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United States Department of Agriculture

Agricultural Research Service

Research Project: Novel Disease Control Strategies for Cellular and Sub-Cellular Pathogens

Location: Molecular Plant Pathology

2013 Annual Report

1a.Objectives (from AD-416):
Objective 1: Develop improved virus expression vectors through characterization of plant viral genomes and investigation of plant virus-host interactions. Objective 2: Evaluate plant and pathogen gene function in host/pathogen interactions and identify and test candidate disease resistance sequences for plant pathogen control. Objective 3: Develop new practical strategies for the production in plants of proteins for prevention, treatment, and control of animal diseases.

1b.Approach (from AD-416):
In Objective 1, we will.
1)characterize the genome expression strategy of Maize rayado fino virus and develop into a gene expression tool for maize, and.
2)develop novel plant virus-based expression vectors for plants using organelle targeting sequences derived from viroids and viruses. In Objective 2, we will.
1)determine the role of membrane-associated, phospholipid-signaling phosphorylation pathways in virus and viroid infection, and.
2)evaluate genes with potential for conferring resistance to bacterial pathogens in plants. In Objective 3, we will.
1)express novel functional proteins in plants for treatment and control of animal diseases, and.
2)develop multi-component vaccines and diagnostic reagents by expression of diverse molecules on the surface of a plant virus-like particle.

3.Progress Report:
In 2013, we constructed a number of modified full-length infectious cDNA clones of Maize rayado fino virus (MRFV), a virus that replicates in both insect and plant hosts and causes significant losses in corn yields in Central and South America, in order to study its interaction with its hosts and to develop it into a virus-based vector for monocots. We demonstrated packaging of highly represented heterologous RNAs by wild-type and modified MRFV virus-like particles in plants, and additionally examined protein requirements for particle formation, expanding the use of these particles for nanotechnology applications. We demonstrated the ability of the capsid protein encoded by MRFV to act as a novel silencing suppressor of heterologous gene expression in tobacco. We constructed modified plant virus-based vectors to express bacteriophage lysin proteins (normally toxic to plant cells when expressed in the cytoplasm) in targeted plant cell organelles where accumulation of small amounts of the protein allows for further testing of their antimicrobial activities against animal and plant bacterial pathogens. Within the scope of collaboration with the Research Institute for Biological Safety Problems RK ME&S/RK NBC, the Republic of Kazakhstan on “Development of methods to construct recombinant prophylactic means for sheep pox with use of transgenic plants”, we produced several sheep pox virus (SPPV) antigens in bacteria, obtained antibody specific to these antigens, and demonstrated that the antibodies can neutralize sheep pox virus in cell culture. Follow-up studies for production of SPPV antigens in plants were conducted.

1. Developed chemically-modified MRFV virus-like particles produced in plants for the display of diverse molecules, such as vaccine epitopes and fluorophores. Agricultural losses due to plant and animal diseases necessitate the development of reagents for detection and control of the pathogens that cause the disease. Plant viruses and virus-like particles are able to assemble themselves in unique ways and provide useful templates for this purpose, and can be applied to both plant and animal disease control. We produced Maize rayado fino virus engineered spherical virus-like particles in plants which were chemically-modified to serve as platforms for the display of diverse molecules. These particles can be used to generate reagents which have multiple uses for applications in pathogen detection and vaccine production. These results are of interest to plant and animal virologists who are studying virus particle assembly and disease specialists who are developing methods for disease detection control. The novel application of this technology will also be of interest to scientists, action agencies, and producers who are faced with emerging diseases or those that are difficult to diagnose.

Review Publications
Natilla, A., Hammond, R. 2013. Analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in Nicotiana benthamiana plants and cross-linking of peptides to VLPs. Journal of Visualized Experiments. 72:e50084.

Last Modified: 7/30/2014
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