Source: OKLAHOMA STATE UNIVERSITY submitted to
EVOLUTION OF VIRUSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0097848
Grant No.
(N/A)
Project No.
OKL01789
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Melcher, U. K.
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Biochemistry & Molecular Biology
Non Technical Summary
Understanding virus evolution allows us to consider prediction of future outbreaks of disease caused by viruses. However, such efforts are complicated by the lack of knowledge of the biodiversity of viruses. Almost all viruses known to man are viruses of humans, our animals and our plants. Viruses of other animals and plants are virtually unknown but will likely serve as the source of emerging pathogens as changes occur in cropping patterns, land usages and climate and the changes combine to alter selective forces acting on viral populations in the less-well studied organisms. Important to prediction of disease outbreaks is an understanding of the role of viruses in shaping the community of plants and animals in which they exist and understanding how the relationships in that community influence virus fitness and virulence. These issues are just beginning to be examined and are important for agriculture, wildlife conservation, ecological restoration and related efforts. It is unfortunate that we now live in a time of international suspicion and intrigue and need to be concerned about the intentional release of pathogens. Although viruses are only one of several microbial pathogen groups, their properties lend themselves well as models for strategies to protect homeland security against bioterrorists and biocriminals. For example, upon recognition of an outbreak, it is critically important to determine quickly whether the outbreak is natural or intentional. Decision tools to aid in that determination need to be developed and tested. Outbreaks of plant viruses present opportunities for the testing of such tools since such outbreaks can be created intentionally for comparison with natural occurrences, while use of animals and humans in such an endeavor is ethically impossible. One factor important in a decision tool is whether the causative agent is known to exist in the area. Knowledge of the distribution of potential crop pathogens is important to assess more rapidly whether law enforcement should become involved in the investigation. Should an intentional release be indicated, it will be important also to have detailed information about the field isolate of the pathogen. Capabilities of distinguishing one isolate from another are important microbial forensics tools for legal attribution or exclusion. The project will contribute data and concepts to interdisciplinary efforts to understand the diversity, distribution and environmental influences of viruses associated with plants. The information will be used to assist predictions useful to agriculture, to land restoration efforts and to homeland security.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2060780110150%
2060780107040%
2060780104010%
Goals / Objectives
Previous work has led to numerous new and exciting hypotheses which will be addressed in this project. 1) Viruses of non-cultivated areas are distinct, with few exceptions, from viruses characterized from cultivated plants. 2) Intriguing levels of polymorphism have been found both in native and wheat plants suggesting an important role. 3) There is considerable variation among viruses in their distribution in natural environments. 4) The unexpected presence in some plants of sequences from bacteria and fungi raises the question of the effects of these organisms on virus success and the effect of viruses on these organisms. 5) Fundamental research on virus evolution has immediate practical impacts on homeland security. 6) The paths of virus evolution during the evolution of plant species may be traceable. Objective 1 is to characterize more completely several viruses whose signatures have been identified in TPP samples. Such characterization includes completion of nucleotide sequence determination, construction of partial and full-length genomic clones, characterization of the infectivity of the genome to a range of plant species, and identification of mode of transmission. The objective is aimed at providing tests of the hypothesis that viruses from natural environments differ in some essential properties from those affecting cultivated species. Objective 2 is to better understand nucleotide polymorphisms of sequence in TPP viruses. One aim is to test the degree of linkage of polymorphic nucleotides in the genome population within a plant. A second aim is to test whether the polymorphisms are sensitive to environmental conditions in which plants are grown. The objective is aimed at testing the hypothesis that polymorphisms are essential elements of viral fitness allowing the virus to continue to replicate under a variety of external conditions. Objective 3 is to determine whether viruses found in the TPP are found in places outside of the preserve and to chart temporal variations. The objective tests the hypothesis that, absent human-mediated transport, viruses are localized to relatively small areas bounded by geographical-ecological barriers to their spread. Objective 4 is to characterize bacterial, archaeal and viral populations associated with individual plants. Comparisons of these populations among individual plants should provide a test of the hypothesis that viruses in plants affect the distribution of other microbes in the plant and that in turn, these microbes affect the virus levels. Objective 5 is to apply techniques developed for exploration of biodiversity, ecology, and evolution to needs of homeland security. The hypothesis is that basic research can lead to applications of significant societal impact. Objective 6 is to identify, in the sequences of existing viral genomes, evidence of changes in selective forces over the course of their evolution. The hypothesis is that the phylogenetic record contains evidence of environmental changes (natural or human made) that have shaped virus evolution. Expected outputs include several complete viral genome sequence database entries, publications in virus ecology and evolution.
Project Methods
1. To complete sequences of the genomes of chosen viruses, RT-PCR with primers from known sequence will amplify fragments spanning gaps in the sequence. Templates for amplification will be from already prepared cDNA, viral nucleic acid, and frozen plant tissue as previously described. Products will be directly sequenced and also cloned in bacterial plasmid vectors. The comparison of direct and after-cloning sequences will explore polymorphisms in viral genomes. 2. Three regions of a viral genome will be targeted for RT-PCR amplification with high fidelity enzymes and the products sequenced directly and cloned also in bacterial plasmids. Inserts of 20 plasmids per region will be sequenced. Results will be evaluated for linkage. We will continue perfecting a microarray-based method of evaluating. We will use one or both of these methods to compare polymorphism profiles of populations to test whether profiles respond to environmental variables. Three levels of testing are contemplated: plant growth chamber, environmental plots, diverse collections. 3. We will explore metagenomic survey approaches. Various agents and processes can serve as methods of sampling multiple plants: surface water run off from areas with plant litter; frass of herbivores; and the contents of plant-feeding insects. Methods will be developed to enrich each of these sources for viral particles. These will be then processed by previously developed methods to obtain nucleic acid preparations enriched in viral genomes. These will be targets for hybridization to microarrays containing signature sequences of TPP viruses. 4. To characterize plant associated bacteria and, samples of targeted plants will have their leaf surfaces sterilized and DNA prepared from them by the CTAB method. Specific primers for rDNA of large taxonomic groupings of bacteria will be used in Q-PCR to assess the amounts of each broad category of microbe in the sample. Further profiling of the populations will be by terminal restriction fragment length polymorphism to discover the taxonomy of abundant microbes. 5. The potential use of profiles of nucleotides at polymorphic positions (whether determined by SNaP shot or microarray) as sensitive markers for virus isolates and their possible sensitivity to environmental variables has already been mentioned in the procedures to address objective 2. A pan-virus microarray is being developed by colleagues for plant introduction screening for viruses. This laboratory will participate in the validation of the microarray hybridization method. In the future, it may be possible and advisable to incorporate into such an array probes for signs of genetic engineering as well as bacterial and fungal pathogens. 6, This laboratory will collaborate in the recovery and characterization of viral nucleic acids from herbarium material. We will receive samples and, under conditions carefully controlled to avoid contamination, process them for virus RNA detection and eventual sequencing. Positive samples will be sent for corroboration. Success of these efforts will be evaluated by numbers of literature citations of publications arising from the work.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: ACTIVITIES Nucleic acid extracts of plant tissue infected separately with Plum pox and bean golden mosaic viruses and spiroplasma were obtained and prepared for DNA sequencing. Resulting sequences were converted to searchable databases. Procedures for testing the statistical significance of BLAST hits by e-probes of the databases were evaluated. Additional e-probes specific for strains of PPV were designed and tested against actual and simulated infected plant sequence databases. Optimal parameters for searching sequence databases were established. E-probes specific for Serratia marcescens, for green fluorescent protein (GFP) and for antibiotic resistance genes were designed. Nucleic acid was extracted from zucchini tissue, tissue infected with S. marcescens, and infected with S. marcescens carrying a GFP plasmid. Nucleotide sequences of these preparations were probed with the e-probes. Amplified 16S rDNA from endophytic bacteria of plants of the Tallgrass Prairie Preserve (TGP) was prepared and sequenced by the 454 method. The sequences were analyzed computationally to identify the number and kinds of operational taxonomic units. Phylogenetic analysis of a large number of grapevine fanleaf virus strains was completed. Plant material from the TGP, likely containing viruses related to Curvularia thermal tolerance virus (CThTV) were obtained and tested. Primers for detection of CThTV relatives were designed and tested. TGP sequences of flexuous viruses were characterized. RNA extracts from Texas watermelons were labeled for hybridization to Universal Plant Virus Microarrays (UPVMs), hybridized and the results analyzed to determine viruses present. The number and genomic locations of variant nucleotides in Asclepias asymptomatic virus from 100 TGP plants were analyzed. Hypotheses to explain distributions were constructed and tested. Support vector machines were explored to distinguish host plant from bacterial and from fungal sequences. Graduate students A. Stobbe, T. Ding, M. Dutta and postdoctoral fellow R. Verma were mentored. Mentoring was also provided two OSU faculty members. Three undergraduate students and one predoctoral student received research experiences. Seven students received instruction in Molecular Genetics and 10 in Nucleic Acid and Protein Synthesis. EVENTS UM co-organized the 5h Plant Virus Ecology Network (PVEN) workshop at Lawrence KS and a Plant Virology workshop at the SR Noble Foundation. Group members attended them and also attended conferences: the Ann. Meetings of the American Phytopathological Society, 13th Genome Standards Consortium Workshop, MCBIOS Annual Meeting, SWARM-AAAS meeting, US Culture Collections Network Workshop. SERVICES: Grant proposal review for Louisiana EPSCoR, ANR (France), California AgNatRes, NSF, and Texas Higher Education Board; manuscript review for 7 journals; evaluation of faculty of other institutions for promotion; evaluation of OSU faculty for awards; strong contributions to NIMFFAB. PRODUCTS: 1 PhD graduate; 2 workshops, UPVM, an enhanced database of virus-enriched TGP sequences, databases and scripts for selecting and testing e-probes, and nucleic acid extracts of TGP plants. PARTICIPANTS: INDIVIDUALS Ulrich Melcher, PI. Planning, synthesis, writing manuscripts, proposals, advising, coordination. Tao Ding, PhD student. Bacterial endophyte investigation. Anthony Stobbe, PhD student, e-probes for viruses and multiple parallel sequencing. Mukat Dutta, PhD student.Virus exploration. *Ruchi Verma, postdoctoral fellow. Support vector machines and probes for genetically engineered organisms. *Suraj Sukumar, MS student, database construction and maintenance. *Paige Lenaburg, undergraduate student, virus exploration. *Kayla Mislick, predoctoral student, Universal plant virus microarray. *Ariel Clark, undergraduate, virus evolution. * no direct OAES support. PARTNER ORGANIZATIONS Michigan State University, University of Kansas, USDA-ARS, University of Tulsa, SR Noble Foundation, NIMFFAB. NEW COLLABORATIONS AND CONTACTS Marilyn J. Roossinck, Brendan Rodoni, Robin MacDiarmid, Akhtar Ali, William Schneider, Nemat Sokhandan Bashir, Lucy Stewart, Richard Winegar, Ravi Jain. TRAINING & PROFESSIONAL DEVELOPMENT T. Stobbe interned at the USDA-ARS lab in Fort Detrick. He also heled conduct a 4-H workshop on DNA forensics. Mentoring was provided for the individual listed. TARGET AUDIENCES: TARGET AUDIENCE The immediate target audience for the research in this project is agricultural scientists including phytosanitary officials, plant virologists, epidemiologist, diagnosticians, plant ecologists, evolutionary biologists, vector entomologists, plant breedres. Broader scientific audience includes all evolutionary biologists, virologists, ecologists and related disciplines. Societal groups are indirectly affected via the immediate targets except for the educational and training aspects of the project. EFFORTS: Summer internship experiences were provided for two individuals. Courses in Molecular Genetics and Nucleic Acids and Protein synthesis were taught, the former associated with a self-authored webtext. T. Stobbe helped conduct a 4-H workshop on DNA forensics for HS students and their teachers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
KNOWLEDGE EDNA is an efficient method of screening plant material for the presence of undesirable viral and eukaryotic pathogens. Application to bacterial phytpathogens requires overcoming an antibacterial bias in sequence amplification. Analyzing predicted amino acid sequences of databases of plant derived nucleotide sequences using models constructed using support vector machines has potential for detecting proteobacterial sequences associated with plants. UPVM assay of plant extracts correctly identified whether extracts had viruses detectable by dot immunoassay, and many cases supported the serological identification suggesting that it may be used in screening plant material for virus infection. The first glimpse into endophytic bacteria of non-cultivated plants revealed that several thousand species of bacteria are intimately associated with leaves of five plant species from the TGP. Most are proteobacteria, but bacteriodetes and actinobacteria are also frequent. The taxonomic profiles of such endophytic bacteria are highly dependent on the host species. Iranian strains of GFLV diverged from one another much earlier than those of most other strains suggesting that the origin of GFLV may have been in Persia. Alpha- and betaflexivirdae are represented in the TGP sample by only 8 species, fewer than for many plant viral groupings. Pair-wise distances between AsAV sequence from individual A. viridis plants had a narrow uniform distribution at 3.5% sequence difference not correlated with geographical position, supporting the hypothesis that this virus is a long term resident of the tallgrass prairie and may exemplify a stable virus-host relationship. ACTIONS: Those responsible for plant biosecurity in various countries and jurisdictions now consider the implications of the project's findings of the existence of many more viruses than are currently recognized, as witnessed by a manuscript shortly to be submitted. The UPVM probes have been used as e-probes of metagenomic databases from plants carrying a disease of unknown etiology and have resulted in some identifications, aiding disease diagnosis and hence treatment and/or prevention. CONDITIONS: Students who have graduated from the team have assumed societal positions in national security, biotechnological infrastructure, and international healthcare with the potential of having beneficial impacts in these fields. The PVEN founded by our team (with C. Malmstrom of MSU) has established plant virus ecology as a significant valid scientific field. Multiple laboratories have taken our lead in exploration of the uncharted virus world. Plant diagnosticians around the world are considering microarrays and database e-probes as means to solve challenging diagnostic problems. Microarray hybridization for virus detection is being used commercially and is being contemplated for use by regulatory government laboratories

Publications

  • Carpane, P., Melcher, U., Wayadande, A., de la Paz Gimenez Pecci, M., Laguna, G., Dolezal, W. & Fletcher, J. (2012) An analysis of the genomic variability of the phytopathogenic mollicute Spiroplasma kunkelii Phytopathology, in press. Verma, R. & Melcher, U. (2012) A Support Vector Machine based method to distinguish proteobacterial proteins from eukaryotic plant proteins. BMC Bioinformatics 13 (S15) S9. Sokhandan-Bashir, N., Melcher, U. (2012) Population genetic analysis of Grapevine fanleaf virus. Arch. Virol., 157(10): 1919-1929. Thapa, V., Melcher, U., Wiley, G. B., Doust, A.,.Palmer, M. W, Roewe,K.,. Roe, B.A., Shen, G., & Roossinck, M.J. (2012). Detection of members of the Secoviridae in the Tallgrass Prairie Preserve, Osage County, Oklahoma USA, Virus Research, 167: 34-42. Rogers, S.M., Payton, M., Allen, R.W., Melcher, U., Carver, J., & Fletcher, J. (2012) Validation of a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus. Investigative Genetics 3:10. Melcher, U. (2012) Virus Operation Control Centers. In Biocommunication of Plants, G. Witzany, editor, Springer Verlag, pp. 231-254. Stobbe, A.H., Melcher, U., Palmer M.W., Roossinck, M.J., & Shen, G. (2012) Co-divergence and host-switching in the evolution of tobamoviruses. J. Gen. Virol. 93: 408 - 418. Melcher, U. & Comer, J. (2012) Plectrovirus. In Springer Index of Viruses. Part 40, 749-755. Min, B.-E., Feldman, T., Wiley, G.B., Muthukumar, V., Roe, B.A., Palmer, M.W., Ali, A., Roossinck, M.J., Melcher, U. and Nelson, R.S. (2012). Molecular characterization, ecology and epidemiology of a novel tymovirus in Asclepias viridis (milkweed) from the Tallgrass Prairie Preserve of Oklahoma. Phytopathology 102(2): 166-176. Dutta M., Melcher U. K. and Bashir N. S. (2012) Ambrosia asymptomatic virus 1. A novel Mandarivirus. Phytopathology 102:S4.33


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: ACTIVITIES Detection methods for SNP alleles in virus populations were investigated. Nucleotide sequencing compared the variabilities within Wheat streak mosaic samples from natural and human-induced outbreaks. Samples generated for testing by microarray hybridization to universal plant virus microarrays (UPVMs) were hybridized. UPVM oligonucleotide sequences were tested as BLAST queries (e-probes) of the database of virus-enriched sequences from the Tallgrass Praire Preserve (TGP). E-probes were also developed for Bean golden mosaic and Plum pox viruses and for Spiroplasma citri for testing as queries of mock databases. Optimal parameters for creating e-probes of pathogens and mock databases and of searching same for probe presence were studied. The incidence of Secoviridae member Asclepias virus TGP2 in plants of six species harvested in 2009 was assessed by RT-PCR. More sequences of Secoviridae members were extracted from a database of non-assembled sequences from the TGP. Phylogenetic placement of TGP Tombusviridae members and analysis of polymorphisms in their sequence were completed. Similarities between phylogenetic trees of tobamoviruses and of natural host orders were tested. The sequence of internal regions of the Ambrosia asymptomatic virus, a member of the Alphaflexiviridae, was completed and viruses from different plants were compared. Samples from herbarium specimens were tested for Barley yellow dwarf and Wheat streak mosaic viruses. The characterization of variations in the endophytic bacterial populations in five TGP plant species at three locations and at three dates was completed by t-RFLP analysis. Statistical analysis of the t-RFLP data was completed and DNA was prepared for sequencing. Support vector machines were explored to distinguish host plant`from foreign sequences. Graduate students T. Brown, A. Stobbe, T. Ding, M. Dutta and postdoctoral fellow R. Verma were mentored. Mentoring was also provided three OSU faculty members and one from Tulsa Univ. Four undergraduate students and a Junior College faculty member received research experiences. Seven students received instruction in Molecular Genetics. EVENTS Training was obtained at Michigan State Uni. by MD in RNA isolation from herbarium samples and at USDA-ARS-BARC by UM in microarray hybridization. UM co-organized the 4th Plant Virus Ecology Network (PVEN) workshop at Montpellier and organized a Molecular Virology Symposium for the Oklahoma Academy of Science. Group members also attended conferences: the Ann. Meetings of the American Phytopathological Society and American Society for Microbiology, the International Congress of Virology, a BARD-sponsored Workshop on Microarrays and Next Generation Sequencing, Plant Virology Retreat, DTRA meeting SERVICES: Grant proposal review for the Texas Higher Education Review Board; judging senior HS science projects for the Oklahoma Junior Academy of Science; strong contributions to the development of NIMFFAB. PRODUCTS include: One PhD graduate (TB); the PVEN, UPVM, an enhanced database of virus-enriched sequence from the TGP, databases and scripts for selecting and testing e-probes, and nucleic acid extracts of TGP plants. PARTICIPANTS: INDIVIDUALS Ulrich Melcher, PI. Planning, synthesis, writing mansucripts, proposals, advising, coordination TeeCie West Brown, PhD student. Microbial Forensic methods for virus outbreaks Tao Ding, PhD student. Bacterial endophyte investigation Anthony Stobbe, PhD student. Multiply parallel sequencing, tobamovirus evolution Ruchi Verma*, PostDoc fellow. Bioinformatics multiply parallele sequencing Mukta Dutta, PhD student. Herbarium viruses of cereals JulieAnna Rohde*, EPSCoR ROA fellow, seitchgrass viruses and yield Jennifer Cobb*, BS student, Wheat streak mosaic virus assay Brad VanCleve*, BS INBRE summer intern UCO Asclepias virus tissue distribution study Nitin Kamath*, BS summer intern, Virus incidence measurement Chester Hendershot*, BS summer intern, assisted TeeCie Brown Paige Lenaburg*, Herbarium viruses of cereals * no direct OAES support PARTNER ORGANIZATION Michigan State University, USDA-ARS, Crop Protection Institute CR, University of Tulsa, SR Noble Foundation, NIMFFAB COLLABORATIONS AND CONTACTS Jacqueline Fletcher, Carla Garzon, Francisco Ochoa-Corona, Peter Hoyt, Kay Scheets, Michael Palmer, Andrew Doust TRAINING & PROFESSIONAL DEVELOPMENT PI attended UPVM workshop. M. Dutta received training in handling of herbarium specimens for PCR at Michigan State University. T. Stobbe interned at the USDA-ARS lab. He also helped conduct a 4-H workshop on DNA forensics. Mentoring provided for individuals listed. TARGET AUDIENCES: TARGET AUDIENCES The immediate target audience for the research in this project is agricultural scientists including plant virologists, epidemiologists, diagnosticians, plant ecologists, evolutionary biologists, vector entomologists, plant breeders. Broader scientific audience includes allevolutionary biologists, virologists, ecologists and related disciplines. Societal groups are indirectly affected via these immediate target, except for the educational and training aspects of the project. For example, PhD graduate TeeCie Brown is a Native American. EFFORTS Summer internship type experiences were constructed and administered to four individuals (see individuals section). Molecular Genetics, a first year graduate course was taught by the PI. A. Stobbe helped conduct a 4-H workshop on DNA forensics for HS student and their teachers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
KNOWLEDGE Important hypotheses received observational support, putting them on the road to becoming general knowledge. These include: known plant-associated virus species represent but a small fraction of extant plant viruses; viruses of wild plants contain unusual genes and genome features; some viral lineages have codiverged with the lineages of their hosts, suggesting that viruses play essential roles in ecosystems; viruses are not necessarily pathogenic; in silico probing of DNA sequencing results efficiently screens deep sequencing data for signs of pathogen presence; community t-RFLP profiles of bacterial endophytes depend principally on plant host species and secondarily on location and season. On the other hand, we learned that several hypothetically promising methods for detection of SNPs cannot be used in viral forensics for lack of easy reproducibility. We also noticed that virus variants can show year-to-year variation, reappearing after absence, in later years. ACTIONS Discouraging results led us to cease testing applicability of several microarray SNP methods for forensic analysis of viral outbreaks. We will be recommending deep nucleotide sequencing as an alternative for comparing outbreak and laboratory populations of a pathogen and further developing E-DNA and UPVM. We participated in a workshop training USDA virologists in UPVM microarray hybridization. In an additional, BARD-sponsored, workshop, our experience in microarray hybridization and plant virus discovery drove the discussion. Through additional international scientific meetings (PVEN, International Congress of Virology) and publications we have publicized the likelihood that tobamoviruses codiverged with their host lineages. These observations also drive our pursuit of cereal viruses from herbarium specimens, and motivate a study of mummified onions. A hypothesis describing events in speciation of viruses has been generated. Funds to pursue the speciation hypothesis and the ecological role of viruses have been applied for. The t-RFLP data on bacterial endophytes allow us to further pursue identification of the members of the community by deep sequencing of selected communities. CONDITIONS Students who have graduated from the team have assumed societal positions in national security, biotechnological infrastructure, and international healthcare with the potential of having beneficial impacts in these fields. PVEN, founded by our team (with C. Malmstrom of MSU), has established plant virus ecology as a significant valid scientific field and delineated its distinction from epidemiology. Multiple laboratories have taken our lead in exploration of the uncharted virus world. Plant diagnosticians around the world are considering microarrays and database e-probes as means to solve challenging diagnostic problems. Microarray hybridization for virus detection is being used commercially and is being contemplated for use by regulatory government laboratories. Our finding of over 300 previously not described viruses solely by sequence identification has caused virologists to reevaluate the standards for virus designation and naming. A 2012 conference will address this issue.

Publications

  • Melcher, U. (2012) Virus Operation Control Centers. In Biocommunication of Plants, G. Witzany, editor, Springer Verlag, in press
  • Stobbe, A.H., Melcher, U., Palmer M.W., Roossinck, M.J., Shen, G. (2012) Co-divergence and host-switching in the evolution of tobamoviruse. J. Gen. Virol., in press
  • Melcher, U. and Comer, J. (2012) Plectrovirus. In Springer Index of Viruses. Part 40, 749-755.
  • Min, B.-E., Feldman, T., Wiley, G.B., Muthukumar, V., Roe, B.A., Palmer, M.W., Ali, A., Roossinck, M.J., Melcher, U. and Nelson, R.S. (2011). Molecular characterization, ecology and epidemiology of a novel tymovirus in Asclepias viridis (milkweed) from the Tallgrass Prairie Preserve of Oklahoma. Phytopathology, in press.
  • Scheets, K., Blinkova, O., Melcher, U., Palmer, M.W., Wiley, G.B., Ding, T. and Roe, B.A. (2011). Detection of members of the Tombusviridae in the Tallgrass Prairie Preserve, Osage County, Oklahoma USA. Virus Research 160(1-2): 256-263.
  • Thresh, J.M., Fereres, A., Bosque-Perez, N.A., Melcher, U. and Valkonen, J. (2011). Guest Editorial. Virus Research 159: 83.
  • Malmstrom, C.M., Melcher, U. and Bosque-Perez, N.A. (2011). The expanding field of plant virus ecology: Historical foundations, knowledge gaps, and research directions. Virus Research 159: 84-94.
  • Mutaqin, K., Comer, J.L., Wayadande, A.C., Melcher, U. and Fletcher, J. (2011). Selection and characterization of Spiroplasma citri mutants by random transposome mutagenesis. Can. J. Microbiol. 57(6): 525-532.
  • Melcher, U., and Grover, V. (2011) Genomic Approaches to Discovery of Viral Species Diversity of Non-cultivated Plants in Recent Advances in Plant Virology, Caranta, C., Aranda, M.A., Tepfer, M. and Lopez-Moya, J.J. Caister Academic Press, Norwich, pp. 321-342.
  • Mello, A.F.S., Yokomi, R.K., Melcher, U., Chen, J.C., Civerolo, E., Wayadande, A.C. and Fletcher, J. (2010). New perspectives on the epidemiology of citrus stubborn disease in California orchards. Plant Health Progress.
  • Daniels J., A. Stobbe, A. Espindola, W. Schneider, J. Fletcher and F.M. Ochoa-Corona. 2011. Massively parallel sequencing as a diagnostic tool for bacterial plant pathogens. DTRA Chem-Bio Defense Annual Meeting, Las Vegas, NV.
  • Daniels, J., T. Stobbe, A. Espindola, W. Schneider, J. Fletcher and F.M. Ochoa-Corona. 2011. In silico simulation of massively parallel sequencing as a diagnostic tool for bacterial phytopathogens. APS Annual Meeting, Honolulu, HI.
  • Espindola A., T. Stobbe, J. Daniels, J. Fletcher, W. Schneider and C. Garzon . 2011. Query based detection of eukaryotic plant pathogens Puccinia graminis and Phytophthora ramorum in computer-generated pyrosequencing databases. DTRA Chem-Bio Defense Annual Meeting, Las Vegas, NV.
  • Espindola, A., J. Daniels, T. Stobbe, J. Fletcher, C. Garzon and W. Schneider. 2011. Design and validation of queries for the detection of Puccinia graminis in simulated metagenomes. APS Annual Meeting, Honolulu, HI.
  • Espindola, A., J. Daniels, T. Stobbe, J. Fletcher, C. Garzon and W. Schneider. 2011. Design and validation of queries for the detection of Phytophthora ramorum in simulated metagenomes. APS Annual Meeting, Honolulu, HI.
  • Stobbe, A., J. Daniels, A. Espindola, W. Schneider, J. Fletcher, U. Melcher. 2011. Massively parallel sequencing used a diagnostic tool. DTRA Chem-Bio Defense Annual Meeting, Las Vegas, NV. Abstract.
  • U. Melcher 2010 Filling Phylogenetic Gaps Virus Evolution Workshop, Ardmore OK, October 21-24, 2010
  • Schneider, W.L., A. Stobbe, J. Daniels, A. Espindola, R. Verma, T. Blagden, J. Fletcher, F. Ochoa-Corona, C. Garzon, P. Hoyt, and U. Melcher. 2011. Finding the diagnostic needle in a deep sequence data haystack: E-probe Diagnostic Nucleic acids Analysis (EDNA). Binational Agricultural Research & Development Conference on Microarrays and Next Generation Sequencing for Detection and Identification of Plant Viruses; Beltsville, MD.
  • Stobbe, A., J. Daniels, A. Espindola, W. Schneider, J. Fletcher, U. Melcher. 2011. Massively parallel sequencing used a diagnostic tool. DTRA Chem-Bio Defense Annual Meeting, Las Vegas, NV.
  • Stobbe, A. Melcher, U. 2011. Massively Parallel Sequencing used as a Diagnostic Tool. Oklahoma State University, Biochemistry and Molecular Biology Graduate Student Symposium 2011
  • Stobbe, A. Fletcher, J. Schneider, W. Melcher, U. 2011. Massively Parallel Sequencing used as a Diagnostic Tool. American Phytopathology Society, Annual Meeting Honolulu, HI
  • Stobbe, A. Melcher, U. 2011. Massively Parallel Sequencing used as a Diagnostic Tool. Plant Virology Retreat, Ardmore OK 2011.
  • Ding, T. 2011.How are microorganisms distributed among plants --- A study of plant endophytic bacteria using T-RFLP. 2011 OSU Research Symposium.
  • Ding, T., Palmer, M.W. and Melcher, U. 2011, Community Terminal Restriction Fragment Length Polymorphisms Reveal insights into the Diversity and Dynamics of Endophytic Bacteria. 2011 Annual Meeting, American Society for Microbiology, New Orleans, May 2011
  • Ding, T. 2011. How are microorganisms distributed among plants --- A study of plant endophytic bacteria using T-RFLP. 2011 BMB Graduate Research Symposium, OSU, Stillwater.
  • Rohde, J. and Melcher, U. 2011. A study of virus type and biomass correlation in Panicum virgatum. Oklahoma Academy of Science Annual Technical Meeting, Durant OK, November 11
  • Melcher, U. 2011. Covariation of nucleotide residues in the evolution of viral taxa. 4th Plant Virus Ecology Network Workshop, Montpelier FRANCE 2 June 2011.
  • Melcher, U. 2011. Covariation of nucleotide residues in the evolution of viral taxa. International Congress of Virology, Sapporo, Japan, 13 September 2011.
  • Rogers, S. M., Payton, M., Allen, R. , Melcher, U. Fletcher, J. 2011. Validation of a single nucleotide polymorphism genotyping assay for Wheat streak mosaic virus. APS Annual Meeting, Honolulu, HI. Dutta, M., Melcher, U. and Malmstrom, C. 2011. Evolution Patterns of BYDV. 2011 BMB Graduate Research Symposium, OSU, Stillwater.
  • Rogers, S.M. Payton, M., Hunger, R., Fletcher, J. and Melcher, U.. 2011. CBIIAT: Crop Bioagent Introduction Intent Assessment Tool Using Wheat Streak Mosaic Virus as a Model. DTRA Chem-Bio Defense Annual Meeting, Las Vegas, NV.
  • Melcher, U., Brown, T.W., and Grover, V. 2011 UPVM probes in testing for relatives of known viruses. Binational Agricultural Research & Development Conference on Microarrays and Next Generation Sequencing for Detection and Identification of Plant Viruses; Beltsville, MD. November 2011


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Activities: Comparison of phylogenetic trees of tobamoviruses with those of the host plants from which they were isolated. Assistance in the design of an oligonucleotides-based microarray with 9000 features targeting all plant viruses. Completion of classification of all viral sequence contigs from the Plant Virus Biodiversity and Ecology (PVBE) project. Identification of PVBE singleton sequences for viruses in four taxa . Phylogenetic analysis of TGP comovirin 1. Sequence gap closing for Ambrosia asymptomatic virus 1. RT_PCR assay of plant extracts for AsAV 1 and TGP Totivirid 5. Assay of stem sections of Ruellia humilis by in situ hybridization for the location of TGP Totivirid 5 and by staining for the presence of fungus . A test of seed transmission of a Totivirid. Generation of nucleotide sequence of one region of the coat protein gene of Wheat streak mosaic virus (WSMV) from field-infected and naturally infected plants. Comparison of the utilities of microarray hybridization strategies for analyzing polymorphisms of WSMV in plant extracts. Development of a theory, invoking fractals, accounting for the difference between evolution within a viral species and the evolution of species and higher taxa. Events: Organization, planning and convening of the third Plant Virus Ecology Network (PVEN) Workshop jointly with the International Plant Virus Epidemiology Symposium. Invited presentations to institutions (OSU Botany Dept., Univ. California-Davis Plant Pathology Dept.). Invited speaker at symposium within the American Phytopathological Society Annual Meeting (AM). Attendance and presentations at AMs of the Midsouth Computational Biology and Bioinformatics Society, the Noble Foundation Plant Virology, and the Oklahoma Academy of Science (OAS). Services: Presiding over the OAS to 2009-12-31. Judging high school science presentations at the Oklahoma Junior Academy of Science. Judging college student presentations and assisting in the operation of the OAS AM. Products: Total nucleic acid extracts from 20 specimens of each of 6 plant species harvested from TPP in 2009 to complement similar ones from 2005-2008 . A protocol for the analysis of diversity of bacterial endophytes by t-RFLP. DNA extracts of 5 species of plants, 4 sites and 3 or 4 harvest dates, suitable for bacterial endophyte diversity analysis. Preliminary data on diversity. A plasmid clone encoding the cysteine-rich protein encoded by TGP Tobamovirus 1. An oligonucleotide array targeting cereal viruses. Computer scripts to create mock datatbases of nucleotide sequence as generated by various sequencing technologies. Mock databases created, using these scripts, to simulate sequence results from uninfected and pathogen infected plants. Metrics to measure the confidence with which a plant can be judged to be healthy or pathogen infected. Training provided 4 graduate students, a visiting professor (Iran), a technical school undergraduate (Germany), 3 Oklahoma undergraduates. PARTICIPANTS: INDIVIDUALS supported by the agency: Ulrich Melcher (PI/PD); Anthony Stobbe, (Graduate Research Assistant), Veenita Grover (Graduate Research Assistant). PARTNER ORGANIZATIONS: Samuel Roberts Noble Foundation (SRNF), National Science Foundation, USDA-NIFA, OSU Foundation (Sirny Professorship & Plant Virus Evolution Research fund), University of Tabriz, Deutsche Akademische Austausch Dienst. COLLABORATORS AND CONTACTS, within OSU: Jana Comer, Peter Hoyt, Jacque Fletcher, Astri Wayadande, Kay Scheets, Vaskar Thapa, Michael Palmer, Andrew Doust, Mostafa Elshahed, Francisco Ochoa-Corona, Carla Garzon; outside OSU: Carolyn Malmstrom (Michigan State University), Marilyn Roossinck (SRNF) , Rick Nelson (SRNF), Akhtar Ali (Univ. Tulsa), Bruce Roe (Oklahoma Univ.), Jiban Kundu Kumar (Crop Disease Institute, Prague, Czech Repulic), Nemat Sokhandan Bashir (Univ. Tabriz, Tabriz, Iran); William Schneider (USDA-ARS), Claude Fauquet (Danforth Institute), Kael Fisher (Univ. Utah), David Wang (Washington Univ.), Keith Perry (Cornell Univ.), John Hammond (USDA-ARS). TRAINING OR PROFESSIONAL DEVELOPMENT: Anthony Stobbe, Veenita Grover. Other group individuals not supported by salary, wages or a stipend from the agency:, Teecie Brown (Graduate Research Assistant), Tao Ding (Graduate Research Assistant), Nemat Sokhandan Bashir (Visiting Professor), Whitney Lewis (Undergraduate Researcher), Margaret Ye (Undergraduate Researcher), Stefanie Seelk (Undergraduate Researcher), Amanda Wilson (Undergraduate Researcher), Jennifer Cobb (Undergraduate Researcher). TARGET AUDIENCES: TARGET AUDIENCES: 1) beginning career scientists (undergraduate students, graduate research assistants, postdoctoral fellows); 2) Basic scientists in the fields of virus evolution, molecular biology, plant pathology, virology, epidemiology, vector biology and ecology; 3) government scientists responsible for biosecurity measures and antiterrorism activities. EFFORTS. Beginning career scientists were provided laboratory research experiences, weekly (or more frequent) consultation, oral and written scientific communication training. Some of these efforts were in the form of internships. Basic scientists and government scientists were reached through presentations at seminars (OSU Botany Department; Univ. Calif. Davis Plant Pathology Dept.), and at regional, national and international meetings (OAS, Midsouth Computational Biology and Bioinformatics Society, American Phytopathological Society and the Plant Virus Retreat, Virus Evolution Symposium, Roscoff, France) and through publications. Submissions of grant proposals also reached some of these individuals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Knowledge: The PI's view that evolution within a viral species differs substantially from the evolution of species and higher taxa was solidified. The PI's view that viruses must not be thought of as pathogenic was also solidified. Others are beginning to consider these views as evidenced by an increase in the number of invitations to speak. Actions: A growing awareness of plant virologists of the importance of surveying non-cultivated plants for virus diversity is developing as evidenced by an invitation to write a chapter on the subject for Recent Advances in Plant Virology. Requests for collaboration from scientists in Iran, the Czech Republic, the Ukraine, China and Australia indicate desire to adopt virus biodiversity efforts developed in this project. Similar surveys of plants for viruses have been initiated by others, for example in the Great Smokey Mountain National Park. Conditions: The field of plant virus ecology has become stronger as evidenced by the number of new members of PVEN. Several students and other scientists have acquired diverse laboratory skills related to this project.

Publications

  • Melcher, U. (2010). Assessing constancy of substitution rates in viruses over evolutionary time. BMC Bioinformatics 11, S3.
  • Fletcher, J., Barnaby, N.G., Burans, J., Melcher, U., Nutter, F.W., Jr., Thomas, C. and Ochoa-Corona, F.M. (2010). Forensic plant pathology. In: B. Budowle, S. Schutzer, R. Breeze, P. Keim and S. Morse (Eds), Microbial Forensics, Elsevier, Amsterdam pp. 89-105. A
  • llen, M. S., R. G. Hamilton, U. Melcher, and M. W. Palmer (2009). Lessons from the Prairie: Research at The Nature Conservancy's Tallgrass Prairie Preserve. Oklahoma Academy of Science, Edmond OK.
  • Hackett, J., Muthukumar, V., Wiley, G.B., Roe, B.A. and Melcher, U. (2009). Viruses in Oklahoma Euphorbia marginata (Pursh). Proc. Okla. Acad. Sci., 89:57-62.
  • Comer, J. and Melcher, U. (2009). The Plectroviruses. In Springer Index of Viruses


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The PI supervised the research of three graduate students and three undergraduate intern students as well as himself conducting some analyses of evolution of viruses in the Mastrevirus genus and further analyzing data from the Plant Virus Biodiversity and Ecology (PVBE) project. He taught the graduate course Molecular Genetics, and judged scientific presentations by high school students in the Oklahoma Junior Academy of Science competition. He participated in a training workshop for Sub-Saharan plant health professionals in virus detection at the IITA in Ibadan, Nigeria. There, he also presented a talk on plant virus biodiversity and ecology and consulted with IITA scientists. He presided over the Oklahoma Academy of Science and ran its annual technical meeting. He participated in an AAAS panel evaluation of the PVBE project. He served on a search and screen committee for a faculty member in food safety to join the National Institute For Microbial Forensics and Food and Agricultural Biosecurity (NIMFFAB). He brought together a large group of investigators to prepare a full proposal to the National Science Foundation for a Science and Technology Center in Plant Virus Ecology. Additional cooperative proposals were prepared to the USDA for National Needs Fellowships in agricultural biosecurity and to the USDA for research funding investigating the use of megasequencing in plant quarantine and security endeavors. Results of the work from the project were presented at several locations to diverse groups of scientists: Virus Evolution retreat at the Noble Foundation, Ardmore OK; Workshop on Emerging Viral Diseases, Roscoff, France; and the American Society for Virology meeting in Vancouver BC Canada. The second meeting of the Plant Virus Ecology Network, co-coordinated by the PI, was organized by the co-coordinators and held at the International Center for Genetic Engineering and Biotechnology outside of Venice, Italy. As an Associate Member of the NIMFFAB, he advised NIMFFAB leaders on issues regarding pathogen detection methods. PARTICIPANTS: Individuals: Ulrich Melcher, PI (only one to be funded by agency) Veenita Grover, graduate student, virus microarray analysis TeeCiee West, graduate student, SNP typing of pathogens Vijay Muthukumar, technician, plant virus biodiversity Tao Ding, graduate student, bacterial endophytes of plants Nicole Cabalo, undergraduate intern, virus characterization Kimberly Roewe, undergraduate intern, virus characterization Nemat Sokhandan Bashir, visiting professor, virus characterization Partner Organizations: University of Oklahoma Partner Organizations: Tulsa University, International Institute of Tropical Agriculture, Michigan State University, Oklahoma EPSCoR. Collaborators: Jacqueline Fletcher, Recombinant DNA/Protein Resource Facility (Steven Hartson), OSU Microarray Core Facility/Bioinfosu (Peter Hoyt), Francisco Ochoa-Corona, National Institute for Microbial Forensics and Food and Agricultural Biosecurity.. Training and development: Graduate and undergraduate students, Plant health professionals at IITA Workshop, High Scool science judging OJAS. TARGET AUDIENCES: See "Outputs" PROJECT MODIFICATIONS: A revised proposal project has been submitted. That document details all the changes in direction contemplated. Briefly, it states the following objectives: Objective 1. The objective is to characterize more completely several viruses whose signatures have been identified in TPP samples. Such characterization includes completion of nucleotide sequence determination, construction of partial and full-length genomic clones, characterization of the infectivity of the genome to a range of plant species, and identification of mode of transmission. The objective is aimed at providing tests of the hypothesis that viruses from natural environments differ in some essential properties from those affecting cultivated species. Objective 2. The objective is to better understand nucleotide polymorphisms of sequence in TPP viruses. One aim is to test the degree of linkage of polymorphic nucleotides in the genome population within a plant. A second aim is to test whether the polymorphisms are sensitive to environmental conditions in which plants are grown. The objective is aimed at testing the hypothesis that polymorphisms are essential elements of viral fitness allowing the virus to continue to replicate under a variety of external conditions. Objective 3. The objective is to determine whether viruses found in the TPP are found in places outside of the preserve and to chart temporal variations. The objective tests the hypothesis that, absent human-mediated transport, viruses are localized to relatively small areas bounded by geographical-ecological barriers to their spread. Objective 4. The objective is to characterize bacterial, archaeal and viral populations associated with individual plants. Comparisons of these populations among individual plants should provide a test of the hypothesis that viruses in plants affect the distribution of other microbes in the plant and that in turn, these microbes affect the virus levels. Objective 5. The objective is to apply techniques developed for exploration of biodiversity, ecology, and evolution to needs of homeland security. The hypothesis is that basic research can lead to applications of significant societal impact. Objective 6. The objective is to identify, in the sequences of existing viral genomes, evidence of changes in selective forces over the course of their evolution. The hypothesis is that the phylogenetic record contains evidence of environmental changes (natural or human made) that have shaped virus evolution.

Impacts
Recent inventories of the biodiversity of viruses in non-cultivated plants sampled plants without regard to symptoms. The Plant Virus Biodiversity and Ecology (PVBE) project developed several hypotheses from the accumulated data about the biodiversitysuch viruses. First, viruses are frequent in non-cultivated plants. Between a quarter and a half of all specimens and all species tested in PVBE had evidence of virus presence. Second, previously unknown viruses are abundant. Of several hundred potential viral species detected, only three represented viruses whose sequences were in the general GenBank/DDBJ/EMBL database. Third, as originally suggested by Brian Harrison in 1981, the taxonomic distribution of the prominnent viruses of non-cultivated plants differs from that of crop viruses. Virus taxa, such as Potyviridae and Geminiviridae, abundant among those causing crop disease, were relatively absent in the PVBE samples. Fourth, viruses of non-cultivated plants often have features not found in their crop relatives. For example, several PVBE putative viruses had additional open reading frames (ORFs) overlapping standard ORFs. Last, viruses naturally exhibit a limited number of types of distributions among plants and plant species. The project undertaken at the Area de Conservacion Guanacaste (ACG) in Costa Rica by Roossinck and colleagues provides support for many of these hypotheses, except that one particular crop virus was found with high frequency, Zucchini yellow mosaic virus, a member of the Potyviridae. In addition to these conclusions about virus diversity, evolution and ecology from the PVBE project, several other results have been solidified in the past year. That work can also be summarized in terms of hypotheses. First, Intriguing levels of polymorphism have been found both in non-cultivated plants of the tallgrass prairies and in wheat plants suggesting that nucleotide polymorphisms may play an important role in virus biology. Second, the PVBE data demonstrate considerable variation among viruses in their distribution in natural environments. In several cases, one plant species acts as a primary reservoir, but other species are also infected, yet do not accumulate the same high levels that reservoir species have. In other cases, each plant species appears to have its own variety of virus species of selected genera. Third, the unexpected presence in some plants of sequences from bacteria and fungi raises the question of the effects of these organisms on virus success and the effect of viruses on these organisms. We hypothesize tat interactions between plants and bacteria are bilaterally species specific. Fourth, he fundamental research on virus evolution has immediate practical impacts on homeland security. Fifth, the paths of virus evolution during the evolution of plant species may be traceable.

Publications

  • Grover, V., Pierce, M.L., Hoyt, P., Zhang, F., and Melcher, U. (2009). Oligonucleotide-based microarray for detection of plant viruses employing sequence-independent amplification of targets. J. Virol. Methods, Epub before print dx.doi.org/10.1016/j.jviromet.2009.08.023.
  • Wu, B., Melcher, U., Guo, X., Wang, X., Fan, L. and Zhou, G. (2008). Assessment of codivergence of mastreviruses with their plant hosts. BMC Evolutionary Biology 8:335 doi:10.1186/1471-2148-8-335.
  • Muthukumar, V., Melcher, U., Pierce, M.L., Wiley, G.B., Roe, B.A., Palmer, M.W., Thapa, V., Ali, A. & Ding, T. (2008). Non-cultivated plants of the Tallgrass Prairie Preserve of northeastern Oklahoma frequently contain virus-like sequences in particulate fractions. Virus Res. 149:169-173.
  • Hackett, J., Muthukumar, V., Wiley, G.B., Roe, B.A. and Melcher, U. (2009) Viruses in Oklahoma Euphorbia marginata (Pursh). Proc. Okla. Acad. Sci., in press.
  • Fletcher, J., Luster, D.G., Melcher, U., & Sherwood, J.L. (2010) Microbial forensics and plant pathogens: attribution of agricultural crime. In Wiley Handbook of Science and Technology for Homeland Security, J. Voeller, Ed., Wiley & Sons, NY, in press.
  • Melcher, U., & Grover, V. (2010) Genomic approaches to discovery of viral species diversity of non-cultivated plants. In Plant Virology, Horizon Press, pending.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: An NSF-funded Research Coordination Network, the Plant Virus Ecology Network was established in 2007 and held its first workshop in the spring of 2008. This network brought together plant virologists, entomologists and ecologists from around the world to plan and coordinate research in this emerging field at the interface of virology and ecology. One result of this coordination is the submission of a pre-proposal to the NSF for a multi-institutional Science and Technology Center (STC). The plant virus biodiversity studies of the project are part of Oklahoma's NSF-EPSCoR research infrastructure improvement award. The PI was coordinator for this theme area and coordinated the activities of the group (faculty searches at three institutions in the state, organization of an annual conference, group communication). Expertise developed in the project led, in addition to the STC preproposal, to cooperative roles in proposals to the USDA Agricultural Biosecurity research program, one to use megasequencing to test for threat agents in plant material and the other to develop microarrays for screening quarantine plants for viruses. The latter proposal will be funded. Four graduate students and five undergraduate students received direct training working on this project. One graduate student successfully completed an MS degree. A PhD was awarded to a student who was advised on studying markers to distinguish P. syringae strains. The knowledge of plant viruses developed during this project has contributed to the training of two graduate students in Entomology and Plant Pathology and one student in Forensic Science. Presentations related to this project have also been made at the regional American Society for Virology meeting, The Oklahoma Academy of Science Annual Technical Meeting, Tallgrass Parairie Preserve Retreat (organized by the PI), and a Plant Virology Retreat at the SR Noble Foundation. Two publications have appeared, and two others are in press. The PI serves as President of the Oklahoma Academy of Science and had to use that office to lobby against efforts to water down science teaching by including religion in science classes. The PI was named Regents Professor at his university. PARTICIPANTS: Participants Individuals (Agency supported) Ulrich Melcher Marlee Pierce Individuals (other support) Veenita Grover Vijay Muthukumar Tao Ding Yan Song TeeCie West Partner Organizations: Samuel Roberts Noble Foundation Oklahoma University Michigan State University University of Tulsa University of Oklahoma Health Science Center Oklahoma State University Center for Health Sciences Oklahoma State Regents for Higher Education Oklahoma EPSCoR National Science Foundation USDA Tulsa University Collaborators and Contacts: Jacqueline Fletcher Astri Wayadande Jeanmarie Verchot-Lubicz Kay Scheets Michael Palmer Peter Hoyt Robert Allen Akhtar Ali Training or professional development Two summer research internships Several school year undergraduate employees and research students Seven graduate students impacted by the work TARGET AUDIENCES: Effort: a 2 hour unit on viruses by the Stillwater Children's Museum Target Audience: fifth and sixth graders in the Stillwater School System Effort: Summer internship training Target Audience: undergraduate science students at four-year colleges and at minority colleges (HBCU) PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The project continues to explore how viruses of plants on non-cultivated lands could lead to emergence of crop disease outbreaks. Many hundreds of plant specimens representing a large diversity of plants present in the Tallgrass Prairie Preserve (TPP) have been processed for analysis of virus content. In this analysis, subcellular fractions of plant material that should contain virus-like particles (VLP) were prepared and the nucleic acid extracted from them. That nucleic acid was amplified, and sequenced. The probable viruses or organisms contributing those sequences are being identified by database search. In the past year, the approach has yielded further support for the following major concepts. 1) Viruses found in environments like the TPP are distinct from known viruses (only two exceptions to date) and include viruses of novel genera. 2) Plants are naturally associated with bacteria and fungi, as well as with viruses. 3) Nucleotide sequences of a virus found in different plants of the same species exhibit considerable nucleotide sequence variation. 4) Viruses are frequent in plants in natural settings. 5) Plants often harbor multiple viruses. 6) Some virus species are found in multiple diverse plant species, yet are only present in high titer in one of those species. The project is refining a microarray method in which oligonucleotides specific to conserved regions of TPP viruses are spotted on glass slides to be used as probes of fluorescently labeled DNA obtained by amplification from the total nucleic acid extract of the plant samples. Optimization experiments resulted in a protocol in which fluorescently tagged RNA molecules from a VLP fraction of plant material are hybridized to 50 nt long oligonucleotide probes. In a separate study, cooperative with Chinese scientists, phylogenetic analysis of nucleotide sequence was used to examine evolution of viruses of the Mastrevirus genus of the Geminiviridae family. Data are consistent with divergence of wheat, barley and oat dwarf viruses along side with the divergence of their host plants, suggesting a much slower rate of overall evolution than has been observed for other viruses. Expertise developed in this laboratory on evolution of viruses and prokaryotes was used to examine pathogen variability in corn stunt disease in maize, citrus stubborn disease in citrus and in Pseudomonas syringae. It is also being applied to microbial forensics. How can a bioterrorism agent be attributed to an individual or group perpetrator Answers require understanding of variation and the rates of evolution. Methods for surveying isolates for single nucleotide polymorphism profiles are being evaluated. Knowledge of the background of viruses in the world is needed to exclude a natural cause for an outbreak. The analysis of the complexity of microbes in natural plants may lead to a holistic approach to plant health.

Publications

  • Melcher, U., Muthukumar, V., Wiley, G.B., Min, B.E., Palmer, M. W., Verchot-Lubicz, J., Nelson R.S., Roe, B.A., Ali, A., Thapa, V., & Pierce, M.L. (2008) Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya. J. Virol. Methods 152: 49-55.
  • Mello, A.F.S., Yokomi, R.K., Melcher, U., Chen, J.C., Wayadande, A.C. & Fletcher, J. (2008). Genetic diversity of Spiroplasma citri strains from different regions, hosts, and isolation dates. Phytopathology, 98: 960-968.


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: An NSF-funded Research Coordination Network in Plant Virus Ecology has been established. This network seeks to bring together plant virologists and ecologists from around the world to plan and coordinate research in this emerging field at the interface of virology and ecology. Plant virus ecology, an emerging field, examines the ecological roles of plant-associated viruses in managed and unmanaged ecosystems, and investigates the reciprocal influence of ecosystem properties on the distribution and evolution of plant viruses. The Network has been publicized at national meetings of virologists, plant pathologists, and ecologists and to other Research Coordination Networks at a meeting in Arlington VA. As a result of the Network and the plant biodiversity work going on in the laboratory, the PI has participated as invited speaker in an International Plant Pathology Symposium in Huang Zhou, China, and the International Plant Virus Epidemiology Conference at ICRISAT in Hyderabad, India. The studies on plant virus biodiversity are part of Oklahoma's NSF-EPSCoR research infrastructure improvement award. The PI is coordinator for this theme area and coordinates the activities of the group (faculty searches at three institutions in the state, organization of an annual conference, group communication). The PI was also a keynote speaker at the annual meeting of the Midsouth Computational Biology and Bioinformatics Society in New Orleans. The PI served as scientific advisor for a project of the Stillwater Children's Museum to reveal viruses to 5th and 6th graders. Three graduate students and five undergraduate students are receiving direct training working on this project. The knowledge of plant viruses developed during this project has contributed to the training of two graduate students in Entomology and Plant Pathology and one student in Forensic Science. Presentations related to this project have also been made at the regional American Society for Microbiology meeting, The Oklahoma Academy of Science Annual Technical Meeting, an Ecological Genomics Workshop, Tallgrass Parairie Preserve Retreat (organized by the PI), Plant Virology Retreat at the SR Noble Foundation. The PI served on an NSF grant review panel on Microbial Interactions and Processes. A web-accessible key for the identification of plant viruses has been developed and will be made generally available shortly. PARTICIPANTS: Individuals (Agency supported): Ulrich Melcher, Marlee Pierce. Individuals (other support): Veenita Grover, Vijay Muthukumar, Tao Ding, Shashank Bhide, Yan Song, Hua Weng. Partner Organizations: Samuel Roberts Noble Foundation, Oklahoma University, Michigan State University, University of Tulsa, University of Oklahoma Health Science Center, Oklahoma State University Center for Health Sciences, Oklahoma State Regents for Higher Education, Oklahoma EPSCoR, National Science Foundation, USDA. Collaborators and Contacts: Jacqueline Fletcher, Astri Wayadande, Jeanmarie Verchot-Lubicz, Kay Scheets, Michael Palmer, Peter Hoyt, Robert Allen. Training or professional development: Two summer research internships; Several school year undergraduate employees and research students; Seven graduate students impacted by the work. TARGET AUDIENCES: Effort: a 2 hour unit on viruses by the Stillwater Children's Museum; Target Audience: fifth and sixth graders in the Stillwater School System. Effort: Summer internship training; Target Audience: undergraduate science students at four-year colleges and at minority colleges (HBCU). Effort: Presentations at conferences, workshops and meetings, publications; Target audience: scientists in this and related fields.

Impacts
A complete picture of virus evolution requires knowledge of the diversity of existing viruses. The laboratory is contributing to this knowledge in several ways. Over 500 plant specimens representing a large diversity of plants present in the Tallgrass Prairie Preserve (TPP) are being processed for analysis of virus content. In one approach, a subcellular fraction that should contain virus-like particles is prepared and the nucleic acid extracted from it. That nucleic acid is amplified, cloned in plasmid vectors, and the inserts sequenced. The probable viruses or organisms contributing those sequences are identified by database search. To date, the approach has supported the following major changes in concepts in virology. 1) Viruses found in environments like the TPP are not the same as viruses that have been studied before and include viruses of novel genera. 2) Plants are naturally associated with bacteria and fungi, as well as with viruses. The presence of viruses, fungi and bacteria associated with the same healthy appearing plant suggests complex interorganismal interactions that maintain a balance beneficial to all. 3)Nucleotide sequences of a virus found in different plants of the same species exhibit considerable nucleotide sequence variation. To complement the virus-like particle approach, the laboratory is refining microarray methods in which oligonucleotides specific to conserved regions of known viral families are spotted on glass slides to be used as probes of fluorescently labeled DNA obtained by amplification from the total nucleic acid extract of the plant samples. In this area, we have identified the need to design the detecting microarray so that the detecting sequences are lifted off of the surface by taiing the oigonucleotides with oligo dT. We have also challenged the conventional wisdom that sense of the detecting sequence is unimportant. A second area of research has been the interaction of plectroviral genomes with the bacterial host genomes of Spiroplasma citri and kunkelii. This research is facilitated by the availability of near complete genome sequences of both species of spiroplasma. Large proportions of the genomes are made up of sequences derived from at least four plectroviruses. The results are making scientists realize that viruses may be heavily involved in the evolution of bacterial chromosomes. Expertise developed in this laboratory on evolution of viruses and procaryotes is being used to examine pathogen variability in corn stunt disease in maize and citrus stubborn disease in citrus. It is also being applied to microbial forensics. How can a bioterrorism agent be attributed to an individual or group perpetrator? Answers require understanding of variation and the rates of evolution. They also require knowledge of the background of viruses in the world to exclude a natural cause for an outbreak. The analysis of the complexity of microbes in natural plants may lead to a holistic approach to plant health.

Publications

  • Comer, J., Fletcher, J., Davis, R.E., & Melcher, U. (2007) Evolution of the Spiroplasma P58 multigene family. Biochem. Genet., 45 (1):25-32.


Progress 10/01/05 to 09/30/06

Outputs
A complete picture of virus evolution requires knowledge of the diversity of existing viruses. The laboratory is contributing to this knowledge in several ways. In a major project, over 500 plant specimens representing a large diversity of plants present in the Tallgrass Prairie Preserve (TPP) are being processed for analysis of virus content. In one approach, a subcellular fraction that should contain virus-like particles is prepared and the nucleic acid extracted from it. That nucleic acid is amplified, cloned in plasmid vectors, and the inserts sequenced. For the first five plants, three were identified as containing sequences of recognizable viruses, two tymoviruses and one chrysovirus. One of the tymoviruses appeared to be present in all three plants (a monocot, a dicot and a fern). These results indicate that most TPP plant viruses have not previously been characterized by nucleotide sequencing, and that most virus infections of plants are inapparent. The technique also revealed the presence of fungal and bacterial organisms in the samples, mostly derived from copurification of the ribosomes of these organisms. The presence of viruses, fungi and bacteria associated with the same healthy appearing plant suggests complex interorganismal interactions that maintain a balance beneficial to all. To complement the virus-like particle approach, the laboratory is refining microarray methods in which oligonucleotides specific to conserved regions of known viral families are spotted on glass slides to be used as probes of fluorescently labeled DNA obtained by amplification from the total nucleic acid extract of the plant samples. A second area of research is the interaction of plectroviral genomes with the bacterial host genomes of <i>Spiroplasma citri</i> and <i>kunkelii</i>. This research is facilitated by the availability of near complete genome sequences of both species of spiroplasma. Large proportions of the genomes (12.9% and 17.5%, respectively, for <i>S. citri</> and </>S. kunkelii</>) are made up of sequences derived from at least four plectroviruses. The junctions between viral and non-viral sequences are being identified and characterized. In the majority of cases the circular DNA genome of each of the four viruses has been interrupted at a specific position. For two of the viruses, a 21 or 26 nt direct repeat of viral DNA occurs at each end of the insertion. A considerable number of solo direct repeats, where non-viral sequences flanked both sides were found. For the other two, the interruption occurs in the middle of an inverted repeat sequence. Some of the junctions between viral and host DNA were identical in the two bacterial species (19% of <i>S. kunkelii</i> and 30% of <i>S. citri </i> junctions) suggesting that these resulted from insertions prior to speciation of the hosts. Because of the large number of viral insertions in these genomes and because of their apparent varying lengths, the use of viral sequences for the design of oligonucleotide primers for PCR discrimination among isolates of both spiroplasma species has been investigated. A few such primers exhibit a limited degree of polymorphism.

Impacts
Expertise developed in this laboratory on evolution of viruses and procaryotes is being used to examine pathogen variability in corn stunt disease in maize and citrus stubborn disease in citrus. It is also being applied to microbial forensics. How can a bioterrorism agent be attributed to an individual or group perpetrator? Answers require understanding of variation and the rates of evolution. They also require knowledge of the background of viruses in the world to exclude a natural cause for an outbreak. The analysis of the complexity of microbes in natural plants may lead to a holistic approach to plant health.

Publications

  • Comer, J., Fletcher, J., Davis, R.E., & Melcher, U. 2006. Evolution of the Spiroplasma P58 multigene family. Biochem. Genet., in press (expected Sept.)
  • Wren, J. D., Roossinck, M. J., Nelson, R. S., Scheets, K., Palmer, M. W. & Melcher, U. 2006. Plant virus biodiversity and ecology. PLoS Biology 4(3):e80.


Progress 10/01/04 to 09/30/05

Outputs
A complete picture of virus evolution requires knowledge of the diversity of existing viruses. The laboratory is contributing to this knowledge in several ways. DNA microarray analysis, in which short oligonucleotides representing relatively conserved regions of viral genomes are deposited on a glass slide and asked to bind fluorescently labeled DNA molecules derived from samples, is being adapted to a survey of plant viruses. Over 1200 oligonucleotide sequences capable of recognizing viruses from all viruses associated with plants and fungi have been designed. Methods are being developed to produce labeled targets from nucleic acid extracts of plants. Methods are under development to obtain viral nucleic acid molecular clones from subcellular fractions of plant homogenates. Over 500 plant specimens representing a large diversity of plants present in the Tallgrass Prairie Preserve are being processed for analysis. A database has been designed and implemented to handle this data. To improve the quality of data in presently public databases, viral entries in GenBank/DDBJ/EMBL without information about the isolates were identified and authors asked to provide data for entry into the database of the International Committee for the Taxonomy of Viruses. The laboratory maintains a database of oligonucleotides used in virus detection. Collaboration with a text-mining expert has resulted in the ability to recover oligonucleotide sequences from electronic versions of journal articles, a capability that will greatly accelerate data entry. Tests were performed of the hypothesis that coat proteins of both major types of helical virions, rod-shaped and filamentous, have a common ancestor. Nucleocapsids of envelope viruses may also have such structures for their subunits. Adding to models made in previous years, the laboratory explored representatives of these groups. The rigid rod viruses were represented by the Furoviruses and the Hordeiviruses, the flexuous by the Closteroviruses and the Potyviridae, and the nucleocapsid by the Filoviruses, Rhabdoviridae, and the Paramyxoviridae. Molecular modeling based on secondary structure prediction-informed amino acid sequence alignments resulted in models of coat protein structure for these groups. All but some nucleocapsid proteins failed to be modeled as four-helix bundles. The significance of these observations is under exploration. Viruses played a role in the evolution of Spiroplasma citri and kunkelii, plant pathogenic mollicutes. The complete nucleotide sequence of pBJS, a plasmid of S. citri was completed and compared to that of a similar plasmid from S. kunkelii. A short virus-derived sequence is in homologous positions in both plasmids. About 80% of the sequences of the two plasmids are highly similar. The remaining 20% shares substantially lower levels of similarity, suggesting recombination events. Recombination events were also implicated in evolution of P58-like genes in S. citri. Two genes of the family were previously sequenced. The third gene was obtained by polymerase chain reaction and sequenced. Comparison of these genes with each other and seven such genes in S. kunkelii is nearing completion.

Impacts
Expertise developed in this laboratory on evolution of viruses and procaryotes is being used to consider aspects of microbial forensics. How can a bioterrorism agent be attributed to an individual or group perpetrator? Answers require understanding of variation and the rates of evolution. They also require knowledge of the background of viruses in the world to exclude a natural cause for an outbreak.

Publications

  • Joshi, B. D., Berg, M., Rogers, J. Fletcher, J., & Melcher, U. (2005). Sequence comparison of plasmids pBJS-O of Spiroplasma citri and pSKU146 of S. kunkelii: implications for plasmid evolution. BMC Genomics 6:175.
  • J. Fletcher, C. Bender, B. Budowle, W. T. Cobb, S. E. Gold, C. A. Ishimaru, D. Luster, U. Melcher, R. Murch; H. Scherm, R. C. Seem, J. L. Sherwood, B. W. Sobral, & S.A. Tolin (2006). Plant pathogen forensics: capabilities, needs and recommendations. MMBR, submitted.
  • Wren, J. D., W. H. Hildebrand, S. Chandrasekaran, U. Melcher (2005). Markov Model recognition and classification of DNA/protein sequence patterns within large text databases. Bioinformatics 21:4046-4053.
  • Zhang, Q., U. Melcher, L. Zhou, F. Z. Najar, B.A. Roe, & J. Fletcher. (2005). Genomic comparison of plant pathogenic and non-pathogenic Serratia marcescens strains using suppressive subtractive hybridization. Appl. Environ. Micro. 71:7716-7723.
  • Te, J., U. Melcher, A. Howard, & J. Verchot-Lubicz. (2005). Soilborne wheat mosaic virus (SBWMV) 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing. Virology J. 2:18.
  • Fletcher, J., Melcher, U., & Wayadande, A. (2005) The phytopathogenic spiroplasmas. In "The Prokaryotes" (ed. M. Dworkin), in press.


Progress 10/01/03 to 09/30/04

Outputs
A complete picture of virus evolution requires knowledge of the diversity of existing viruses. To facilitate detection and identification of all viruses in environmental (or other) samples, we have turned to hybridization to oligonucleotide microarrays. Our previous efforts were extended to evaluate plant virus-specific oligonucleotides on several solid supports. Hybridization of these probes to targets derived from turnip vein-clearing and cauliflower mosaic viruses identified a polycarbodiimide slide as the best type of support and revealed more consistent results, with good recall and precision, than experienced with influenza viruses. As an adjunct to oligonucleotide use in virus detection, our VirOligo database has undergone the final phase of a major revision. The phase includes facilitating links from NCBI resources, notification of authors of articles that information from their article has been entered, and an improved curation process. Preliminary experiments of automatic data mining have yielded promising results. Monitoring of links made from NCBI to VirOligo reveal a steady approximately 250 contacts per month, indicating that a useful service is being provided. The focus on evolution concentrated on two plant virus proteins (movement (MP) and capsid (CP) proteins) and two of mollicutes. A phylogenetic analysis of MPs related to those of furoviruses revealed that the rate of divergence in all lineages of this subset of the 30K superfamily was very similar. It also revealed that the rate of appearance of new lineages was biphasic. Relative to CP, the hypothesis is that CPs of both major types of helical virions, rod-shaped and filamentous, have a common ancestor. Molecular modeling based on secondary structure prediction-informed amino acid sequence alignments resulted in models of CP for a tobravirus (rod-shaped), a potexvirus and a carlavirus (filamentous). The models, based on known structures of tobamoviruses, were consistent with several known properties of these viruses. All contained a core bundle of four helices. A ring of aromatic residues characteristic of the outer end of the tobamovirus bundles was also found in the CP of the obravirus CP. In the CP of filamentous virions instead, the aromatic residues were concentrated on the inner end of the bundles. Viruses played a role in the evolution of Spiroplasma citri, a plant pathogenic mollicute. The complete nucleotide sequence of pBJS, a plasmid of S. citri, has been completed. It has genes for SARP1, a protein implicated in adherence to cells of its insect vector, for proteins involved in plasmid partition and mobilization and for some as yet unknown functions. A small region with similarity to the sequence of SpV1 phage hints at a role for viruses also in the evolution of this plasmid. P58A is a S. citri protein first identified as also potentially involved in insect transmission. Its gene is a member of a multigene family. The sequences of these genes and their proteins have undergone recombination in their evolution and may have an origin in bacteriophage terminase genes.

Impacts
Knowing when and where viruses can be found is key to knowing how they will evolve. Hybridization to oligonucleotides obtained from the VirOligo database provides a method for finding and identifying viruses. Understanding capsid protein structure may provide means of intervening in the assembly of viral particles and inhibiting spread of infection. Spiroplasmas serve as models for the effects of viruses on host genomes. Understanding viral evolution will lead to better prediction of future viral disease outbreaks.

Publications

  • Fletcher, J., Melcher, U., & Wayadande, A. (2005) The phytopathogenic spiroplasmas. In The Prokaryotes (ed. M. Dworkin), in press
  • Onodera, K., & Melcher, U. (2004) Selection for 3 end triplets for polymerase chain reaction primers. Mol. Cell. Probes 18(6), 369-372.
  • Pair, S.D., Bruton, B. D., Mitchell, F., Fletcher J., Wayadande, A. & Melcher, U. (2004) Overwintering squash bugs harbor and transmit the causal agent of cucurbit yellow vine disease. J. Econ. Entomol. 97(1): 74-78.


Progress 10/01/02 to 09/30/03

Outputs
A complete picture of virus evolution requires knowledge of the diversity of existing viruses. To facilitate detection and identification of all viruses in environmental (or other) samples, we devised a virus signature hybridization chip. Overall, the results, previously reported, demonstrate positively the potential of microarray-based oligonucleotide hybridization for multiple virus detection. Recently, improvements to the statistical analysis and the ability to discriminate positive from negative reactions were made. VirOligo is a database that contains oligonucleotides reported to be specific for the detection of specific viruses. This database had consisted of two tables, one for oligonucleotides and the other for information about the article and the conditions of reaction used. VirOligo was enhanced by dividing the information into more tables: for the article, the virus target sequence, the conditions of reaction, and the oligonucleotide. Since the entry interface has been revised, new entries are being made into the new structure. The retrieval interface is under development. Meanwhile, oligonucleotides used in microarray analysis have been added, as have oligonucleotides specific for the SARS coronavirus. A colaboration has been initiated to explore the possibilities of automatic entry via text mining algorithms. Earlier work had identified size variants of cauliflower mosaic virus capsid proteins in isolated virions. Since degraded products were not found in isolated inclusion bodies, we concluded that degradation occurs during isolation. A survey of available preparations was undertaken to identify those with size variants. Size variants were examined by MALDI-TOF to determine whether the method could suggest the cleavage points. Fewer variants were found than expected. We had shown that viral DNA strands are asymmetric in base composition, in contrast to strands of bacterial DNA. We analyzed the correlation of triplet frequencies in bacterial genomes with each other and with base composition. We detected high correlations among triplets that are members of the same quartet group and among triplets with extreme %GC contents. Poorly correlated with frequencies of other triplets were those for GAT and ATC. Correlations of frequencies for AAA and GGG with %GC content were, respectively, higher and lower than expected. %GC content may be the primary factor determining triplet frequency, but a few triplets, probably having special functions, exhibit anomalous behavior. Viruses have played a large role in the evolution of spiroplasma genomes. We had previously established that pBJS is a novel Spiroplasma citri plasmid that encodes the possible adhesion related protein SARP1, and shows limited conservation among spiroplasmas and other mollicutes. No clear association of pBJS with insect transmissibility or plant pathogenicity was revealed. Except for arp1 and a few other open reading frames (ORFs), most of the pBJS sequence was so far uncharacterized. Hence, current efforts were focused on sequencing and characterizing the unknown portion of pBJS. This, almost complete, is being accomplished by primer walking.

Impacts
Key to knowing how viruses will evolve is knowing when and where they can be found. Hybridization to oligonucleotides obtained from the VirOligo database provides a method for finding and identifying viruses. Some oligonucleotides are conserved signatures due to roles in fundamental processes. Spiroplasmas serve as models for the effects of viruses on host genomes. Understanding viral evolution will lead to better prediction of future viral disease outbreaks.

Publications

  • An, H., Melcher, U., Doss, P., Payton, M., Guenzi, A. C. & Verchot-Lubicz, J. (2003). Evidence the Soilborne wheat mosaic virus 37K protein is a viral movement protein. J. Gen. Virol. 84, 3153-3163.
  • Sengupta, S., Onodera, K., Lai, A. & Melcher, U. (2003) Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization. J. Clin. Micro. 41(10), 4542-4550.
  • Rascoe, J.E., Berg, M., Melcher, U., Mitchell, F.L., Bruton, B.D., Pair, S.D. & Fletcher, J. (2003) Identification, phylogenetic analysis and biological characterization of strains of Serratia marcescens causing cucurbit yellow vine disease. Phytopathology 93(10), 1233-1239.
  • Zhang, Q., Weyant, R., Melcher, U., Bruton, B. D., Pair, S. D., Mitchell, F., & J. Fletcher (2003) Genotyping of Serratia marcescens strains associated with cucurbit yellow vine disease. Phytopathology 93(10), 1240-1246.
  • Melcher, U. (2003) Turnip vein-clearing virus, from pathogen to host expression profile. Molec. Plant Pathol. 4 (3), 133-140.
  • Bruton, B. D., Mitchell, F., Fletcher, J., Pair, S.D., Wayadande, A., Melcher, U., Brady, J., Bextine, B. & Popham, T. H. (2003) Serratia marcescens, a phloem-colonizing, squash bug-transmitted bacterium: causal agent of cucurbit yellow vine disease. Plant Disease, 87(8) 937-944.


Progress 10/01/01 to 09/30/02

Outputs
A complete picture of virus evolution requires knowledge of the diversity of existing viruses. To facilitate detection and identification of all viruses in environmental (or other) samples, we devised a virus signature hybridization chip. A collection of 480 influenza virus-specific oligonucleotides were spotted on glass slides as probes. Viral RNAs, extracted from test influenza A viruses, were reverse transcribed, amplified by PCR and labeled with cyanine dyes. The presence of influenza virus and its identification was determined by hybridization. Intensities of probe spots known to be completely complementary to target sequences varied from background to saturation without any clear correlation with base composition, nucleotide sequence, or potential internal secondary structures. Nevertheless, normalized intensities of specific oligonucleotides correctly identified the test viruses as influenza A viruses and identified their HA subtypes. Overall, the results demonstrate positively the potential of microarray-based oligonucleotide hybridization for multiple virus detection. Most oligonucleotides spotted on the microarray were obtained from VirOligo, a database created to contain oligonucleotides reported in the literature to be specific for the detection of specific viruses. The database now contains complete coverage for 22 viruses. The database contains information on the oligonucleotides themselves and on the conditions that were employed for their use in virus detection. It has links to sequence entries, literature references and taxonomic virus descriptions in other databases. Computational analysis suggested that only certain sequences could function to signal read through of termination during translation of viral genes. The ability of various sequences surrounding read through stop codons to program readthrough in mammalian cells was assessed. The assay used a construct in which ORFs for two distinguishable luciferases were separated by a potential read-through sequence. Intriguing differences in the ability of oligonucleotides altered at single positions to program read-through were noted. Whether these differences were a characteristic of the organism used or were generally true for eukaryotes was addressed using tobacco protoplasts. Assays for the two luciferases revealed that the pattern of read through propensities mirrored those found with the mammalian cells closely. Viruses have played a large role in the evolution of spiroplasma genomes. The near complete genome sequence of Spiroplasma kunkelii revealed the presence of about 100 dispersed sequence elements derived from SpV1 viruses, accounting for 18% of the genome sequence. This and another laboratory have focused on genes for two proteins, P58 and SARP1, that may be involved in the transmission of S. citri by insects. The genes for both proteins are near potentially mobile sequences. Nucleic acid hybridization of the distribution of the genes in related organisms reveals that the genes have a limited distribution, consistent with their mobility and their plant pathogenic role.

Impacts
Key to knowing how viruses will evolve is knowing when and where they can be found. Hybridization to oligonucleotides obtained from the VirOligo database provides a method for finding and identifying viruses. Some oligonucleotides are conserved signatures due to roles in fundamental processes. Spiroplasmas serve as models for the effects of viruses on host genomes. Understanding viral evolution will lead to better prediction of future viral disease outbreaks.

Publications

  • Melcher, U. (2002) Viruses and Viroids. In Magill's Encyclopedia of Science: Plant Life, Salem Press, Pasadena CA, pp. 1045-1047
  • Harrell, L., Melcher, U., & Atkins, J. F. (2002). Predominance of six different hexanucleotide recoding signals 3' of read-through stop codons. Nucleic Acids Res. 30(9), 2011-2017.
  • Hii, G. Pennington, R., Hartson, S., Taylor, C.D., Lartey, R., Williams, A., Lewis, D.,& Melcher, U. (2002) Isolate-specific synergy in disease symptoms between cauliflower mosaic and turnip vein-clearing viruses. Arch. Virol., 147, 1371-1384.
  • Onodera, K. & Melcher, U. (2002) VirOligo: a database of virus-specific oligonucleotides. Nucleic Acids Res. 30(1), 203-204.
  • Onodera, K., d'Offay, J., & Melcher, U. (2002). Nylon-membrane immobilized PCR for detection of bovine viruses. BioTechniques, 32(1), 74-80.


Progress 10/01/00 to 09/30/01

Outputs
A complete picture of virus evolution requires knowledge of the diversity of existing viruses. To facilitate detection and identification of all viruses in environmental (or other) samples, the laboratory is devising a virus signature hybridization chip. When completed, this chip will contain spots of oligonucleotides complementary to signature sequences of the genomes of known viral genera so that a labeled nucleic acid derived from the sample will be bound by hybridization to only those spots whose oligonucleotides have complements represented in the sample. Three efforts related to this goal have been initiated. First, the VirOligo databse was created to contain oligonucleotides reported in the literature to be specific for the detection of specific viruses. Currently, the database contains viruses implicated in bovine respiratory disease and influenza virus. The database contains information on the oligonucleotides themselves and on the conditions that were employed for their use in virus detection. It has links to sequence entries, literature references and taxonomic virus descriptions in other databases. Second, a project to validate the chip approach by attempting to detect and discriminate among influenza virus subtypes has been initiated. Technical hurdles are being addressed. Third, to obtain experience and knowledge in microbial detection strategies, the laboratory has participated with others in design of new methods for detection of food-borne bacterial pathogens. A variety of viruses use the partial suppression of translation termination codons as essential parts of their gene expression strategy. To determine whether this strategy evolved once or multiple times during viral evolution, sequences surrounding the suppressible termination codons (read-through codons) of all known examples of this strategy were analyzed. The sequences fell into six groups based on the six nucleotides following the read-through codon. Some related viruses appeared in different groups, suggesting multiple origins for the read-through strategy. The analysis led to predictions of the effects of nucleotide alterations on read-through that are currently being tested. The findings also shed light on the mechanisms of termination suppression. Viruses have played a large role in the evolution of spiroplasma genomes. In cooperation with another laboratory, focus has been on the genes for two proteins, P58 and SARP1, that may be involved in the transmission of Spiroplasma citri, a plant pathogen, by insects. The genes for both proteins are near potentially mobile sequences. An ongoing study by nucleic acid hybridization of the distribution of the genes in related organisms reveals that the genes have a limited distribution, consistent with their mobility and their plant pathogenic role. An insect transmissible spiroplasma line contains two additional P58-like genes. Their ongoing characterization shows that P58-like proteins are related to bacteriophage-encoded terminases. To better understand the relation of viral sequences to the spiroplasma genome, viral sequences in the S. kunkelii genome have been compiled and are being analyzed.

Impacts
A combination of sequence comparisons and experimental studies are being used to examine how viruses evolve. A VirOligo database will provide a key resource for virus detection for many fields. Factors examined included interactions with their hosts and strategies used for gene expression. Understanding viral evolution will lead to better prediction and handling of future viral disease outbreaks.

Publications

  • Berg, M., Melcher, U., & Fletcher, J. (2001). Molecular characterization of the gene encoding P89 (SARP1), an adhesion related protein of Spiroplasma citri. Gene 275(1), 57-64.
  • de los Reyes, B. G., Taliaferro, C. M., Anderson, M. P., Anderson, J. A., Melcher, U., & McMaugh, S. (2001) Induced expression of class II chitinase during cold acclimation and dehydration of bermudagrass (Cynodon sp.). Theor. Appl. Genet., 103(2/3), 297-306.


Progress 10/01/99 to 09/30/00

Outputs
Because viruses are obligate intracellular parasites, their evolution must be influenced by interaction with their hosts. The reverse transcriptase of cauliflower mosaic virus (CaMV) was tested for interaction with host hsp90 proteins, an interaction seen in hepadnavirus infection. None was found. Post-transcriptional gene silencing (PTGS) is an RNA destruction mechanism of the host that may protect the host against viruses or may lead to host disease symptoms on triggering by viruses. Recognition of 21-23 long oligonucleotide RNAs is required. A survey of viral and host genomes revealed no excess or deficit over expectation of virus and host-common oligonucleotides of these lengths or longer, suggesting that PTGS has not played a great evolutionary role in shaping viral sequences. To identify genes changes in whose expression are early events in infections it would be ideal to compare two Arabidopsis thaliana infecting viruses, only one of which causes symptoms. Experiments demonstrated that turnip vein-clearing and tobacco mosaic tobamoviruses (TVCV and TMV) were not suitable since the time courses of their systemic infection differed markedly. Viruses also interact with one another when resident in the same host. Such interaction can lead to recombination, which sequence analysis suggested occurred frequently during propagation of a line of rice tungro bacilliform virus. The competitive ability of a recombinant tobamovirus was observed to increase as a result of passage in tobacco at high multiplicity of infection, whereas it decreased after a series of bottleneck local lesion transfers. Experiments also addressed the gene region of CaMV DNA that was required for a synergistic interaction with TVCV in turnips that resulted in severely diseased plants. Virion stability should govern longevity in the environment and thus influence evolution. Differences in tobamovirus virion stabilities were analyzed by investigating the accessibility of the RNA in particles to RT-PCR. The finding that the genome of the plectrovirus SVTS2 was only half homologous to those of other plectroviruses raised new questions about plectrovirus evolution. Sequence comparisons on the 30K superfamily of plant virus movement proteins were completed and defined a group of proteins related in sequence and, presumably, in evolutionary origin. Diverse groups of viruses use suppression of translation termination codons as part of their gene expression strategies. Signals around these suppressible stop codons were classified in four groups. The groups bore no relation to the taxonomic affinities of the viruses, suggesting multiple events of evolution of termination codon suppression. Understanding viral evolution requires knowledge of what viruses are present in the environment. Two techniques were explored that may provide that knowledge. ViSA cards amplify by PCR pairs of primers immobilized on a solid support, allowing amplification only at the location of the primer pair spot. ViSH chips are hybridization probes consisting of virus-specific oligonucleotide arrays. Technical improvements in both techniques were made.

Impacts
A combination of sequence comparisons and experimental studies were used to examine how viruses evolve. Factors examined included interactions with their hosts, interactions with other viruses, and stability of the particles. Understanding viral evolution should lead to better abilities to predict and handle future viral disease outbreaks.

Publications

  • Melcher, U. 2000 Historical importance of TMV. Trends in Plant Science, 5(6), 268.
  • Melcher, U. 2000 The 30K superfamily of viral movement proteins. J. Gen. Virol., 81, 257-266.
  • Sha, Y., Melcher, U., Davis, R. E., & Fletcher, J. 2000 Common elements of Spiroplasma plectroviruses revealed by nucleotide sequence of SVTS2. Virus Genes, 20 (1), 47-56.
  • Bost, S. C., Mitchell, F., Melcher, U., Pair, S. D., Fletcher, J., Wayadande, A., & Bruton, B. D. 1999 Yellow vine of watermelon and pumpkin in Tennessee. Plant Disease, 83 (6), 587.
  • Cabauatan, P. Q., Melcher, U., Ishikawa, K., Omura, T., Hibino, H., Koganezawa, H., & Azzam, O. 1999 Sequence changes in six variants of rice tungro bacilliform virus and their phylogenetic relationships. J. Gen. Virol. 80 (8) 2229-2237.


Progress 10/01/98 to 09/30/99

Outputs
Understanding virus evolution requires knowledge of what viruses are present in the environment. Two techniques are explored that may provide that knowledge. Bridge PCR is an amplification technique in which pairs of primers are immobilized on a solid support allowing amplification only at the location of the primer pair spot. As a result, one assay with a large array of spots should be able to determine which viruses are present and which are absent in each sample. Use of bridge PCR with primer arrays requires that the primer attachment to the solid support be stable to the denaturing temperatures used in PCR. Since amino-linked oligonucleotides bound to silylated glass are not stably attached, we explored the use of nylon membranes as solid supports. As test material we examined the specificity of detection of bovine viral diarrhea virus (BVDV) and bovine herpes virus (BHV). Nylon membranes with two spots of UV-immobilized primer pairs, one for BVDV-1 and one for BHV-2 were used in amplification with both corresponding templates, with each template singly and with no template. When amplification was detected by chemiluminescent detection of incorporated DIG-nucleotides, the expected amplification patterns were obtained. Hybridization of a labeled probe derived from an environmental sample to an array of oligonucleotides representing signatures of known viruses is a second approach. This approach was demonstrated to be successful with the above viruses. One determinant of evolutionary success of a virus is its stability in the environment. To understand determinants of the stability of viral particles, we determined whether RT-PCR can be used as a direct estimator of thermal stability. Tobacco mosaic virus (TMV) and turnip vein-clearing virus (TVCV) each required a 50 to 65 C treatment to make their RNA genomes available for accurate RT-PCR. Analysis of aberrant products formed at lower temperature revealed that the TVCV origin of virion assembly was unavailable for amplification. Prior heat treatment was not needed for success with a recombinant tobamovirus, TVCVrMP. The distinguishing feature of this virus was the presence of two origins of assembly. The results suggest that the origin of assembly is an important contributor to virion stability. Examination of genomic sequences of Spiroplasma citri revealed important roles for integrated viral sequences in the evolution of the host genome. Additional work on virus evolution is reported in OKL02302.

Impacts
Successful development of a microarray-based assay for the detection of viral sequences could lead to a new level of understanding of the distribution of viruses in the environment, to the identification of new viruses related to known viruses, and to the development of rapid diagnostic tools for plant, animal and human medicine.

Publications

  • Melcher, U., Sha, Y., Ye, F., & Fletcher, J. (1999). Mechanisms of Spiroplasma genome variation associated with SpV1-like viral DNA's inferred from sequence comparisons. Micro. Comp. Genomics, 4 (1), 29-46.
  • Melcher, U., & Fletcher, J. (1999). Sources of genetic variation in Spiroplasma citri. Eur. J. Plant Pathol. 105(6) 519-533.


Progress 10/01/97 to 09/30/98

Outputs
Interaction between viruses coinfecting a plant influences their evolution in many ways. The synergistic effect on symptom severity of coinfection with cauliflower mosaic virus (CaMV) and turnip vein-clearing virus (TVCV) correlated with increases in the level of TVCV caused by the presence of CaMV. In contrast, the level of CaMV was not affected by the presence of TVCV. Related viral genomes residing in the same host interact by recombination. This was illustrated by the detection of sections of rice tungro bacilliform virus genomes that originated from other genomes of the same virus. New methods to explore the molecular phylogeny of viruses at the suprageneric level were developed. An iterative alternation of sequence alignment and parsimony analysis was applied to consensus sequences of 30K superfamily movement proteins. The resulting alignment revealed conserved elements of secondary structure and placed movement proteins known to form tubules on the same branch of a tree of relatedness. It also allowed the assignment of proteins encoded by viruses of several other genera to this superfamily. The known three-dimensional structure of the RNA-dependent RNA polymerase encoded by poliovirus RNA was used to align the sequences of these enzymes encoded by several genera of plant viruses with the aim of improving phylogenetic analysis based on these highly conserved sequences. Evolution of the Inoviridae was also explored with the completion of the nucleotide sequence determination of SVTS2, a plectrovirus obtained from Spiroplasma melliferum. The sequence reveals a 4 knt section homologous to other sequenced plectroviruses and a 3 knt unique section. The sequences had diverged too far from those of inoviruses to assign homologous regions.

Impacts
(N/A)

Publications

  • Sha, Y., Melcher, U., Davis, R. E., & Fletcher, J. (1998) SpV1-like Spiroplasma virus essential features suggested by SVTS2 DNA sequence. Phytopathology, 88 , S80.


Progress 10/01/96 to 09/30/97

Outputs
Phylogenetic analysis of protein sequences encoded by RNAs of tobamoviruses revealed three subgroups. For odontoglossum ringspot virus, phylogenetic trees for three domains suggested a different origin for this virus than was suggested by two others. Recombination must have occurred to generate the precursor to this virus. Recombination as a contributing mechanism to evolution is only feasible when two viruses infect the same plant. Thus, influences of one virus on another were studied. The movement protein (MP) gene of turnip vein-clearing virus (TVCV), as a transgene, did not complement movement of cauliflower mosaic virus (CaMV) in rape plants. However, CaMV did complement both local and systemic movement of TVCV defective in its MP function in turnips and Arabidopsis. In addition, in Arabidopsis mutants of TVCV and tobacco mosaic virus (TMV) defective in MP due to a frameshift in the MP gene established local, but not systemic infections. TMV does not infect turnips. The limitation of TMV infection of turnips is not due to its MP since TVCV with its MP gene replaced by that of TMV efficiently infected turnips systemically. TVCV exacerbated symptoms in turnips caused by the Cabbage S isolate of CaMV, but not those caused by other isolates. Tests with CaMV chimeras revealed that the coat protein gene was probably responsible for the symptom synergy. CaMV increased the replication level of TVCV. The effect was greater with the Cabbage S isolate.

Impacts
(N/A)

Publications

  • MELCHER, U. (1996). Inferring viral evolution by phylogenetic analysis. In S. G. Pandalai (Ed.), Recent Research Developments in Plant Pathology (pp. 101-111). Trivandrum, India: Research Signpost.
  • LARTEY, R. T., VOSS, T. C., & MELCHER, U. (1997). Completion of a cDNA sequence of a tobamovirus pathogenic to crucifers. Gene, 188
  • YE, F., MELCHER, U., WAYADANDE, A. C., & FLETCHER, J. (1997). Molecular characterization of a gene encoding a membrane protein of Spiroplasma citri. Gene, 189 (1), 95-100.


Progress 10/01/95 to 09/30/96

Outputs
Comparisons of tobamoviral protein sequences revealed three clusters: viruses infecting solanaceous species (subgroup 1), cucurbits and legumes (subgroup 2) and crucifers (subgroup 3). The orchid-infecting odontoglossum ringspot virus was subgroup 1 by its coat and movement protein (CP and MP) sequences, but subgroup 3 by the remainder, suggesting that it is the progeny of a recombinant. For 4 of 5 genomic regions, subgroup 1 and 3 were equidistant from a subgroup 2 genome suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences, but had a different root, suggesting codivergence tempered by rare colonizations of new plant families.Phylogenetic analysis of the overlap between MP and CP genes was consistent with a model in which a no or short overlap organization was ancestral. Understanding the importance of recombination between unrelated viruses requires knowledge of interchangability of genes between viruses. Cauliflower mosaic virus (CaMV) facilitated the intercellular and long distance movement in turnips of turnip vein-clearing tobamoviruses (TVCV) defective in MP genes. Transviral complementation of movement was also observed when the CaMV MP gene replaced the TVCV MP gene. Between related viruses, such as TVCV and TMV, recombinational exchanges resulted in less virulent genomes. The 3' non-translated region was an important determinant of lowered virulence.

Impacts
(N/A)

Publications

  • Williams, A., Chenault, K.D., and Melcher, U. 1995. Graphic representations of amino acid sequences. In "Visualizing Biological Information" (C.A. Pickover, Ed.), pp. 6-14. World Scientific, River Edge NJ.
  • Lartey, R.T., Voss, T.C. and Melcher, U. 1996. Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications. Mol. Biol. Evol. 13: 1327-1338.
  • Melcher, U. 1996. Overturned 'truths'. The Scientist 10 (19), 12.
  • Melcher, U. 1996. Recombination in the evolution of tobamoviruses. Phytopathology 86. (Suppl.), S18.
  • Ye, F., Melcher, U., and Fletcher, J. 1996. Spiroplasma citri BR3 encodes a protein with homology to mycoplasma adhisins. Phytopathology 86 (Suppl.), S96.
  • Ye, F., Melcher, U., Rascoe, J.E., and Fletcher, J. 1996. Extensive chromsome aberrations in Spiroplasma citri strain BR3. Biochem. Genet. 34: 269-286.
  • Lartey, R.T., Voss, T.C., and Melcher, U. 1995. Completion of a cDNA sequence of a tobamovirus pathogenic to crucifers. Gene 166: 331-332.


Progress 10/01/94 to 09/30/95

Outputs
Understanding evolution of plant viral genomes was approached by analysis of movement protein structure-function and host specificity and evolution of gene organization in the subgroups of tobamoviruses. A third subgroup was proposed based on the 25 codon overlap previously identified. Secondary structure predictions were obtained for aligned sequences of movement protein sequences. These led to a model with a central region delimited by two helices with five to six beta-elements in between. The predicted common secondary structure regions improved the alignment of the central domain. TMV-TVCV chimeric tobamoviruses were constructed to identify genes that limited TMV infection of turnips. The P126/183 and the movement protein regions were found responsible for the limitation. Homogenates of turnip leaves inoculated with chimeric viruses in which TMV replicase or movement protein gene components were present did produce a small number of lesions when bioassayed on Xanthi tobacco. Thus, limited replication and movement of the chimeras occurred and the limitation may have been due to a host defense response. Virus movement may be easier to assess if the virus bears a tag that is easily recognized. Thus, derivatives of TMV and TVCV cDNA and of CaMV DNA were engineered to produce the jellyfish green fluorescent protein. Each of three cDNA plasmid constructs produced transcripts that were infectious to tobacco and produced lesions that fluoresced green. The stability of the GFP gene in these construc.

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    Progress 10/01/93 to 09/30/94

    Outputs
    A 25 codon overlap in the tobamovirus, turnip vein-clearing virus (TVCV), of genes for movement and coat proteins (MP and CP) is not found in tobamoviruses of other subgroups. The overlap is present in genomes of two other viruses related to TVCV. Analysis of the overlap regions of these genes established that the CP gene was the original gene. Nucleotides encoding the CP N-termini were inferred to have evolved to encode also the C-termini of the MPs. Phylogenetic analysis suggests that the overlap organization is derived from an ancestor with a smaller or no overlap. A full-length clone of TVCV cDNA has been assembled in a plasmid vector. RNA transcripts produced in vitro from this clone were infectious to tobacco and Arabidopsis thaliana. A chimeric transcribable plasmid clone was constructed that contains the 5' replicase genes of tobacco mosaic virus (TMV) and the 3' movement and CP genes of TVCV. This chimera infected tobacco (host for both viruses) but was unable to infect turnip (host for TVCV but not for TMV) suggesting that the limitation of TMV infection was not due to incompatibility of the TMV MP with turnip tissue. TMV genomes with part or all of the MP gene replaced with the analogous segments of the MP gene of cauliflower mosaic virus were tested. All infected MP-transgenic tobacco plants. Two chimeras underwent changes during propagation in tobacco.

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      Progress 10/01/92 to 09/30/93

      Outputs
      The nucleotide sequence of turnip vein-clearing virus (TVCV) RNA has been completely determined. It revealed four open reading frames. They corresponded to those previously identified in the RNAs of other tobamoviruses, except that the movement protein gene overlapped considerably with the coat protein gene. The predicted amino acid sequence of the TVCV coat protein and the phylogenetic position of the 126K component of the replicase suggested that TVCV was a strain of ribgrass mosaic virus. For many viruses, virus-encoded proteins responsible for intercellular movement of infection have similar amino acid sequences. The more conserved central regions of these sequences are similar to lentiviral protease sequences. Phylogenetic analysis of the central regions identified two groups. No correlation of phylogenetic position with movement mechanism (RNA-mediated or tubule-facilitated) or with polymerase supergroups was found, suggesting considerable shuffling of coding regions during virus evolution. The identity of residues at almost invariant positions was found by in vitro mutagenesis to be crucial for activity in movement. Adaptation of a virus to a new host plant may be a major factor in virus evolution, and movement proteins may be important participants in the adaptation. We exchanged parts of tobacco mosaic virus and cauliflower mosaic virus movement protein genes. In non-common hosts, the chimeras did not replicate.

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        Progress 10/01/91 to 09/30/92

        Outputs
        The sequence of turnip vein-clearing virus (TVCV) (1) RNA has been 65% determined. This sequence will help us explore the evolution of host specificity of viral infection of plants. Turnip and tobacco are hosts to TVCV, while only the latter is host for tobacco mosaic virus (TMV). Arabidopsis is a host for TMV (2) and TVCV. By probing skeletons of inoculated turnip leaves through hybridization (3), we established that TMV is restricted in intercellular movement in turnips (4)8 probably causing turnip resistance to TMV. Intercellular movement is mediated by the p30 protein encoded in TMV RNA. A similar p38 protein encoded by cauliflower mosaic virus (CaMV) was shown to serve a similar function by the detection in inoculated leaves of small quantities of encapsidated DNA mutant in the p38 gene. The movement proteins encoded by many viruses have similar amino acid sequences. The central regions are similar to those of lentiviral proteinases (5). This similarity has been used to assign movement function to unknown reading frames of newly sequenced viral genomes of badna-, tospo- and capilloviruses. TVCV is also of interest since with some CaMV isolates, but not others, it cooperates to produce a more severe stunting of the plants than caused by either virus alone (6). Comparisons of the sequences of CaMV isolates (7-9) revealed evidence of recombination events during evolution, evidence for two sequence groups of and an asymmetric substitution profile.

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          Progress 10/01/90 to 09/30/91

          Outputs
          We work to understand evolution of plant viruses by identifying nucleotides thatconfer selective advantages on their genome and by characterizing sources of sequence variation. We devised a structural model for viral movement proteins based on their amino acid sequence similarity with lentiviral proteinases. The model allows the identification of residues important to the function of the proteins. In this process, we further developed computer applications that assist in alignment of amino acid sequences and proposed a structurally informative display of sequences. The structural model identified genes for movement proteins in other virus groups. A new tobamovirus, turnip vein clearing virus (TVCV) was identified in turnips. Its comparison with tobacco mosaic virus (TMV) may identify sequences selected by the turnip host. In turnips, TVCV did not complement systemic movement of TMV nor of gene I mutants of cauliflower mosaic virus (CaMV). To characterize the relative importance of sources of variation, we completed the nucleotide sequence determination of the DNAs of three isolates of CaMV. Comparison of the known sequences of eight CaMV isolates will follow. Because CaMV replicates by reverse transcription, CaMV RNA templates are possible sources of variation. We found that no major RNAs other than 19S and 35S RNAs in turnip leaves of varying ages at two different times post inoculation and in protoplasts infected in vitro.

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            Progress 10/01/89 to 09/30/90

            Outputs
            A PCR technique to detect CaMV and TMV nucleic acids in infected tissue was successfully developed. Its use confirmed that CaMV adapts to replication in tobacco. A different TMV strain than that used in the PCR studies was a contaminant in turnips. Its discovery marks the first report of TMV infection of turnips and opens the way to host-specificity studies of both CaMV and TMV in each turnip and tobacco. Alkaline denaturation-renaturation of CaMV in the presence of labeled DNA preferentially bound single-stranded DNA, though double-stranded DNA (psoralen cross-linked) was bound weakly. DNA in complexes was partially exposed to DNAse I. The instabilities of extraneous DNA inserts in the large intergenic region were shown to be due frequently to recombination at direct repeats. The length of the deleted sequence was directly related to the length of the repeats, suggesting a copy-choice mechanism of deletion. Cross-protection, the prevention of infection of a plant by a CaMV isolate by previous infection with CaMV has been confirmed and also shown to occur at the cell level. Protoplasts from diseased plants were resistant to multiplication of the challenge virus. Alignment of amino acid sequences of plant viral proteins putatively involved in cell-to-cell transport has proceeded to over 30 sequences from about 10 groups. The proteins of three viral supergroups are included in the alignment. Macintosh versions of the program used have been written.

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              Progress 10/01/88 to 09/30/89

              Outputs
              Dialysis of alkali-denatured CaMV against a pH 7 buffer resulted in the incorporation of exogenous DNA into virus-like particles. At low total DNA concentrations, complexes incorporated both CaMV and unrelated DNAs. In the presence of calf thymus DNA, selective incorporation of restriction fragments into complexes allowed the identification of three regions of CaMV DNA containing sequence specifically recognized by the CaMV coat protein. A multiple amino acid sequence alignment strategy that allows the statistically significant alignment of groups of distantly related sequences was devised & used to find a previously unrecognized relationship between the 3a proteins of tripartite viruses and the ORFI proteins of caulimoviruses. We have modified CaMV DNA's large intergenic region to answer whether the intergenic region is dispensable and how nucleotide sequences are deleted. A variety of restriction fragments were inserted in the unique cloning sites of pCML1. Most, but not all, DNA inserts were unstable in plants. Nucleotide sequencing of the progeny identified RNA splicing and homologous recombination of two mechanisms contributing to the instability of inserts. Nucleotide sequencing of the DNA of the NY8153 isolate of CaMV is now 85% complete. A new isolate of CaMV has been obtained from a Pak Choi plant. Nucleotide sequencing of junctions of chimeras produced by in planta recombination has been completed.

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                Progress 10/01/87 to 09/30/88

                Outputs
                Phytoalexins formed in plant tissues in response to bacterial, fungal and viral infections are active in combatting bacterial and fungal infections. With M. Essenberg and J. Sun we have shown that the cotton phytoalexin, dihydroxycadalene, inactivates in a light-dependent reaction the infectivity of cauliflower mosaic virus (CaMV). The phytoalexin cross-links the viral coat proteins to the viral DNA and induces single strand breaks in double-stranded DNA. Only limited use has been made of the DNA of CaMV as a vehicle to carry extraneous DNA sequences into plant cells. One of the difficulties of using CaMV DNA vectors has been overcome by the discovery of a site between genes VI and I that tolerates modification. A vector with three unique restriction sites for cloning of extra DNA replicates in plants without loss of the added sites. Selective loss of one of two coinoculated variants of CaMV during replication in plants was found to be due to a better competitive ability of one of the variants. Evaluation of the effects of exchanging DNA segments between variants suggests that more than one genetic factor is responsible for this competition. Previous alignments of the amino acid sequences of viral proteins thought to be responsible for transport of the infection from cell to cell were subjectively arrived at. A more objective method has been devised and applied to the alignment of these sequences.

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                  Progress 10/01/86 to 09/30/87

                  Outputs
                  Amino acid sequence comparisons of virally encoded proteins probably responsiblefor cell-to-cell movement of infections suggested that caulimoviruses evolved at a much slower rate than other plant viruses. Statistically significant sequence similarity of caulimoviral ORFI proteins to similar proteins of several diverse plant viruses was detected while little significant similarity was found between other distantly related virus sequences. Allele dominance, the selective loss of one of two co-inoculated sequence alleles, required sequence identity on only one side of the allele and depended on the nature of the allelic sequence difference. These properties are consistent with a role for allele dominance in limiting evolutionary rates. The sequences of junctions between parental DNAs in chimeric viruses created by recombination in vivo suggest that recombination occurs by template switching during DNA synthesis and by homologous crossing over, mechanisms which can also contribute to a slower rate of evolution. To assist in the comparison of sequences, a novel representation of nucleotide sequences has been devised. The Puppy representation has allowed the identification of a potentially significant stem-loop structure in caulimoviral RNAs.

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                    Progress 10/01/85 to 09/30/86

                    Outputs
                    Mutations constructed by recombinant DNA techniques in each of the protein-coding regions of CaMV DNA except that for the aphid-transmission factor abolished the ability of the DNA to replicate or spread in mechnically inoculated turnip leaves. An analysis of the temporal and spatial distribution of a variety of symptoms caused by different CaMV isolates on turnip and Arabidopsis thaliana plants suggests that virus infection alters the expression of genes important for normal development. CaMV was shown to replicate and spread locally but not systemically in cotton leaves. A cotton phytoalexin inactivated the infectivity of CaMV to turnip, probably by cross-linking of protein to DNA. The selective loss of one of two coinoculated restriction site markers during growth in turnip was prevented by heterologies elsewhere in the DNA. The position of junctions between parental DNAs in chimeric viruses created by recombination in vivo were analyzed and have caused a reevaluation of current models of CaMV DNA replication. Interference with wild-type DNA infection by mutant CaMV DNA was documented. Preliminary determinations of the presence of CaMV DNA in progeny Arabidopsis thaliana from infected plants suggest that CaMV DNA integration in the host genome may occur, but not frequently.

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