VIRUS INDUCED REPROGRAMMING OF THE CELLULAR ENVIRIONMENT: A MECHANISM FOR DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0214037
• Grant No.: 2008-35319-19168
• Proposal No.: 2008-00688
• Project Start Date: Jul 1, 2008
• Project End Date: Jun 30, 2011
• Grant Year: 2008
• Program Code: [51.8B]- Microbial Biology (B): Microbial Associations with Plants

Recipient Organization
UNIVERSITY OF MARYLAND
(N/A)
BALTIMORE,MD 21201

Performing Department
BIOTECHNOLOGY INSTITUTE

Non Technical Summary
The capacity of a viral pathogen to cause disease is often attributed to its ability to reprogram its hosts cellular machinery to facilitate its own replication and spread. To investigate the importance of cellular reprogramming in disease development a tomato host pathosystem was used to identify an interaction between a host-encoded NAC family transcription factor, ATAF2, and the replicase protein of Tobacco mosaic virus (TMV). This interaction was confirmed in planta and found to affect virus accumulation. NAC domain proteins represent a large family of transcription factors linked to plant development and defense responses. Uniquely, ATAF2 in strongly induced upon TMV infection, suggesting that this transcription factor may be associated with host defense responses. Efforts in this study will focus on determining if the virus alters the function of ATAF2 as a means to reprogram its host's transcriptome. Additional studies will investigate the specific advantages conferred by TMV-induced disruption of ATAF2 functions. Does this interaction promote virus replication, movement or the avoidance of host defense responses? Answers to these questions will significantly advance our long-term goal of understanding how plant viruses modulate their host's cellular environment to promote infection and induce disease. In addition, understanding the mechanisms involved in these disease processes should aid in the development of new strategies aimed at reducing the economic impact of plant viruses.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1460	1040	50%
212	4030	1040	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato; 4030 - Viruses;

Field Of Science
1040 - Molecular biology;

Keywords

plant disease

virus resistance

transcriptome

plant defense

virus-host interaction

cell reprogramming

Goals / Objectives
Within a tomato host pathosystem we identified an interaction between a host-encoded NAC-domain transcription factor (ATAF2) and the replicase protein of Tobacco mosaic virus (TMV). This interaction appears to modulate virus accumulation and spread. Four specific objectives in this study will address the mechanism responsible for this modulation and its role in the induction of disease. 1. ATAF2 induced affects on virus accumulation. Preliminary studies indicate that overexpression of ATAF2 significantly reduces the accumulation of TMV, suggesting that TMV targeted disruption of ATAF2 is an important factor in establishing an infection. However, we do not know if ATAF2 induction affects virus replication or movement. Efforts in this objective will determine if the observed affect on TMV accumulation is due to alterations in virus replication and/or movement. Information from these studies should provide insight into the mechanism(s) whereby ATAF2 modulates virus infection. 2. TMV directed modulation of ATAF2 expression. We speculate that TMV interacts with ATAF2 as a means to block its transcriptional function, potentially disrupting its reported role as a regulator of plant defense processes. To investigate these possibilities we will examine the timing of ATAF2 induction and accumulation in relation to virus replication and movement. This study should provide new insights into how TMV modulates ATAF2 activity during virus infection. 3. What are the target genes under the control of ATAF2 in tomato? We speculate that TMV disrupts ATAF2 function to modulate basal defense responses and promote further systemic infection. This possibility suggests that genes under the control of ATAF2 may interfere in the infection process. To identify ATAF2 regulated genes we will perform a series of microarray experiments aimed at examining gene expression in tomato leaf tissue induced to express ATAF2. 4. Identification of additional tomato transcription factors that are functionally regulated by TMV. The ability of the TMV replicase to interact with and alter the function of other host factors will be investigated. Specifically, an identified interaction with a heat shock transcription factor will be investigated for its activity in tomato. Additionally, a tandem affinity purification method will be used to identify additional tomato proteins that interact with the virus replicase. Combine these studies will further our understanding of how viruses modulate their hosts environment and induce disease.

Project Methods
The capacity of a viral pathogen to cause disease is often attributed to its ability to reprogram its host's cellular machinery to facilitate its own replication and spread. Reprogramming cellular processes is likely driven by multiple virus-host interactions that combine to produce a successful infection and subsequent disease. However, the complexity of virus-host interactions has hindered efforts to understand the molecular mechanisms whereby viruses commandeer their hosts cellular machinery. The approach we have taken to address this complexity is to identify individual virus-host interactions and the role each plays in infection and disease development. In this study, the interaction between the replicase protein of Tobacco mosaic virus and a host transcription factor, ATAF2, will be investigated for its role in promoting disease. A range of experimental approaches will be used to investigate the role of ATAF2 in virus infection. These include the development of transient expression systems and transgenic plant lines to investigate the affect ATAF2 has on virus replication and movement. In addition, the transcriptional induction of ATAF2 during virus infection and its affect on the hosts transcriptome will be explored using a microarray approach. Microarray analysis will subsequently be used to identify potential gene targets regulated by ATAF2. Significantly, we will investigate transcriptional changes in relation to the induction of ATAF2 both in inoculated and systemically infected plant tissues. Investigating both inoculated and systemically infected tissue for effects conferred by the association of ATAF2 with the virus replicase provides a means to understand how the plant initially responds to a virus and how that virus modifies the host response to promote it own replication and spread.

Progress 07/01/08 to 06/30/11

Outputs
OUTPUTS: Project activities have included an extensive survey into the transcriptional targets of the plant defense associated transcription factor ATAF2. This work has been presented at events that include the USM Virology Symposia 2009, the 10th Annual Maryland Plant Biology Symposium 2009 and the national meeting of the American Society of Virology, Bozeman MT. Efforts in this project also include the mentoring of two post-doctoral scientists directly supported by this work as well as a graduate and undergraduate student who have participated in this study. PARTICIPANTS: The PI Dr. Culver was involved in experimental design and the analysis of data. Drs. Wang and Hsieh were involved in experimental design, implementation and analysis of experiments to identify and characterize the effects of the plant defense transcription factor ATAF2 on virus biology. Ms. Tami Fischer is a graduate student and Mr. Nick McKnight is an undergraduate student. Both have received training opportunities on this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Scientific studies supported by this award determined the virus targeted degradation of the plant defense associated transcription factor ATAF2 is an important counterdefense mechanism used by the virus to disarm host derived resistance mechanisms. For plant viruses this counterdefense mechanism is involved in suppressing defense signals that could limit the systemic cellular spread of the virus. A survey to identify the transcriptional targets or genes under the control of ATAF2 has revealed the presence of a variety of host factors involved in stress/defense responses, transcriptional regulation, protein degradation, metabolic functions, and unknown processes. In addition, the potential ATAF2 binding site in the promoter of these genes was identified. Knowledge of the ATAF2 promoter binding site will permit additional basal defense related genes to be identified based on the presence of the binding site with their promoters. Taken together, these studies provide new insights into virus-induced alterations in host gene expression and its role in host defense and pathogen counterdefense responses.

Publications

• Wang X, Goregaoker SP, and Culver JN. 2009. Interaction of the Tobacco mosaic virus replicase protein with a NAC domain transcription factor is associated with suppression of systemic host defenses. J. Virol. 83, 9720-9730.
• Wang X and Culver JN. 2009. Tobacco mosaic virus interferers with the defense associated function of a plant transcription factor, ATAF2. ASV Bozeman MT, W11-1.

Progress 07/01/08 to 06/30/09

Outputs
OUTPUTS: Ongoing activities have included an extensive survey into the transcriptional targets of the plant defense associated transcription factor ATAF2. This work has been presented at events that include the USM Virology Symposia 2009 and the 10th Annual Maryland Plant Biology Symposium 2009. Efforts in this project also include the mentoring of Post-doctoral Scientists directly supported by this work as well as graduate and undergraduate students who have participated in this study. PARTICIPANTS: The PI Dr. Culver was involved in experimental design and the analysis of data. Drs. Wang and Hsieh were involved in the design, implementation and analysis of experiments to identify and characterize the effects of the plant defense transcription factor ATAF2 on virus biology. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Scientific studies supported by this award have determined the virus targeted degradation of the plant defense associated transcription factor ATAF2 is an important counterdefense mechanism used by the virus to disarm host derived resistance mechanisms. For plant viruses this counterdefense mechanism is involved in suppressing defense signals that could limit the systemic cellular spread of the virus. A survey to identify the transcriptional targets or genes under the control of ATAF2 has revealed the presence of a variety of host factors involved in stress/defense response, transcriptional regulation, protein degradation, metabolic functions, and unknown processes. In addition, the potential ATAF2 binding site in the promoter of these genes was identified. Taken together, these studies provide new insights into virus-induced alterations in host gene expression and its role in host defense and pathogen counterdefense responses. Ongoing efforts are directed toward understanding the effects of ATAF2 target genes on virus biology with the goal of using this information to develop novel virus resistance strategies.

Publications

• Wang X, Goregaoker SP, and Culver JN. 2009. Interaction of the Tobacco mosaic virus replicase protein with a NAC domain transcription factor is associated with suppression of systemic host defenses. J. Virol. 83, 9720-9730.