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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
Objective 1: Characterize plant viral genomes and develop novel plant virus-based vectors for expression of foreign gene sequences in plants.

Objective 2: Evaluate plant and pathogen gene function in host/pathogen interactions and disease resistance (functional genomics), and candidate sequences for plant disease control.

Objective 3: Develop new practical strategies for the production of vaccines and other biomedical products in plants for prevention, treatment, and control of animal diseases.

1b.Approach (from AD-416)
In Objective 1, we will.
1)develop novel plant virus-based gene expression vectors, and.
2)develop novel vector strategies based on foreign gene expression from defective interfering RNAs and subgenomic RNAs.

In Objective 2, we will.
1)develop virus-induced gene silencing to suppress pathogenic and disease-related genes of cellular and subcellular pathogens,.
2)determine the role of plant protein phosphorylation signaling pathways in host/pathogen interactions, and.
3)evaluate genes with potential for conferring resistance to cellular and subcellular pathogens and modulation of biochemical pathways involved in host/pathogen interactions and innate immunity.

In Objective 3, we will 1)test the ability of an epitope presentation system based on Cucumber Mosaic Virus coat protein expressed from a Potato Virus X vector to produce novel vaccines and applications for nanotechnology,.
2)develop a new strategy for treatment and control of coliform mastitis in dairy cows by utilizing a plant-manufactured antimicrobial agent,.
3)develop stable transient expression modules for production of useful biomedical products in plants, and.
4)develop multi-component vaccines and diagnostic reagents by expression of multiple foreign epitopes on the surface of a plant virus-like particle.

3.Progress Report
Plant diseases caused by viruses, viroids, and bacteria affect crop yield and quality, and the development of improved disease control measures against these pathogens is a continuing and long-term goal of our project. In 2011, we focused on two viruses that cause losses to grain and tomato crops worldwide. Maize rayado fino virus (MRFV) causes a disease in corn resulting in yield losses up to 100%, is widely distributed throughout Latin America, and has been found in the southern U.S. We constructed full-length cDNA copies of MRFV which will be used by MPPL scientists and collaborators in the U.S. to study its interaction with its corn and leafhopper-vector hosts. A second virus, tomato chlorosis virus, is an emerging, insect-transmitted plant virus that causes serious disease and loss in vegetable crops worldwide. Rapid, accurate, and sensitive detection of tomato chlorosis virus in field and greenhouse samples is important for control of the disease. In this study, we developed a rapid, reproducible, and sensitive molecular assay for the detection of this virus in tomato leaf samples. Our assay provides a useful tool for the reliable detection and quantitation of Tomato chlorosis virus on a large scale. The technology was transferred to cooperators in Costa Rica for validation and to study the epidemiology of the virus in Costa Rica. Another ongoing project in 2011 was a study of the response of plants to virus infection at the gene expression level. Analysis of currently available microarray data revealed that genes whose expression levels change in response to virus infection can be grouped or clustered at the chromosome level, suggesting coordinated regulation of those genes. Finally, agricultural losses due to plant and animal diseases necessitate the development of reagents for detection and control of the pathogens that cause the disease. To produce a platform for these reagents, we studied the ability of MRFV coat proteins to reassemble into different types of virus-like particles. We demonstrated that these chemically-modified particles can serve as platforms for the display of diverse molecules such as antigenic peptides and fluorescent dyes. These particles will be used to generate reagents which have multiple uses for applications in pathogen detection and vaccine production. Within the scope of collaboration with the Research Institute for Biological Safety Problems RK ME&S/RK NBC, The Republic of Kazakhstan, supported by the ARS-Former Soviet Union program, to produce vaccines in plants, we expressed sheep pox antigens in bacteria to generate diagnostic antibodies specific to sheep pox virus. Genes encoding the vaccine antigens were cloned into a plant virus-based vector for transient expression in tobacco and clover plants which will be used by the Kazakhstan collaborators to produce oral vaccines against sheep pox virus.

1. Bemisia biotype Q is present in Costa Rica. Whiteflies are among the most important pests in agriculture as they transmit a number of plant viruses and are prevalent worldwide. Until recently, the most important whitefly species in Costa Rica were the greenhouse whitefly and a specific type of the silverleaf whitefly; however, the new, insecticide-resistant type of the silverleaf whitefly has been increasing in incidence in several countries. Using molecular tools, ARS researchers in Beltsville, MD, determined that the new type was present in greenhouses in which tomato and peppers are grown, representing both the first report of the new type in Costa Rica as well as its presence at high altitudes. This information has been communicated to growers in Costa Rica and is being used to assess the incidence of the insect and the economic impact on tomato and pepper production. The results impact U.S. agriculture as this new, insecticide-resistant whitefly type is emerging as a serious threat to vegetable production in North America.

2. Discovered new natural hosts of tomato chlorosis virus in Costa Rica. Tomato chlorosis virus (ToCV) is an emerging whitefly-transmitted crinivirus. In early 2007, severe yellowing and chlorosis were observed in field-grown tomatoes in Costa Rica. In 2008 and 2009, studies were conducted by an ARS scientist in Beltsville, Maryland, and collaborators in Costa Rica to determine if weeds could also serve as reservoirs for the virus. Results of molecular analyses completed in 2011 revealed that ToCV was present in tomatoes and common weeds adjacent to tomato nurseries. This is the first report of these weeds as natural hosts of ToCV in Costa Rica. This information has been communicated to growers in Costa Rica and is being used to assess the incidence of the virus in weeds and the economic impact on tomato production. The results impact U.S. agriculture as ToCV is emerging as a serious threat to tomato production in North America.

3. Multiple metabolite transport systems mediate virus-induced resistance to oxidative stress. It is known that plants can be developed that are tolerant to more than one stress; however, it not known if exposure to one stress can make a plant more tolerant to another. We showed that virus-infected tobacco plants were better adapted to short wave ultraviolet irradiation as compared to non-infected plants. We think that virus infection may trigger common defense mechanisms that could protect against unrelated stress. This study may lead to the development of novel strategies to protect plants against complex environmental stress conditions. These findings will benefit plant pathologists, agronomists and plant researchers in general by providing valuable information on the adverse effects of different environmental factors that limit plant productivity.

4. Pathogen-response genes are clustered in the genome of Arabidopsis thaliana. The mapping of disease-related genes into the genomes of eukaryotes is important for human, animal and plant health. Once these genes are mapped, their functions can be deduced. In higher organisms, genes responsible for similar functions may be located in groups on the chromosomes. We demonstrated that genes, related to plant defense responses against various pathogens can lie close together and form such groups or clusters. Since very little is known about mechanisms of regulation of clustered genes, results of this work will help us to understand how to increase resistance to pathogens in agriculturally important crops. We expect that this study will be of interest to the wide spectrum of plant researchers in academia and government organizations.

Review Publications
Shabala, S.N., Baekgaard, L., Shabala, L., Fuglsang, A.T., Cuin, T.A., Nemchinov, L.G., Palmgren, M.G. 2011. Endomembrane Ca2+ -ATPases play significant role in virus-induced adaptation to oxidative stress. Plant Signaling and Behavior. 6(7):1053-1056.

Shabala, S., Baekgaard, L., Shabala, L., Fuglsang, A.T., Cuin, T.A., Palmgren, M.G., Nemchinov, L.G. 2011. Multiple transport systems mediate virus-induced acquired resistance to oxidative stress. Plant Cell and Environment. 34(3):406-411.

Solozano-Morales, A., Barboza, N., Hernandez, E., Mora-Umana, F., Ramirez, P., Hammond, R. 2011. Newly discovered natural hosts of tomato chlorosis virus in Costa Rica. Plant Disease. 95:497.

Last Modified: 4/24/2014
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