Biological Sciences, School of


Date of this Version



A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biological Sciences, Under the Supervision of Professor James R. Alfano. Lincoln, Nebraska: July, 2012

Copyright (c) 2012 Emerson Crabill


Pseudomonas syringae is a Gram-negative plant pathogen whose virulence is dependent upon its type III secretion system (T3SS), a nanosyringe that facilitates translocation, or injection, of type III effector (T3E) proteins into eukaryotic cells. The primary function of P. syringae T3E proteins is suppression of plant immunity. Bacterial proteins called translocators form a translocon that forms a pore in the host plasma membrane which is traversed by T3Es. HrpK1, a putative P. syringae translocator, is a type III-secreted protein important for virulence and T3E injection, but not secretion of T3Es. Harpins are a group of proteins specific to plant pathogens that are also important for T3E translocation. P. syringae pv. tomato DC3000 has 4 harpins – HrpZ1, HrpW1, HopAK1, and HopP1. Here, HrpK1 is confirmed to be a translocator. HrpK1 had a greater impact on T3E translocation than the harpins. HrpK1 and HrpZ1 disrupted liposomes. Both proteins interacted with phosphatidic acid which interfered with T3E translocation. HrpJ, a type III-secreted protein required for HrpZ1 secretion, was also required for secretion of HrpK1, HrpW1, and HopAK1. A hrpJ mutant secreted elevated levels of the Hrp pilus protein HrpA1. HrpJ appears to control transition from Hrp pilus secretion to translocator secretion. Secretion was complemented by secretion incompetent HrpJ derivatives indicating that HrpJ controls secretion from inside the bacteria. The hrpJ mutant expressing secretion incompetent HrpJ was reduced in virulence but was complemented by HrpJ expressed inside plant cells. Additionally, transgenic Arabidopsis plants expressing HrpJ were reduced in their immune responses indicating that HrpJ can suppress plant immunity. Plants pretreated with an inducer of pathogen-associated molecular pattern-triggered immunity are unable to produce an HR. Plants, as an immune response, have evolved the ability to block T3E translocation when plant immunity has been induced prior to bacterial inoculation. This is especially true in non-host interactions whereas virulent bacteria appear to be able to attenuate injection restriction in host plants via T3E activity.

Adviser: James R. Alfano