Glycosylation of flagellin contributes to swimming and swarming motilities, adhesion ability, and consequently virulence in Pseudomonas syringae pv. tabaci 6605. Glycans attached to six serine residues are located in the central region of the flagellin polypeptide. The glycan structure at position Ser 201 was recently revealed to consist of two L-rhamnoses and one modified 4-amino-4,6-dideoxyglucose (viosamine). To clarify the mechanisms for glycosylation of modified viosamine, genes encoding dTDP-viosamine aminotransferase (vioA), dTDP-viosamine acetyltransferase (vioB), and viosamine-derivative transferase (vioT) were isolated and defective mutants were generated. MALDI-TOF-MS analysis of a lysyl endopeptidase-digested peptide including all six glycosylation sites from each flagellin indicated that the molecular masses of the three flagellin mutants were reduced with highly heterogeneous patterns at regular intervals of 146 Da in the mass range from m/z 13,819 to 15,732. The data indicated that the glycopeptides obtained from mutants had glycans consisting only of deoxyhexose instead of the flagellin glycans including the viosamine derivatives determined previously. The motility and virulence on host tobacco leaves were strongly impaired in the Delta vioA mutant and were weakly reduced in the Delta vioB and Delta vioT mutant strains. These results suggest that the genes vioA, vioB, and vioT are essential for glycosylation of flagellin, and accordingly are required for bacterial virulence.

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The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.