Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: Salicylic acid induces a variety of defense related genes, many of which have direct effects on the invading pathogen. R gene mediated defense often triggers programmed cell death (PCD), resulting in necrotic lesions and inhibition of further pathogen spread. The induction, and possibly the limitation of PCD around the infection sites, is regulated by reactive oxygen species (ROS). This project explored the roles of SA, alternate oxidase (AOX), and RDR1 in PCD and defense responses to virus pathogens. To analyze the role of alternate respiration on virus susceptibility, transgenic N. tabacum and N. benthamiana expressing 35S driven wt or mutant AOX showing increased and decreased AOX pathway capacity were inoculated with TMV or PVX. We found very little effect on virus accumulation. We further inoculated PVX to SA-treated and untreated transgenic plants expressing wtAOX or AOX-E which over express or under express AOX and found no effect of SA treatment. To determine if PVX TGBp1 suppresses RDR1 resistance to TMV, the TGBp1 and TGBp3 proteins will be introduced into the TMV genome replacing the GFP gene in the 30B infectious clone. TMV-TGBp1 and TMV-TGBp3 were each inoculated to N. benthamiana, antisenseRDR1, and MtRDR1 plants and TMV-TGBp3 showed an obvious increase in virus accumulation. TGBp1 had no impact on TMV infection. PVX infection was also enhanced on the antisense lines. Concurrent research found that TMV expressing TGBp3 induces systemic necrosis. This research was conducted in collaboration with Dr. M. Dickman at Texas A&M University who provided plants expressing anti-apoptotic genes and was willing to conduct certain PCD assays in his laboratory. This study identified the TGBp3 protein as a novel plant viral inducer of ER stress which under prolonged expression either from the heterologous TMV genome or Agrobacterium infiltration leads to cell death. The PVX TGBp3 protein is essential for virus cell-to-cell movement and is normally expressed at low levels from the PVX genome and therefore does not induce death during PVX infection. Mutational analysis revealed that ER association of TGBp3 is essential for cell death and upregulation of certain ER chaperones when expressed from the TMV genome, revealing that the molecular chaperone branch of the unfolded protein response (UPR) is linked to cell death. Chemically induced ER stress interfered with TGBp3 related cell death further demonstrating a role for the ER in eliciting cell death. Histochemical staining reveals ROS production precedes programmed cell death (PCD). Transgenic plants expressing animal antiapoptotic genes Bcl-xl, CED-9, or Op-IAP fail to alleviate PCD showing that the intrinsic PCD pathway may not be operative. SGT1 and SKP1, which contribute to plant ubiquitin related protein degradation and host defenses, were upregulated during TMV-p3 infection. Furthermore, experimentally silencing SGT1 or SKP1 reduced TGBp3 related cell death indicating these genes contribute to cellular defense responses limiting the extent of PCD in TMV-p3 infected leaves. PARTICIPANTS: Jeanmarie Verchot-Lubicz was the lead PI who went on sabbatical to Cambridge University, Dept Plant Science. The coPI and Cambridge host was Dr. John Carr. Dr Verchot-Lubicz provided expertise in plant virus movement and Dr. Carr provided expertise in oxidative stress. Together they explored the impact of AOX on virus spread in plants. This project is likely to result in a publication showing PVX specific effects of AOX and RDR1. After the grant was funded an informal collaboration with Dr. Marty Dickman at Texas A&M University was established to look at the imapct of anti-apoptotic genes on virus infection. These genes also affect oxidative stress responses to virus infection. This project resulted in a manuscrip that is submitted to Genes and Development. While at Cambridge University, Dr. Verchot-Lubicz contacted Dr. Ray Goldstein in the Dept Applied Math and Physics. Dr. Goldstein was not a participant in this project, but the ensuing dialogue resulted in the 2 PIs drafting a manuscript for Trends in Plant Biology reviewing the impact of cytoplasmic streaming on virus spread in plants. This is an indirect outcome of being in Cambridge, and is not directly related to the goals of this project. TARGET AUDIENCES: The outcomes of this research uncovered the first indication that plant viruses suppress host protein degradation machinery to promote cell-to-cell spread. These results will create a new program looking at viral interactions with the host degradation processes and will create new targets for engineering antiviral therapies. The community best served by this work will be the producers who will benefit from these novel strategies to limit virus infection. This research created a new avenue for research and will result in new book chapters and expanded course curriculum in the next year to include comparisons of host degradation processes acting on plant and animal viruses. Such pathways have been identified in animals but were previously not described in plants PROJECT MODIFICATIONS: One important change was expanding our conversation of virus and oxidative stress to include experiments involving Dr. M. Dickman at Texas A&M University. This led to a pilot project which then led us to uncover evidence that a PVX movement protein is a factor that can induce proteasome degradation, oxidative stress, and ER stress. Conducting research in collaboration with both Dr. Carr and Dr. Dickman allowed us to fully explore the relationship of PVX with oxidative stress related pathways and propelled our research forward much more rapidly. THis is the first evidence that a viral movement protein may associate with the ER as a means to suppress host degradation machinery to promote its own movement. The discovery of TGBp3 as an ER and oxidative stress inducer creates new opportunities for future research looking at events during susceptible and resistant interactions. Working with the collaborators, we plan to submit further project proposals for expanding this research program. The current funding enabled us to launch this entire new avenue for research.
Impacts
This sabbatical grant provided new opportunities for the Verchot-Lubicz laboratory to study viral-host interactions and establish legitimacy in the field of host immune responses to viral pathogens. There is one manuscript that is in preparation and more to follow. The second impact is that this research identified a new PCD pathway linking ER stress to pathogen induced cell death. This mechanism has not been previously discovered and appears to be related to the ubiquitin-proteasome pathway. In addition SGT1 and SKP1 are known to play a role in R gene mediated plant immunity and this study established a link to ER stress and viral pathogenesis. This creates new opportunities for understanding a role for the SCF for complex in cell death and disease. Excessive and prolonged ER stress triggers a cell death response in mammals that is often observed in the form of apoptosis (Ciccaglione et al., 2007; Zhang and Kaufman, 2006). In plants, very little is known about ER stress related UPR and many signaling components known to exist in mammals and yeast have not yet been identified in plants (e. g. (Ferri and Kroemer, 2001; Tardif et al., 2005; Xu et al., 2005; Zhang and Kaufman, 2006). In mammals, ER stress and UPR have been linked to hereditary and viral diseases including Parkinsons, West Nile Virus, and Hepatitis virus C. Plant biologists have utilized nutrient depletion or chemicals known to disturb ER and Golgi functions to map the plant signaling pathways relating to ER stress and UPR (Martinez and Chrispeels, 2003). This is the first study to show pathogen induced ER stress related cell death in plants. There are a number of plant viruses that are known to cause systemic necrotic diseases. The nature of the cell death pathways relating to this type of disease has not been described. There are also examples of plant viruses that during co-infection experiments cause more a severe disease than when they occur in single infections. The best studied examples include the PVX and Tobacco etch virus (TEV) synergisms which led to the discovery of viral proteins which suppress the RNA silencing pathway. The TEV HC-Pro silencing suppressor protein promotes infection by suppressing host defense response relating to viral RNA degradation (Kasschau and Carrington, 1998; Kasschau et al., 2003; Llave et al., 2000; Pruss et al., 1997). A synergistic disease involving Tobacco mosaic virus (TMV) and PVX produces a systemic necrosis phenotype (Balogun et al., 2002; Hu et al., 1998). Plants doubly infected with PVX and TMV show reduced growth as well as mosaic symptoms and necrotic lesions on the leaves (Balogun et al., 2002). In this study we provide the first evidence in plants that a single PVX protein, named TGBp3, when expressed from the TMV genome induces prolonged ER stress and systemic necrosis. Systemic necrosis is accompanied by upregulation of ER chaperones and has the hallmarks of programmed cell death (PCD) in plants.
Publications
- Ye, C.-M., Lu, S., Dickman, M. Verchot-Lubicz, J. (2008). A Plant Viral Movement protein functions as an elicitor of ER stress and Programmed Cell Death, Molecular Cell, submitted.
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