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United States Department of Agriculture

Agricultural Research Service

Research Project: APPLICATION OF PLANT-VIRAL BASED VECTORS TO THE DEVELOPMENT OF NOVEL DISEASE CONTROL STRATEGIES
2012 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:
We tested the infectivity of full-length cDNA copies of the genome of Maize rayado fino virus (MRFV), a virus that causes significant losses in corn yields in Central and South America, in order to study its interaction with its maize and leafhopper-vector hosts. We developed chemically-modified MRFV virus-like particles produced in plants for the display of diverse molecules, such as vaccine epitopes and fluorophores. We produced virus-like particles of Lolium latent virus to examine protein requirements for particle formation. We provided expertise and reagents that facilitated the characterization of a Switchgrass mosaic virus, a new virus species associated with yellowing symptoms in the biofuel plant switchgrass. We identified for the first time the presence of the emerging crinivirus Tomato chlorosis virus in sweet pepper in Costa Rica. We generated and charactrerized transgenic plants expressing mutant PKV (protein kinase viroid-induced) genes to evaluate nuclear targeting signals and the role of PKV role in viroid pathogenesis. We developed a functional classification of genes with altered expression levels in virus-infected Arabidopsis plants. 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 antigens in bacteria, obtained antibody specific to these antigens, and demonstrated that the antibodies can neutralize sheep pox virus in cell culture.

The objectives of this project were to develop fundamental knowledge of plant/pathogen interactions and novel strategies and reagents for the prevention, treatment, and control of plant and animal diseases. Over the life of the project, novel plant virus-based vector expression tools were developed and harnessed for the rapid delivery and expression of plant and pathogen gene sequences involved in host/pathogen interactions, and to test nucleotide sequences and proteins for their ability to control plant diseases caused by cellular and subcellular pathogens. Improved diagnostic reagents for plant and animal diseases were developed during virus characterization and vector construction. Functionally active and/or protective molecules were produce using plant virus-based expression systems and their efficacy was tested in collaboration with other ARS facilities working on livestock and poultry diseases.


4.Accomplishments
1. Demonstrated the antimicrobial activity of a novel antimicrobial peptide fusion protein in tobacco and potato plants. Phytopathogenic bacteria and fungi cause significant losses in important agricultural crops and are the primary cause of post harvest diseases of fruits and vegetables. While control of losses can be achieved by chemical and biological control methods, full realization of these strategies under commercial conditions can be hindered by environmental issues, the emergence of new virulent strains of pathogens, resistance to the current chemical and biological defenses, as well as practical difficulties, and warrant development of new and more effective plant protection techniques. Naturally occurring antimicrobial proteins can be used for crop protection applications; however, selection of appropriate proteins for target applications requires the production of large quantities of active proteins. We report the production of functionally active antimicrobial proteins (snakin-1 and defensin-1) in plants. A hybrid antimicrobial protein was constructed for simultaneous expression of snakin-1 and defensin-1 in plant cells and this protein was found to be active against fungi and bacteria in plant protection experiments. These results will be of interest to scientists who are developing novel plant disease control strategies.

2. Developed methods to efficiently produce modified protein cages composed of plant virus capsid proteins for antigen display. 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
Guevara-Coto, J.A., Barboza-Vargas, N., Hernadez-Jimenez, E., Hammond, R., Ramirez-Fonseca, P. 2011. First report of Bemisia tabaci biotype Q in Costa Rica and detection of viruliferous whiteflies in greenhouses. European Journal of Plant Pathology. 131:167-170.

Acosta-Leal, R., Duffy, S., Xiong, Z., Hammond, R., Elena, S. 2011. Advances in plant virus evolution: Translating evolutionary insights into better disease management. Phytopathology. 101:1136-1148.

Vargas, J.A., Hammond, R., Hernandez, E., Barboza, N., Mora, F., Ramirez, P. 2011. First report of Tomato chlorosis virus infecting sweet pepper in Costa Rica. Plant Disease. 95:1482.

Natilla, A., Hammond, R. 2011. Maize rayado fino virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display. Journal of Virological Methods. 178:209-215.

Agindotan, B.O., Gray, M.E., Hammond, R., Bradley, C.A. 2012. Complete genome sequence of switchgrass mosaic virus, a member of a proposed new species in the genus Marafivirus. Archives of Virology. DOI: dx.doi.org/10.1007/s00705-012-1354-3.

Last Modified: 12/29/2014
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