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Doctoral Dissertation Announcement
Candidate: Vanessa Revindran
Doctor of Philosophy
Department: Biological Sciences
Title: Isolation and Characterization of Cellular Targets of HopM1; a Plant Pathogen Effector of Pseudomonas syringae pv. tomato
Dr. John Geiser, Chair
Dr. Bruce Bejcek
Dr. Todd Barkman
Dr. Wendy Ransom-Hodgkins
Dr. William Tedford
Date: Wednesday, March 14, 2012 9:00 a.m. to 11:00 a.m.
2908 Wood Hall
The researchers have created a yeast model system to study the action of the plant pathogen effector HopM1 in Saccharomyces cerevisiae. Pseudomonas syringae, causative agent of bacterial speck in tomatoes, utilizes the type III secretion system to shuttle the effector proteins into the host cell.
When expressed in yeast, HopM1 is lethal on solid media at 21C, but not at 30C and 37C. The same temperature sensitive ability of HopM1 to cause death on solid media is also observed in liquid. As demonstrated by SDS PAGE-Western blot analysis, HopM1 protein is present at 21C, 30C and 37C. At 21C, a full-length protein of 78kDA is observed. At 30C and 37°C, the majority of HopM1 protein exists as degraded fragments. HopM1 containing strains were visualized using the V5 epitope and immunofluorescent microscopy. HopM1 localizes to mitochondria and secretory organelles. This result was confirmed using cellular fractionation and sucrose gradient density centrifugation. When plated on media containing glycerol, we observed no change in expression of HopM1, thus indicating that is it unlikely that binding to mitochondria result in the lethal phenotype.
The researchers have isolated 19 spontaneous suppressor strains that are capable of surviving the HopM1 imposed lethality at 21C. All strains have been examined for (over) HopM1 protein expression, of which 13 express full-length HopM1 at 21C, and five do not. SupM1-16, showed a significant increase in growth rates as compared to the wild type strain expressing HopM1. None of the suppressor strains show a change in localization of HopM1 as compared to wild type. One of the suppressor strains, SupM1-16 was sequenced to identify the gene(s) responsible for the suppression phenotype. Six genes that may be the suppressor gene were identified. The most likely candidate is RSP5; an E3 Ubiquitin Ligase. RSP5 contains a single mutation that changes a Glycine to Valine in the HECT domain. Overall the findings suggest that HopM1 kills the yeast cell by perturbing a secretory pathway regulator and that mutation of RSP5 alters HopM1 effects on this pathway to allow life.