Date of Award

4-2012

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. John R. Geiser

Second Advisor

Dr. Bruce Bejcek

Third Advisor

Dr. Todd Barkman

Fourth Advisor

Dr. Wendy Ransom-Hodgkinis

Abstract

We 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 21°C, but not at 30°C and 37°C. 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 21°C, 30°C and 37°C. At 21°C, a full-length protein of 78kDA is observed. At 30°C and 37°C, the majority of HopM1 protein exists as degraded fragments. HopM1 containing strains were visualized using the V5 epitope and irnrnunofluorescent 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 results in the lethal phenotype.

We have isolated 19 spontaneous suppressor strains that are capable of surviving the HopM1 imposed lethality at 21°C. All strains have been examined for HopM1 protein expression, of which 13 express full-length HopM1 at 21°C, and 5 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 our 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 survival.

Access Setting

Dissertation-Open Access

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