Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae

Pili are required for protein and/or DNA transfer from bacteria to recipien t plant or bacterial cells, based on genetic evidence. However, it has neve r been shown directly that the effector proteins or DNA are localized along or inside the pili in situ. Failure to visualize an association of effecto r proteins/DNA with pili is the central issue in the debate regarding the e xact function of pili in protein and DNA transfer. In this study, a newly d eveloped in situ immunogold labelling procedure enabled visualization of th e specific localization of type III effector proteins of Erwinia amylovora and Pseudomonas syringae pv. tomato along the Hrp pilus, but not along the flagellum or randomly in the intercellular space. In contrast, PelE, a pect ate lyase secreted via the type II protein secretion system, was not associ ated with the Hrp pilus. These results provide direct evidence that type II I secretion occurs only at the site of Hrp pilus assembly and that the Hrp pilus guides the transfer of effector proteins outside the bacterial cell, favouring the 'conduit/guiding filament' model.