Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent for wildfire disease in tobacco. Recently, we found that flagellin, a major constituent in the flagella filament of this pathogen, is a potent elicitor of hypersensitive reaction in non-host plant species. We also found that flagellin is required for virulence against the host plant. In this study, we investigated the biochemical features of the glycosyl moiety of flagellin and the phytopathological role of flagellin glycosylation. DNA sequence analysis of the flagellum gene cluster revealed that two genes (orf1 and Δrf2) encoding a putative glycosyltransferase were located upstream of the fliC gene. To investigate the role of flagellin glycosylation, we generated deletion mutants for orf1 ( orf1) and Δrf2 ( Δrf2) in Pta6605 and pv. glycinea race 4 (Pgl4). The mutants, orf1 and Δrf2, of both pathovars produced nonglycosylated or partially glycosylated flagellins, respectively. Inoculation of host plants with these mutant strains confirmed that orf1 and Δrf2 had reduced ability to cause disease. Biochemical and genetic approaches revealed that a total of six serine residues of FliC were glycosylated in Pta6605. The serine residues were replaced individually with Ala by site-directed mutagenesis. All glycosylation-defective mutants including orf1 and Δrf2 and the six Ser/Ala-substituted mutants of pv. tabaci retained swimming ability but their swarming ability was reduced. The abilities to adhere to a polystyrene surface and to cause disease in host tobacco plants were also impaired in all Ser/Ala-substituted mutants of Pta6605. When tobacco leaves were inoculated with Pta6605 wild-type, bacteria were embedded and formed a biofilm-like structure in the matrix on the tobacco leaf surface. In contrast, mucoid material was rarely detected in the area surrounding the orf1 mutant. These results suggest that glycosylation of flagellin in Pta6605 is required for swarming motility, adhesion, biofilm formation, and bacterial virulence. Furthermore, the ability of nonglycosylated flagellin from Pta6605 to induce a defense response in tobacco cells was greater than that of glycosylated flagellin, suggesting that the glycan moiety of flagellin may mask the elicitor function of the flagellin molecule in its host plant.
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Ichinose, Y., Taguchi, F., Takeuchi, K., Suzuki, T., Toyoda, K., Shiraishi, T. (2008). Role of Flagellin Glycosylation in Bacterial Virulence. In: Fatmi, M., et al. Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-6901-7_18
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