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United States Department of Agriculture

Agricultural Research Service

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2010 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. BL1/BL2-P certified 3/01/2006. 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 an infectious clone of Beet mosaic virus (BtMV), which was sequenced in our laboratory for the first time in 2004. RNA transcripts, synthesized from the BtMV clone successfully infected an indicator host, tobacco species Nicotiana benthamiana L. as well as the natural host, Beta vulgaris L. The BtMV clone was engineered to express a reporter gene, Green fluorescence protein of jellyfish, Aequorea victoria and a putative protein of interest, a capsid protein of the emerging Porcine circovirus (PCV), a cause of postweaning multisystemic wasting syndrome (PMWS) in a swine. We developed and further tested a genomic approach for studying pathogen-host interactions in plants by means of EST (heterologous expressed sequence tags) mining. Using this approach, we provided direct evidence that genes associated with defense response in Arabidopsis thaliana are physically clustered and are co-expressed and showed that induction of clustered genes is influenced by basic mechanisms of genetic resistance to pathogens. For the first time, we applied non-invasive ion selective microelectrodes (MIFE) technique for early recognition of specific virus-host interactions and demonstrated that MIFE can be effectively used as a rapid screening tool for early diagnostics of virus-host compatibility. We conducted collaborative research with the University of Costa Rica to determine the prevalence and molecular variability of Tomato chlorosis crinivirus (ToCV), an emerging plant virus associated with a severe yellowing disease, in Costa Rican tomatoes and found that this virus is widely prevalent in certain regions of the country. We developed novel plant virus-like particles carrying reactive molecules that will allow us to attach functional proteins for drug targeting and vaccine delivery.

4. Accomplishments
1. Discovered a novel 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 discovered that PKV interacts 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.

2. 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. A domain of the viral HPAIV H5N1 M2 protein (M2e epitope) engineered into the coat protein of Cucumber mosaic virus (CMV) was expressed in plants through a Potato virus X (PVX) vector. The recombinant CMV capsids reacted with specific antibodies produced to synthetic M2e domain of the animal virus. To our knowledge, our report was the first one on the expression of potentially neutralizing epitope of avian influenza virus in plants. After this initial study, we continued our effort to develop a plant-derived vaccine candidate against AIV, by expressing the same M2e epitope of AIV as an terminal fusion to the coat protein of the PVX. Both crude plant extracts and purified chimeric virus particles (CVPs) were immunoreactive with PVX antibodies and antibodies specific to M2e epitope. Chickens immunized with purified CVPs developed an M2e-specific response. 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;

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.

4. 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.

5. Developed a genomic approach to study pathogen and/or virus-host interactions in plants by means of EST (heterologous expressed sequence tags) mining. Gene identification by EST mapping allows efficient discovery of homologous sequences in evolutionary different species and comparison of gene sets on the whole genome scale. Using computer-assisted EST-driven prediction, we identified 4,935 genes or 16% of the total Arabidopsis genome which, according to the origin of EST sets, were associated with plant defense responses. To experimentally support computer data, we used real-time PCR to analyze expression levels of arbitrarily selected genes detected by EST profiling, during infection with yellow strain of Cucumber mosaic virus, a compatible pathogen of Arabidopsis. More than half of the chosen genes were up-regulated in virus-infected plants in accordance with the EST-generated profile. Several genes were further screened in virus-induced gene silencing (VIGS) assay to determine their possible function and/or role in virus-host interactions. In summary, we demonstrated that EST mapping and profiling, although limited by the number of EST collections in databases, represents a valuable alternative to microarrays and may be efficiently used to reveal genes involved in host defense responses to a variety of plant pathogens, including viruses.

6. Demonstrated that virus infection may trigger common defense mechanisms that could protect against unrelated stress. In this work, we investigated the phenomenon of acquired cross-tolerance to oxidative (UV-C and hydrogen peroxide) stress in Nicotiana benthamiana plants infected with Potato virus X (PVX) and examined the functional expression of transport systems in mediating this phenomenon. By combining multiple approaches, we show that virus-infected plants have a better ability to control UV-induced elevations of free Ca2+ and prevent structural and functional damage of chloroplasts. Although specific mechanisms of such “cross-protection” against unrelated stress factors remain obscure, we demonstrate that it could be relevant to some common signaling pathways involved in stress tolerance, particularly reactive oxygen species (ROS)-signaling and Ca2+ signaling during plant defense responses. This study may lead to the development of novel strategies to protect plants against complex environmental stress conditions.

7. Demonstrated the antimicrobial activities of a novel antimicrobial peptide fusion protein in vitro and in vivo. 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) using a bacterial expression system. 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.

8. 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 spherical virus-like particles that are capable of incorporating RNA. We discovered that the amino terminus of the capsid protein is involved in capsid stability and encapsidation of viral 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.

9. Developed an infectious clone of Beet mosaic virus (BtMV). Beet mosaic virus (BtMV) was identified almost five decades ago. It is a member of the economically important Potyvirus group, a non-persistently aphid-transmitted virus infecting mainly sugar beet and its close relatives world-wide. We have recently determined, for the first time, the complete nucleotide sequence of BtMV genomic RNA and have developed specific molecular means for its diagnosis. This work has been further continued toward the development of a BtMV-based virus-vector system intended for expression of anti-pathogenic peptides specifically in sugar beet crop. Several full-length cDNA clones of the virus were engineered to incorporate multiple cloning sites located at the nuclear inclusion b (NIb)-coat protein (CP) junction. The clones were sequenced, screened for unneeded mutations disrupting open reading frame, corrected and tested for infectivity. Infectious clones were identified and engineered to express a reporter gene, Green fluorescence protein. Next, a synthetic gene encoding capsid protein of the emerging Porcine circovirus (PCV), a cause of postweaning multisystemic wasting syndrome (PMWS) in a swine was assembled and incorporated into newly designed BtMV vector. Recombinant vectors are currently undergoing infectivity and protein expression tests.

10. Developed and applied a genomic approach to study pathogen and/or virus-host interactions in plants by means of EST (heterologous expressed sequence tags) mining. Gene identification by EST mapping allows efficient discovery of homologous sequences in evolutionary different species and comparison of gene sets on the whole genome scale. Using computer-assisted EST-driven prediction, we previously identified 4,935 genes or 16% of the total Arabidopsis genome which, according to the origin of EST sets, were associated with plant defense responses. Several unannotated genes from the EST-derived profiles were further screened in virus-induced gene silencing (VIGS) assay to determine their possible function and role in host-pathogen interactions. Our results indicated that two sequences, which are homologous to Arabidopsis genes At5G53940 and At5G49530 and represent Yippee - and SIN - like novel gene families, participate in host antiviral defense. The sequences of the corresponding EST fragments were extended to produce tentative open reading frames for both genes in N. benthamiana. Since a concept and consequences of gene clusters in eukaryotic genomes are largely unknown and their role in biological processes such as defense reactions is poorly studied, we performed computer-generated analysis of the chromosomal distribution of genes associated with defense response in Arabidopsis thaliana. This analysis revealed numerous clustered genes whose co-regulation may be related to the defense responses. The genes were .distributed among all chromosomes of A. thaliana. Experimental assessment of arbitrarily selected clusters using quantitative real-time PCR indicated that clustered genes are expressed in a constitutive manner but their induction may be regulated by the basic mechanisms of genetic resistance to pathogens in the form of R (resistance) genes.

11. Demonstrated that microelectrode non-invasive potassium flux measurements may be a potential tool for early recognition of plant virus-host compatibility. Knowing whether plants are susceptible to pathogen or not may determine and shape all complex measures associated with production of the major crops, such as introduction, management and potential yield. Even though there is an extensive knowledge on major viruses infecting major cultivated crops, a host range of already known pathogens is constantly expanding. Reliable host-range studies as before are mainly done by mechanical inoculation to an array of plant species following by observations of the development of disease symptoms and back-inoculation to a known host. At the moment, it seems to be an unavoidable, time-consuming and labor-extensive procedure. Its reduction would undoubtedly be beneficial for all aspects of plant virology as well as plant pathology in general. A quick test of potential virus-host compatibility may be a valuable tool both in practical field virology and in the study of molecular host-pathogen interactions. We used non-invasive ion selective microelectrodes (MIFE) to measure net ion fluxes from mesophyll tissue of host and non-host plants in response to infection with Potato virus X, the most widespread of all the potato viruses that infects several Solanaceous crops including potato, tomato, and tobacco. Our results demonstrate that using MIFE technique to measure changes in membrane-transport activity in plant tissues can be an effective screening tool for early diagnostics of virus-host compatibility.

Review Publications
Hammond, R., Hammond, J. 2009. Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli. Virus Research. 147:208-215.

Castro, R.M., Hernandez, E., Mora, F., Ramirez, P., Hammond, R. 2009. First Report of Tomato Chlorosis Virus in Tomato in Costa Rica. Plant Disease. 93:970.

Crosslin, J., Hamm, P.B., Kirk, W.W., Hammond, R. 2010. Complete genomic sequence of a tobacco rattle virus isolate from Michigan-grown potatoes. Archives of Virology. 155:621-625.

Salyaev, R.K., Rekoslavskaya, N.I., Stolbikov, A.S., Hammond, R., Shchelkunov, S.N. 2009. Retention of the ability to synthesize HIV-1 and HBV antigens in generations of tomato plants transgenic for the TBI-HBS gene. Doklady Biochemistry and Biophysics. 425:120-123.

Shabala, S.N., Nemchinov, L.G. 2010. Microelectrode non-invasive potassium flux measurements as a potential tool for early recognition of plant virus-host compatibility. Planta. 232(4):807-815.

Last Modified: 2/23/2016
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