Immunogold labeling of Hrp pili of Pseudomonas syringae pv. tomato assembled in minimal medium and in planta

Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseu domonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible to mato and Arabidopsis thaliana plants and to elicit the hypersensitive respo nse in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, Hr pW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in h rp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilu s structural protein on the basis of copurification and mutational analyses , In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labe ling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilu s antibody revealed a characteristic lineup of gold particles around bacter ia and/or at the bacterium-plant contact site. These results confirm that H rpA is the major structural protein of the Hrp pilus and provide evidence t hat Hrp pili are assembled in vitro and in planta.