resulted in a loss of pathogenicity for grapefruit. The loss of pathogenicity of a galU mutant resulted from its inability to grow in planta rather than from the effect on virulence genes. Quantitative reverse transcription-PCR assays indicated that mutation of galU did not impair the expression of key virulence genes, such as pthA of X. citri subsp. citri. Although D12 had a growth rate similar to that of the wild-type strain in nutrient broth, no D12 population became established in the intercellular spaces of citrus leaves. Coinoculation of a galU mutant with the wild-type strain did not promote growth of the galU mutant in planta. Defects in EPS and CPS production, pathogenicity, and growth in planta of the galU mutant were complemented to the wild-type level using plasmid pCGU2.1 containing an intact galU gene. These data indicate that the galU gene contributes to X. citri subsp. citri growth in intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation.