Location:2008 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. Objective 4: Delineate mechanisms of global adaptive responses of plants to pathogen attack using engineered plant viruses as a molecular tool.
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. In objective 4, we will 1) determine the role of membrane-bound receptor and phospholipid pathways in host response to pathogen infection using virus-based vector expression of silencing RNAs and dominant-negative mutant genes, and 2) study physiological and molecular mechanism of plant defense signaling by virus-based expression of genes associated with two forms of plant innate immunity-basal and avr-induced resistance.
3. Progress Report
We developed Potato virus X-based vectors containing the coat protein of Cucumber mosaic virus (CMV) fused with neutralizing epitopes of the avian Newcastle disease virus (NDV). The recombinant proteins formed CMV virus-like particles and immunoelectron microscopy revealed the immunoreactivity of the NDV epitope with anti-NDV antisera. The candidate vaccines and antisera raised to the vaccines are currently being tested for efficacy of neutralizing challenge NDV infection in animal cell and whole animal studies. We further modified the purification protocol of recombinant virus-like particles and demonstrated that chickens immunized with purified plant-derived virus preparations developed an antigen-specific response. We developed modified plant virus-based constructs encoding recombinant single-chain antibodies to control diseases caused by the wide-host range Cucumber mosaic virus (CMV), which can be devastating in field situations. We found that although the foreign genes were stably expressed in the plants, the single-chain antibody proteins were difficult to detect. We conducted research to dissect the protein phosphorylation cascade involved in the pathogenicity on tomato of Potato spindle tuber viroid (PSTVd). The tomato protein kinase, PKV, previously found to be transcriptionally activated in tomatoes infected with PSTVd, was shown to induce stunting when over-expressed in tobacco. We verified the role of PKV in causing stunting, abnormal root development, and reduced fertility by introducing the gene into tobacco, a model experimental plant used by others to study plants genes associated with dwarfing. We are testing the over-expression of PKV in tomato to verify its role in disease development resulting from viroid infection. We discovered that PKV intereacts with a second protein kinase, PDK1, and mutations that disrupt the interaction modify the phenotype of plants engineered with the mutant PKV, thereby demonstrating that the interaction is critical for activity. We conducted collaborative research with the University of Costa Rica to determine the prevalence and molecular variability of Beet pseudo yellows crinivirus (BPYV), an emerging plant virus associated with a severe yellowing disease, in Costa Rican cucurbits. We found that BPYV in cucurbits is currently limited to an isolated region of the country. We continued our study to determine the nuclear targeting sequence motifs present in the PSTVd molecule. A whole plant nuclear targeting assay was used to analyze four fragments representing different portions of the PSTVd upper RNA strand. The study demonstrated that a structural hairpin within the PSTVd molecule was able to target a chimeric RNA molecule to the nucleus, whereas, others were not, thereby identifying a molecular tool that can be used to target heterologous RNAs to the nucleus to modulate plant gene expression. (Research Component IV –Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors- of NP 303; Problem statement 2A: Pathogen biology, virulence determinants, and genetics of the pathogen)
1. Developed a plant virus particle antigen display system for an avian influenza virus epitope. Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 normally occurs in domestic fowl but has also been found in migratory birds, different mammals and in humans. Recent outbreaks of HPAIV created a threat of pandemic spread and emphasized the need to develop a vaccination strategy that differs from traditional vaccination. Plant-derived vaccines offer promise of greater availability in developing countries, lower production costs, increased safety and new concepts of vaccine construction and properties. This initial study demonstrates the feasibility of producing an HPAIV potential vaccine candidate in plants. These results will be of interest to plant and animal researchers, and representatives of industry, academia, and government organizations with an interest in plant-based systems for production of vaccine, immunology and veterinary health. (Research Component IV –Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors- of NP 303) Problem statement 2A: Pathogen biology, virulence determinants, and genetics of the pathogen.
2. Demonstrated the function of a nematode anti-apoptotic gene in plants. Specific details of the mechanisms of plant cell death in plant-pathogen interactions remain elusive. Using plant virus-based technology, we transiently expressed in plants the anti-apoptotic gene CED-9 from Caenorhabditis elegans and showed that the expression increased plant salt and oxidative stress tolerance. A specific pattern of ion signaling attributed to the mechanisms involved in activity of anti-apoptotic gene and regulation of program cell death in plants was discovered. Understanding the process of apoptosis is crucial to improving plant stress tolerance and pathogen resistance. (Research Component IV –Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors- of NP 303; Problem statement 2A: Pathogen biology, virulence determinants, and genetics of the pathogen).
3. Discovered novel electrophysiological insights into the mechanisms of innate immunity in plants. We demonstrated that initial calcium uptake in response to hypersensitive response-causing pathogen, Pseudomonas. syringae pv syringae, is followed by net calcium efflux initiated at about 12 hrs after the bacterial challenge and sustained for at least another 48 h. These data suggest that calcium acts not only as an important second messenger in the activation of resistance responses but also as a downstream mediator of later cell death acceleration and completion of defense reaction. In a continuation of these two experiments, we demonstrated that transient virus-based expression of anti-apoptotic gene modifies HR (hypersensitive response)-associated defense signaling in plants infected with avirulent pathogen P. syringae pv. syringae 61(Research Component IV –Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors- of NP 303; Problem statement 2A: Pathogen biology, virulence determinants, and genetics of the pathogen).
4. Performed a computer-assisted genome-wide profiling of host defense responses to plant virus infection and identified genes up-regulated during viral pathogenesis. Genes involved in the same biological process, that is, response to viral pathogenesis, may be co-expressed and clustered non-randomly within chromosomes, similarly to the genes involved in metabolic pathways. Our data suggest that one third of the genes responsive to viral infection in plants are clustered together and their expression is coordinated by specific regulatory elements. This research is aimed to understand molecular mechanisms of gene expression in response to pathogen attack and as well as evolution of defense responses in plants in general. (Research Component IV –Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors- of NP 303; Problem statement 2A: Pathogen biology, virulence determinants, and genetics of the pathogen)
5. Demonstrated the antimicrobial activities of a novel antimicrobial peptide fusion protein in vitro. 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 using a bacterial expression system. These results will be of interest to scientists who are developing novel plant disease control strategies.(Research Component IV –Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors- of NP 303; Problem statement 2A: Pathogen biology, virulence determinants, and genetics of the pathogen).
6. Developed methods to efficiently produce spherical protein cages composed plant virus capsid proteins. The development of improved, rapid, and reliable diagnostic methods for plant and animal pathogens is critical to the protection of the nation’s food supply and animal health. For some diseases, detection methods do not exist, or if they do, they may be expensive, time-consuming, and unreliable. The development of nanotechnology contributes to the development of more precise and effective methods disease diagnosis. We expressed maize rayado fino virus capsid proteins in bacteria and plants, resulting in self-assembly of icosohedral virus-like particles that are capable of encapsidating RNA. The particles will be developed for diagnostic applications in plant and animal pathology. The novel application of this technology will be of interest to scientists, action agencies, and producers who are faced with emerging diseases or those that are difficult to diagnose. (Research Component IV –Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors- of NP 303; Problem statement 2A: Pathogen biology, virulence determinants, and genetics of the pathogen).
5. Significant Activities that Support Special Target Populations
Natilla, A., Nemchinov, L.G. 2008. Further improvement of PVX/CMV CP expression tool for presentation of short foreign epitopes. Protein Expression and Purification. 59(1):117-121.