(from the application): Post-transcriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism in which a particular RNA sequence is targeted and destroyed. Although PTGS occurs in diverse organisms, the cellular components of the pathway and its regulation are not well understood. The helper component proteinase (HG-Pro) of plant potyviruses has been shown to suppress PTGS in plants. Using HG-Pro as bait in a yeast two-hybrid system, we have identified a novel calmodulin-related protein (termed rgsCaM) that interacts with this viral suppressor of PTGS. rgs-CaM, like HG-Pro itself, suppresses both initiation and maintenance of PTGS. Our hypothesis is that HC-Pro-suppression of PTGS is mediated by its interaction with rgs-CaM. Here we propose to exploit HC-Pro and rgs-CaM as tools to dissect the mechanism of silencing in plants. The first four aims of the proposal focus on the role of rgs-CaM in PTGS, identifying domains of the protein required to suppress PTGS as well as those involved in the rgs-CaM/HC-Pro interaction. Several approaches will be used to interfere with expression of rgs-CaM and the effect of these manipulations on PTGS and its suppression by HC-Pro will be assayed.
The third aim addresses mechanistic questions, determining how and where HC-Pro and rgs-CaM act with regard to known steps in PTGS. The last two aims focus on the role of other plant proteins in the HC-Pro-mediated suppression of PTGS. We will extend investigations of three other HC-Pro-interacting proteins using the approaches that successfully identified rgs-CaM as a regulator of PTGS. Gene array technology will be used to identify genes that are regulated in response to PTGS or its reversal by HC-Pro. Finally, we will exploit the model genetic organism Arabidopsis thalianato screen for mutants that interfere with suppression of PTGS in response to HC-Pro. The plant silencing system serves as a model to understand similar pathways in animals, and has potential to be exploited for gene therapy applications and manipulation of gene expression. Given the antiviral nature of gene silencing in plants, understanding PTGS in plants could well lead to the development of antiviral strategies in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061014-04
Application #
6712076
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
2001-03-01
Project End
2006-02-28
Budget Start
2004-03-01
Budget End
2006-02-28
Support Year
4
Fiscal Year
2004
Total Cost
$209,525
Indirect Cost
Name
University of South Carolina at Columbia
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Mlotshwa, Sizolwenkosi; Pruss, Gail J; Gao, Zhihuan et al. (2010) Transcriptional silencing induced by Arabidopsis T-DNA mutants is associated with 35S promoter siRNAs and requires genes involved in siRNA-mediated chromatin silencing. Plant J 64:699-704
Endres, Matthew W; Gregory, Brian D; Gao, Zhihuan et al. (2010) Two plant viral suppressors of silencing require the ethylene-inducible host transcription factor RAV2 to block RNA silencing. PLoS Pathog 6:e1000729
Mlotshwa, Sizolwenkosi; Pruss, Gail J; Peragine, Angela et al. (2008) DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis. PLoS One 3:e1755
Pruss, Gail J; Nester, Eugene W; Vance, Vicki (2008) Infiltration with Agrobacterium tumefaciens induces host defense and development-dependent responses in the infiltrated zone. Mol Plant Microbe Interact 21:1528-38