Progress 10/30/02 to 03/13/07
Outputs
Progress Report Objectives (from AD-416) 1. Map genetic determinants of differential host range among strains of WSMV. 2. Map determinants of mite transmission encoded by the WSMV HC-Pro gene. 3. Determine the function of a conserved sequence element in the WSMV P3 gene. 4. Define patterns of nucleotide sequence diversity within a collection of WSMV isolates and determine evolutionary forces affecting diversity in WSMV. 5. Determine the frequency of intermolecular homologous recombination among closely related genotypes of WSMV in the absence of strong selection. Approach (from AD-416) Experiments will be conducted using a cloned cDNA copy of the wheat streak mosaic virus (WSMV) genome from which infectious viral RNA is generated in vitro and tested for biological activity in wheat and other cereal species. The infectious cDNA clone of WSMV will be modified to contain portions of the viral genome derived from different WSMV strains. The resulting chimeric, hybrid WSMV strains will be evaluated for strain- specific differences in host range to determine viral genes required for infection of wheat, corn, and other cereals. The infectious cDNA clone of WSMV also will be subjected to mutagenesis to determine specific domains of the WSMV HC-Pro gene involved in eriophyid mite transmission, and to determine the extent of a newly discovered cis-acting RNA element within the WSMV P3 gene that is required for systemic movement of WSMV within infected wheat plants. To assess the frequency of viral RNA recombination, the infectious clone of WSMV will be modified to contain non-functional GUS reporter gene fragments that will only be capable of expressing active GUS protein following a recombination event between two WSMV RNA molecules such that a complete GUS reporter gene is generated within the genome of the recombinant viral RNA. Evolutionary forces driving diversity and genetic variation of WSMV in the United States will be assessed by applying population genetics theory to the analysis of coat protein gene sequences obtained for a collection of over 50 U. S. isolates of WSMV. BL-2, February 14, 2002. Accomplishments Wheat streak mosaic virus HC-Pro gene is required for mite transmission of virus from plant to plant. The HC-Pro gene was shown to be required for plant to plant transmission of wheat streak mosaic virus by the wheat curl mite. Three cysteine amino acid residues near the beginning of the protein were shown to be essential for mite transmission. Previously, there was no information regarding viral encoded determinants of plant virus transmission by mites. This research may have potential impact on scientists developing models to explain the process of virus transmission. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases) and is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors). Technology Transfer Number of Active CRADAS and MTAS: 13
Impacts
(N/A)
Publications
- Robertson, N.L., French, R.C. 2007. Genetic analysis of a novel Alaska barley yellow dwarf virus in the family Luteoviridae. Archives of Virology. 152(2):369-382.
- Robertson, N.L., French, R.C. 2007. Genetic structure in natural populations of barley/cereal yellow dwarf virus isolates from Alaska. Archives of Virology. 152(5):892-902.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned with National Program 303 (Plant Diseases). Virus diseases of wheat are responsible for substantial and recurrent losses to American agriculture. This CRIS project represents the only ARS research project that focuses on virus diseases of wheat. Current emphasis of the project is on wheat streak mosaic virus, one of the most important viral disease agents of wheat. The research is organized into five objectives: (1) Map genetic determinants of differential host range among strains of wheat streak mosaic virus. (2) Map determinants of mite transmission encoded by the wheat streak mosaic virus HC-Pro gene. (3) Determine the function of a conserved sequence element in the wheat streak mosaic virus P3 gene. (4) Define patterns of nucleotide sequence diversity within
a collection of wheat streak mosaic virus isolates and determine evolutionary forces affecting diversity in wheat streak mosaic virus. (5) Determine the frequency of intermolecular homologous recombination among closely related genotypes of wheat streak mosaic virus. This research is relevant to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases). The project primarily addresses Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors) of National Program 303 (Plant Diseases). Improved understanding of the genetic basis of viral pathogenesis, gene function, vector transmission, RNA recombination, and population genetics will facilitate efforts to ameliorate the effects of viral infection in wheat and other cereal crops.
A major product of this research will be scientific knowledge available in the public domain as published papers in peer reviewed journals and nucleotide sequence data deposited in public databases. Initially, primary customers of the research will be ARS, University, and industry scientists striving to understand plant virus pathogenesis and develop new strategies for control. Ultimately, agricultural producers and consumers will benefit as this newly gained knowledge is applied to generate improved cultivars or cultural practices in which losses due to plant virus infection are reduced. 2. List by year the currently approved milestones (indicators of research progress) Objective 1. Map genetic determinants of differential host range among strains of wheat streak mosaic virus. Year 2 (FY2004) milestones, construct chimeric wheat streak mosaic virus genomes, initiate host range assays; Year 3 (FY2005) milestones, construct chimeric wheat streak mosaic virus genomes, conduct host
range assays, initiate fine mapping; Year 4 (FY2006) milestones, conduct host range assays, fine mapping; Year 5 (FY2007) milestones, conduct host range assays, fine mapping. Objective 2. Map determinants of mite transmission encoded by the wheat streak mosaic virus HC-Pro gene. Year 2 (FY2004) milestones, construct mutants, initiate infectivity assays; Year 3 (FY2005) milestones, construct mutants, conduct infectivity assays, initiate movement and transmission assays; Year 4 (FY2006) milestones, construct mutants, conduct movement and transmission assays, initiate proteinase and synergism assays; Year 5 (FY2007) milestones, complete various assays. Objective 3. Determine the function of a conserved sequence element in the wheat streak mosaic virus P3 gene. Year 1 (FY2003) milestones, construct mutants, initiate translation, infectivity, movement, replication assays; Year 2 (FY2004) milestones, fine mapping and complete various assays. Objective 4. Define patterns of nucleotide
sequence diversity within a collection of wheat streak mosaic virus isolates and determine evolutionary forces affecting diversity in wheat streak mosaic virus. Year 1 (FY2003) milestones, clone and sequence CP genes, initiate phylogenetic analysis; Year 2 (FY2004) milestones, complete phylogenetic analysis. Objective 5. Determine the frequency of intermolecular homologous recombination among closely related genotypes of wheat streak mosaic virus. Year 1 (FY2003) milestones, initiate recombination assays; Year 2 (FY2004) milestones, recombination assays, alter target length; Year 3 (FY2005) revised milestone, initiate statistical analysis of homoplasy; Year 4 (FY2006) revised milestone, complete statistical analysis of homoplasy. 4a List the single most significant research accomplishment during FY 2006. This research addresses Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors) of National Program 303 (Plant Diseases). The
first 3 to 8 amino acids of wheat streak mosaic virus HC-Pro are essential for mite transmission of virus from plant to plant. The HC-Pro gene was shown to be required for plant to plant transmission of wheat streak mosaic virus by the wheat curl mite. Previously, there was no information regarding viral encoded determinants of plant virus transmission by mites. A nested set of deletions were made in the HC-Pro gene of wheat streak mosaic. The results indicated that mite transmissibility was lost upon deletion of amino acids 3 to 8 of HC-pro gene. These studies represent the first step towards development of a model to explain virus-mite interactions required for plant virus transmission. 4b List other significant research accomplishment(s), if any. This research addresses Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors) of National Program 303 (Plant Diseases). The wheat streak mosaic virus HC-Pro gene is not required
for infection of plants. The wheat streak mosaic virus HC-Pro gene was shown to be dispensable for infection and symptom production in wheat, corn, and oat. Previously, potyvirus HC-Pro was thought to be required for viral infection as point mutations resulted in loss of viral replication and/or systemic movement. A wheat streak mosaic virus genome lacking HC-Pro was constructed and evaluated for infectivity on plants, ability to form virus particles, and relative concentration. The results indicated that wheat streak mosaic virus HC-Pro was not needed for virus replication, systemic movement, or maturation of virus particles, but was needed for optimal accumulation of virus. This study represents the first full gene deletion of any member of the potyvirus family and directly contradicts the standard potyvirus model in which the multifunctional HC-Pro gene is required for maintenance of viral replication and systemic movement within plants. The results further indicate that point
mutations in potyvirus HC-Pro may interfere with essential processes of infection rather than directly causing loss of an essential function provided by HC-Pro. 4d Progress report. Objective 1. Twenty-five chimeric genomes containing fragments from the Sidney 81 and El batan strains of wheat streak mosaic virus have been constructed and evaluated for infectivity on wheat and corn. Infectivity assays indicate that that the CI, NIa, and NIb genes must be derived from the Sidney 81 strain to infect corn. Chimeric wheat streak mosaic virus genomes in which only one or two of these genes were derived from Sidney 81 were not able to infect corn but remained infectious to wheat. Objective 2. Infectious wheat streak mosaic virus genomes bearing partial deletions or gene replacements in the HC-Pro gene have been evaluated for transmission by eriophyid mites. Gene replacement experiments indicated that HC-Pro must be derived from wheat streak mosaic virus to retain mite transmissibility.
Mite transmission was abolished when wheat streak mosaic virus HC-Pro was replaced with the corresponding gene from different virus species. Within the wheat streak mosaic virus HC-Pro gene, determinants of mite transmission were mapped to the 5-half of the gene. A wheat streak mosaic virus mutant in which HC-Pro has been completely deleted has been constructed and demonstrated to be infectious. This is a surprising and unanticipated result as the standard potyvirus HC-Pro model predicts that HC-Pro is required for replication maintenance and systemic movement in plants. This outcome further indicates that either wheat streak mosaic virus HC-pro functions differently from potyvirus HC-Pro, or that interpretations based on mutant analyses of potyvirus HC-Pro are incorrect. Objective 3. This objective has been completed. A peer reviewed publication describing the work is available in Journal of General Virology v. 86, p. 2605-2614. Objective 4. This objective has been completed.
A peer reviewed publication describing this work is available in Virology v. 302 p. 58-70; another manuscript describing this work is in review. Objective 5. Experiments conducted during the first two years of the project (FY 2003 and FY 2004) indicated that both the primary and contingency experimental procedures were insufficient to detect recombination in wheat streak mosaic virus. Therefore, in FY 2004 we designed a revised contingency plan. This alternative approach relies on statistical analyses of polymorphism resident within extensive nucleotide sequence data sets of naturally occurring or experimentally derived wheat streak mosaic virus variants. Preliminary analysis of homoplasy (shared substitutions) present in the wheat streak mosaic virus sequence data sets indicated that shared substitutions were present in excess of that expected if solely due to recurrent mutation, satisfying one condition necessary to establish a history of recombination. We plan to complete the
statistical analysis during FY 2007. 5. Describe the major accomplishments to date and their predicted or actual impact. This research addresses Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors) of National Program 303 (Plant Diseases). Sequencing three complete viral genomes. The complete nucleotide sequences of oat necrotic mottle virus (9346 nucleotides), Agropyron mosaic virus (9540 nucleotides) and Hordeum mosaic virus (9463 nucleotides) have been determined and deposited in the public database GenBank. Availability of these complete nucleotide sequences has had actual impact by permitting scientists to correctly classify these pathogenic agents and develop a more accurate and complete evolutionary history of viruses in the potyvirus family. The HC-Pro genes from these three viruses were used to replace the corresponding gene of wheat streak mosaic virus and the resulting chimeras examined for pathogenesis and mite
transmission. Potential impact of this accomplishment includes development and use of PCR-based diagnostic assays for these viruses. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 1 (Pathogen Detection and Diagnosis) and Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors); and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays, initiate movement and transmission assays). Characterization of an RNA element in the wheat streak mosaic virus P3 gene. An RNA element in the wheat
streak mosaic virus P3 gene was identified through functional analysis of synonymous (but not silent) mutations in the P3 gene. Because these synonymous mutations do not alter protein coding information yet dramatically affect viral movement and replication, it appears that important aspects of wheat streak mosaic virus pathogenesis are determined by primary RNA structure. This research may have potential impact on other scientists concerned with potyvirus pathogenesis and disease control as the accomplishment clearly demonstrates that viral-host interactions leading to disease and/or resistance may be controlled not only by viral encoded proteins but also directly by viral RNA sequences. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and
diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 3 of the project Year 1 (FY2003) milestones (construct mutants, initiate translation, infectivity, movement, replication assays) and Year 2 (FY2004) milestones (fine mapping and complete various assays). Functional equivalence of heterologous HC-Pro genes in wheat. HC-Pro of wheat streak mosaic virus strain Sidney 81 was systematically replaced with the corresponding gene derived from four strains of wheat streak mosaic virus, the tritimovirus oat necrotic mottle virus, the rymoviruses Agropyron mosaic virus and Hordeum mosaic virus, or the potyviruses tobacco etch virus and turnip mosaic virus. Surprisingly, wheat streak mosaic virus tolerated each of the gene replacements and remained able to infect wheat. However, HC-Pro derived from distantly related rymoviruses and potyviruses
resulted in a restriction of host range and these gene replacement constructs were unable to infect corn and oat. In some cases, disease severity in wheat was affected. These results implicate HC-Pro as a determinant of viral host range and virulence. It is anticipated that this outcome will impact scientists by demonstrating that plant viruses have the capacity to tolerate significant changes in nucleotide sequence and that the potential for viability of novel pathogenic agents derived from distantly related donors may be greater than previously recognized. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and
Relationship with Hosts and Vectors), and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and project Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays, initiate movement and transmission assays). Wheat streak mosaic virus HC-Pro is required for mite transmission of virus from plant to plant. The HC-Pro gene was shown to be required for plant to plant transmission of wheat streak mosaic virus by the wheat curl mite. Previously, there was no information regarding viral encoded determinants of plant virus transmission by mites. The HC-Pro gene of wheat streak mosaic virus was replaced with the corresponding gene from related viruses that are transmitted by different biological vectors. The results indicated that mite transmissibility was retained upon replacement of the HC-pro gene from different wheat streak mosaic virus strains. In contrast, HC-Pro gene replacements from different
viruses abolished mite transmission of wheat streak mosaic virus. This research may have potential impact on scientists developing models to explain the process of virus transmission. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays, initiate movement and transmission assays). A null mutant of wheat streak mosaic virus retains infectivity and pathogenicity. A complete deletion
of the HC-Pro gene had minimal effect on pathogenicity of wheat streak mosaic virus. Previously, all mutational analysis of potyvirus genomes had been accomplished by mutation or gene replacement. This study represents the first report of a null mutant for any virus in the potyvirus family. The rather surprising result (symptom expression and host range were essentially unaltered and virus concentration only partially reduced) clearly indicates that HC-Pro is not required for infectivity, replication, and systemic movement of virus in plants and directly contradicts the accepted principle that HC-Pro is directly involved in replication and movement of virus. Instead, the results strongly indicate that loss of infectivity with potyvirus HC-Pro mutants is not due to loss of an essential function provided by HC-Pro, but rather due to interference with an essential process by the mutant form of the HC-pro protein. This study is expected to have potential impact on scientists studying
potyvirus gene function, as the large body of work with potyvirus HC-Pro mutants is now subject to new interpretations. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays, initiate movement and transmission assays). Defining genetic variation and population structure of wheat streak mosaic virus in the US. Nucleotide sequences were determined for the coat protein gene of 50+ isolates
of wheat streak mosaic virus from the U. S. Previously, diversity within wheat streak mosaic virus was poorly defined. Analysis of genetic variation resident in the US population of wheat streak mosaic virus indicated that the US population is comprised of closely related viral genomes that have diverged from one another by an average of 2% over the past century. The results further indicated that the U. S. population of wheat streak mosaic virus appears to be well mixed with only limited geographical subdivision. This study will have impact on scientists concerned with breeding wheat for resistance by demonstrating that isolates of the virus used in screening programs do, in most cases, represent the current virus population resident in the field. The study may also impact regulatory agencies by demonstrating that strains of the virus known to occur in Eurasia and Mexico appear not to be present in the U. S. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance
Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 4 of the project Year 1 (FY2003) milestones (clone and sequence CP genes, initiate phylogenetic analysis) and project Year 2 (FY2004) milestones (complete phylogenetic analysis). Defining the tempo and mode of plant virus evolution. Numerous studies have revealed that plant viruses exhibit remarkable genetic stability but the reasons for this were unknown. We have used two different approaches, each essentially measuring the rate of genetic drift, to arrive at estimates effective population size of wheat streak mosaic virus that are remarkably low. Fixation rates in
plants infected with two strains of wheat streak mosaic virus yielded an effective population size ? 4 for systemic infection of wheat tillers, and changes in single nucleotide polymorphism frequencies during passages from plant to plant suggested an effective population size of about 14 to 20. These results may seem paradoxical, given that cells of infected plants each contain more than 100,000 virions, but can be understood by considering the huge variance in reproduction among wheat streak mosaic virus genomes. Only a tiny fraction of viral genomes produced in a cell, or present in ground sap inoculum from infected plants, spread to adjacent cells or initiate new infections. This study will have impact on scientists by providing a new paradigm for understanding of plant virus population genetics and has profound implications for viral epidemiology, population dynamics, and evolution. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the
Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 4 (determine evolutionary forces affecting diversity in wheat streak mosaic virus). 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? For FY 2006, knowledge has been transferred to potential users as four peer reviewed publications, six invited book chapters and two national meeting presentations. It is expected that knowledge transferred will be used primarily by
other scientists, although some of the information presented in book chapters may be utilized by industry and regulatory agencies as basic reference sources.
Impacts
(N/A)
Publications
- Brown, J.K., Idris, A.M., Ostrow, K., Goldberg, N., French, R.C., Stenger, D.C. 2005. Genetic and phenotypic variation of the pepper golden mosaic virus complex. Phytopathology 95:1217-1224.
- French, R.C., Stenger, D.C. 2005. Population structure within lineages of wheat streak mosaic virus derived from a common founding event exhibits stochastic variation inconsistent with the deterministic quasi-species model. Virology 343:179-189.
- Stenger, D.C., Hein, G.L., French, R.C. 2006. Nested deletion analysis of Wheat streak mosaic virus HC-Pro: Mapping of domains affecting polyprotein processing and eriophyid mite transmission. Virology volume 350: 465-474.
- Stenger, D.C., Young, B.A., French, R.C. 2006. Random mutagenesis of wheat streak mosaic virus HC-Pro: noninfectious interfering mutations in a gene dispensable for systemic infection of plants. Journal of General Virology 87: 2741-2747.
- French, R.C., Stenger, D.C. 2006. Estimating of effective population sizes of laboratory and field samples of wheat streak mosaic virus. American Society for Virology Meeting.
- Stenger, D.C., Hein, G.L., Gildow, F.E., French, R.C. 2006. Nested deletion analysis of tritimovirus hc-pro. American Society for Virology Meeting.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Virus diseases of wheat are responsible for substantial and recurrent losses to American agriculture. This CRIS project represents the only ARS research project that focuses on virus diseases of wheat. Current emphasis of the project is on wheat streak mosaic virus, one of the most important viral disease agents of wheat. The research is organized into five objectives: (1) Map genetic determinants of differential host range among strains of wheat streak mosaic virus. (2) Map determinants of mite transmission encoded by the wheat streak mosaic virus HC-Pro gene. (3) Determine the function of a conserved sequence element in the wheat streak mosaic virus P3 gene. (4) Define patterns of nucleotide sequence diversity within a collection of wheat streak mosaic virus isolates and determine evolutionary
forces affecting diversity in wheat streak mosaic virus. (5) Determine the frequency of intermolecular homologous recombination among closely related genotypes of wheat streak mosaic virus. This research is relevant to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases). The project primarily addresses Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors) of National Program 303 (Plant Diseases). Improved understanding of the genetic basis of viral pathogenesis, gene function, vector transmission, RNA recombination, and population genetics will facilitate efforts to ameliorate the effects of viral infection in wheat and other cereal crops. A major product of this research will be scientific knowledge available in the
public domain as published papers in peer reviewed journals and nucleotide sequence data deposited in public databases. Initially, primary customers of the research will be ARS, University, and industry scientists striving to understand plant virus pathogenesis and develop new strategies for control. Ultimately, agricultural producers and consumers will benefit as this newly gained knowledge is applied to generate improved cultivars or cultural practices in which losses due to plant virus infection are reduced. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1. Map genetic determinants of differential host range among strains of wheat streak mosaic virus. Year 2 (FY2004) milestones, construct chimeric wheat streak mosaic virus genomes, initiate host range assays; Year 3 (FY2005) milestones, construct chimeric wheat streak mosaic virus genomes, conduct host range assays, initiate fine mapping; Year 4 (FY2006) milestones, conduct host range assays,
fine mapping; Year 5 (FY2007) milestones, conduct host range assays, fine mapping. Objective 2. Map determinants of mite transmission encoded by the wheat streak mosaic virus HC-Pro gene. Year 2 (FY2004) milestones, construct mutants, initiate infectivity assays; Year 3 (FY2005) milestones, construct mutants, conduct infectivity assays, initiate movement and transmission assays; Year 4 (FY2006) milestones, construct mutants, conduct movement and transmission assays, initiate proteinase and synergism assays; Year 5 (FY2007) milestones, complete various assays. Objective 3. Determine the function of a conserved sequence element in the wheat streak mosaic virus P3 gene. Year 1 (FY2003) milestones, construct mutants, initiate translation, infectivity, movement, replication assays; Year 2 (FY2004) milestones, fine mapping and complete various assays. Objective 4. Define patterns of nucleotide sequence diversity within a collection of wheat streak mosaic virus isolates and determine
evolutionary forces affecting diversity in wheat streak mosaic virus. Year 1 (FY2003) milestones, clone and sequence CP genes, initiate phylogenetic analysis; Year 2 (FY2004) milestones, complete phylogenetic analysis. Objective 5. Determine the frequency of intermolecular homologous recombination among closely related genotypes of wheat streak mosaic virus. Year 1 (FY2003) milestones, initiate recombination assays; Year 2 (FY2004) milestones, recombination assays, alter target length; Year 3 (FY2005) revised milestone, initiate statistical analysis of homoplasy; Year 4 (FY2006) revised milestone, complete statistical analysis of homoplasy. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Construct chimeric wheat streak mosaic virus genomes Milestone Fully Met 2. Conduct host range assay. Milestone Fully Met 3. Initiate fine mapping Milestone Substantially
Met 4. Construct mutants. Milestone Fully Met 5. Conduct infectivity assays. Milestone Fully Met 6. Initiate movement assays Milestone Fully Met 7. Initiate transmission assays. Milestone Fully Met 8. Initiate statistical analysis of homoplasy. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Objective 1. FY2006 (Project Year 4) milestones. Conduct host range assays. Chimeric genomes derived from the Sidney 81 and El batan strains of wheat streak mosaic virus will be evaluated for infectivity on the differential host corn (corn is resistant to El batan but susceptible to Sidney 81). It is anticipated that the results will identify one or more viral genes that control host range. These viral genes may correspond to resistance/susceptibility genes in the host and may impact studies designed to identify resistance
genes in corn. Fine mapping. Differences in viral genes specifying infection of corn will be examined in greater detail to determine which region(s) of the encoded protein(s) control the trait. It is anticipated that these results will provide information regarding the mechanism by which viral strains trigger or avoid host resistance. If successful, the outcome may be a clearer understanding of pathways leading to resistance or susceptibility. FY2007 (Project Year 5) milestone. Conduct host range assays. This milestone represents continuation of the work projected for this objective in FY 2006. Results, outcomes, and impact anticipated as described for FY 2006. Fine mapping. This milestone represents continuation of the work projected for this objective in FY 2006. Results, outcomes, and impact anticipated as described for FY 2006. FY2008 milestones. None. Project expires October, 2007. Objective 2. FY2006 (Project Year 4) milestones. Construct mutants. Site directed
mutagenesis will be employed to generate alter specific amino acid residues of the HC-Pro protein. Mutations in the 5-half of the gene will target conserved cysteine residues known to be involved in aphid transmission of potyviruses. Mutations in the 3-half of the gene will target conserved motifs associated with proteinase activity of potyvirus HC-Pro. Achieving this milestone provides virus mutants that may be analyzed for function as described in the milestone below. Conduct movement assays. Wheat streak mosaic virus genomes bearing site- directed mutations in HC-Pro will be evaluated for infectivity to wheat. Those mutants that are not infectious to wheat will be evaluated for localized infection of inoculated leaves using the GUS reporter gene assay. These results may distinguish between those mutants that are replication defective from those that retain the ability to replicate but are defective in either cell to cell movement or long distance movement within plants. These
results will permit a comparison of wheat streak mosaic virus HC-Pro function to that of potyvirus HC-Pro. Conduct transmission assays. Site directed mutants that retain infectivity and pathogenicity to wheat will be evaluated for transmissibility by the wheat curl mite. These results should identify whether the conserved cysteine residues are needed for vector transmission of wheat streak mosaic virus. An anticipated outcome of these experiments is a better understanding of the similarities and differences in the mode of action by which HC-pro facilitates transmission by different biological vectors. Initiate proteinase and synergism assays. Site directed mutants may be non-infectious for a variety of reasons. One possibility for loss of infectivity is that the mutation has affected the proteinase active site or proteinase cleavage site. It is important to distinguish between those non-infectious mutants that have lost proteinase activity from those that are defective for other
reasons. It is anticipated that the proteinase assays will identify some non-infectious mutants that retain proteinase activity. Such an outcome will demonstrate that wheat streak mosaic virus HC-Pro is multifunctional and indicate that the protein performs another function in addition to vector transmission and processing of mature viral proteins. Those mutants that retain infectivity but have delayed expression of symptoms may be partially or fully debilitated for suppression of post transcriptional gene silencing (a known function of potyvirus HC-Pro). Suppression of gene silencing is thought to be a mechanism by which potyvirus HC-pro contributes to synergistic interactions in coinfection with unrelated viruses. Thus, synergism represents a simple assay for suppression of gene silencing function. Wheat streak mosaic virus bearing site directed mutants in HC- Pro will be coinoculated with a second virus (maize chlorotic mottle virus). Disease severity and virus concentration
will be determined for both viruses in single and mixed infections. It is anticipated that the results will provide strong, albeit indirect, evidence as to whether or not wheat streak mosaic virus HC-Pro serves as a suppressor of gene silencing. A potential impact of this research may be the identification of a silencing suppressor that functions in grain crops. FY2007 (Project Year 5) milestones. Complete various assays. This milestone represents continuation of the work projected for this objective in FY 2006. Results, outcomes, and impact anticipated as described for FY 2006. FY2008 milestones. None. Project expires October, 2007. Objective 3. FY2006 (Project Year 4) milestones. None. Objective completed. FY2007 (Project Year 5) milestones. None. Objective completed. FY2008 milestones. None. Project expires October, 2007. Objective 4. FY2006 (Project Year 4) milestones. None. Objective completed. FY2007 (Project Year 5) milestones. None. Objective completed. FY2008
milestones. None. Project expires October, 2007. Objective 5. FY2006 (Project Year 4) milestones. Complete statistical analysis of homoplasy. Preliminary analysis revealed that the wheat streak mosaic virus nucleotide sequence data sets exhibited excess homoplasy (shared substitutions). Excess homoplasy may result from recurrent mutation or a history of recombination. To distinguish between these two possibilities, the distribution of homoplasy among sequences must be reconciled with the evolutionary history of the variants as determined through phylogenetic analyses. The remaining analyses to be conducted will address this issue. We hypothesize that the primary purpose of recombination in viruses is repair of mutations generated during replication rather than capture of foreign genes. As such, the process is expected to resemble gene conversion in higher organisms. Should this be the conclusion, the potential impact may be a paradigm shift in the understanding of how plant
viruses with error prone replication avoid mutational meltdown and maintain fitness. FY2007 (Project Year 5) milestones. None. FY2008 milestones. None. Project expires October, 2007. 4a What was the single most significant accomplishment this past year? Wheat streak mosaic virus HC-Pro is required for mite transmission of virus from plant to plant. The HC-Pro gene was shown to be required for plant to plant transmission of wheat streak mosaic virus by the wheat curl mite. Previously, there was no information regarding viral encoded determinants of plant virus transmission by mites. The HC-Pro gene of wheat streak mosaic virus was replaced with the corresponding gene from related viruses that are transmitted by different biological vectors. The results indicated that mite transmissibility was retained upon replacement of the HC-pro gene from different wheat streak mosaic virus strains. In contrast, HC-Pro gene replacements from different viruses abolished mite transmission of wheat
streak mosaic virus. These studies represent the first step towards development of a model to explain virus-mite interactions required for plant virus transmission. 4b List other significant accomplishments, if any. The wheat streak mosaic virus HC-Pro gene is not required for infection of plants. The wheat streak mosaic virus HC-Pro gene was shown to be dispensable for infection and symptom production in wheat, corn, and oat. Previously, potyvirus HC-Pro was thought to be required for viral infection as point mutations resulted in loss of viral replication and/or systemic movement. A wheat streak mosaic virus genome lacking HC-Pro was constructed and evaluated for infectivity on plants, ability to form virus particles, and relative concentration. The results indicated that wheat streak mosaic virus HC-Pro was not needed for virus replication, systemic movement, or maturation of virus particles, but was needed for optimal accumulation of virus. This study represents the first full
gene deletion of any member of the potyvirus family and directly contradicts the standard potyvirus model in which the multifunctional HC-Pro gene is required for maintenance of viral replication and systemic movement within plants. The results further indicate that point mutations in potyvirus HC-Pro may interfere with essential processes of infection rather than directly causing loss of an essential function provided by HC-Pro. 4d Progress report. Objective 1. Twenty-five chimeric genomes containing fragments from the Sidney 81 and El batan strains of wheat streak mosaic virus have been constructed and evaluated for infectivity on wheat and corn. Infectivity assays indicate that that the CI, NIa, and NIb genes must be derived from the Sidney 81 strain to infect corn. Chimeric wheat streak mosaic virus genomes in which only one or two of these genes were derived from Sidney 81 were not able to infect corn but remained infectious to wheat. Objective 2. Infectious wheat streak
mosaic virus genomes bearing partial deletions or gene replacements in the HC-Pro gene have been evaluated for transmission by eriophyid mites. Gene replacement experiments indicated that HC-Pro must be derived from wheat streak mosaic virus to retain mite transmissibility. Mite transmission was abolished when wheat streak mosaic virus HC-Pro was replaced with the corresponding gene from different virus species. Within the wheat streak mosaic virus HC-Pro gene, determinants of mite transmission were mapped to the 5-half of the gene. A wheat streak mosaic virus mutant in which HC-Pro has been completely deleted has been constructed and demonstrated to be infectious. This is a surprising and unanticipated result as the standard potyvirus HC-Pro model predicts that HC-Pro is required for replication maintenance and systemic movement in plants. This outcome further indicates that either wheat streak mosaic virus HC-pro functions differently from potyvirus HC-Pro, or that
interpretations based on mutant analyses of potyvirus HC-Pro are incorrect. Objective 3. This objective has been completed. A peer reviewed publication describing the work is available in Journal of General Virology v. 86, p. 2605-2614. Objective 4. This objective has been completed. A peer reviewed publication describing this work is available in Virology v. 302 p. 58-70; another manuscript describing this work is in review. Objective 5. Experiments conducted during the first two years of the project (FY 2003 and FY 2004) indicated that both the primary and contingency experimental procedures were insufficient to detect recombination in wheat streak mosaic virus. Therefore, in FY 2004 we designed a revised contingency plan. This alternative approach relies on statistical analyses of polymorphism resident within extensive nucleotide sequence data sets of naturally occurring or experimentally derived wheat streak mosaic virus variants. Preliminary analysis of homoplasy (shared
substitutions) present in the wheat streak mosaic virus sequence data sets indicated that shared substitutions were present in excess of that expected if solely due to recurrent mutation, satisfying one condition necessary to establish a history of recombination. We plan to complete the statistical analysis during FY 2006. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Sequencing three complete viral genomes. The complete nucleotide sequences of oat necrotic mottle virus (9346 nucleotides), Agropyron mosaic virus (9540 nucleotides) and Hordeum mosaic virus (9463 nucleotides) have been determined and deposited in the public database GenBank. Availability of these complete nucleotide sequences has had actual impact by permitting scientists to correctly classify these pathogenic agents and develop a more accurate and complete evolutionary history of viruses in the potyvirus family. The HC-Pro genes from these three
viruses were used to replace the corresponding gene of wheat streak mosaic virus and the resulting chimeras examined for pathogenesis and mite transmission. Potential impact of this accomplishment includes development and use of PCR-based diagnostic assays for these viruses. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 1 (Pathogen Detection and Diagnosis) and Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors); and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays,
initiate movement and transmission assays). Characterization of an RNA element in the wheat streak mosaic virus P3 gene. An RNA element in the wheat streak mosaic virus P3 gene was identified through functional analysis of synonymous (but not silent) mutations in the P3 gene. Because these synonymous mutations do not alter protein coding information yet dramatically affect viral movement and replication, it appears that important aspects of wheat streak mosaic virus pathogenesis are determined by primary RNA structure. This research may have potential impact on other scientists concerned with potyvirus pathogenesis and disease control as the accomplishment clearly demonstrates that viral-host interactions leading to disease and/or resistance may be controlled not only by viral encoded proteins but also directly by viral RNA sequences. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance
measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 3 of the project Year 1 (FY2003) milestones (construct mutants, initiate translation, infectivity, movement, replication assays) and Year 2 (FY2004) milestones (fine mapping and complete various assays). Functional equivalence of heterologous HC-Pro genes in wheat. HC-Pro of wheat streak mosaic virus strain Sidney 81 was systematically replaced with the corresponding gene derived from four strains of wheat streak mosaic virus, the tritimovirus oat necrotic mottle virus, the rymoviruses Agropyron mosaic virus and Hordeum mosaic virus, or the potyviruses tobacco etch virus and turnip mosaic virus. Surprisingly, wheat streak mosaic virus tolerated
each of the gene replacements and remained able to infect wheat. However, HC-Pro derived from distantly related rymoviruses and potyviruses resulted in a restriction of host range and these gene replacement constructs were unable to infect corn and oat. In some cases, disease severity in wheat was affected. These results implicate HC-Pro as a determinant of viral host range and virulence. It is anticipated that this outcome will impact scientists by demonstrating that plant viruses have the capacity to tolerate significant changes in nucleotide sequence and that the potential for viability of novel pathogenic agents derived from distantly related donors may be greater than previously recognized. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests
and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and project Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays, initiate movement and transmission assays). Wheat streak mosaic virus HC-Pro is required for mite transmission of virus from plant to plant. The HC-Pro gene was shown to be required for plant to plant transmission of wheat streak mosaic virus by the wheat curl mite. Previously, there was no information regarding viral encoded determinants of plant virus transmission by mites. The HC-Pro gene of wheat streak mosaic virus was replaced with the corresponding gene from related viruses that are transmitted by different biological vectors. The results indicated that mite transmissibility was retained upon
replacement of the HC-pro gene from different wheat streak mosaic virus strains. In contrast, HC-Pro gene replacements from different viruses abolished mite transmission of wheat streak mosaic virus. This research may have potential impact on scientists developing models to explain the process of virus transmission. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays, initiate
movement and transmission assays). A null mutant of wheat streak mosaic virus retains infectivity and pathogenicity. A complete deletion of the HC-Pro gene had minimal effect on pathogenicity of wheat streak mosaic virus. Previously, all mutational analysis of potyvirus genomes had been accomplished by mutation or gene replacement. This study represents the first report of a null mutant for any virus in the potyvirus family. The rather surprising result (symptom expression and host range were essentially unaltered and virus concentration only partially reduced) clearly indicates that HC-Pro is not required for infectivity, replication, and systemic movement of virus in plants and directly contradicts the accepted principle that HC-Pro is directly involved in replication and movement of virus. Instead, the results strongly indicate that loss of infectivity with potyvirus HC-Pro mutants is not due to loss of an essential function provided by HC-Pro, but rather due to interference
with an essential process by the mutant form of the HC-pro protein. This study is expected to have potential impact on scientists studying potyvirus gene function, as the large body of work with potyvirus HC-Pro mutants is now subject to new interpretations. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 2 of the project Year 2 (FY2004) milestones (construct mutants, initiate infectivity assays) and Year 3 (FY2005) milestones (construct mutants, conduct infectivity assays, initiate movement and transmission assays). Defining genetic variation and
population structure of wheat streak mosaic virus in the US. Nucleotide sequences were determined for the coat protein gene of 50+ isolates of wheat streak mosaic virus from the U. S. Previously, diversity within wheat streak mosaic virus was poorly defined. Analysis of genetic variation resident in the US population of wheat streak mosaic virus indicated that the US population is comprised of closely related viral genomes that have diverged from one another by an average of 2% over the past century. The results further indicated that the U. S. population of wheat streak mosaic virus appears to be well mixed with only limited geographical subdivision. This study will have impact on scientists concerned with breeding wheat for resistance by demonstrating that isolates of the virus used in screening programs do, in most cases, represent the current virus population resident in the field. The study may also impact regulatory agencies by demonstrating that strains of the virus known
to occur in Eurasia and Mexico appear not to be present in the U. S. This accomplishment contributes to ARS Strategic Plan Goal 3 (Enhance Protection and Safety of the Nation's Agriculture and Food Supply, performance measure 3.2.5 (Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and diseases); is relevant to USDA-ARS National Program 303 Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and was conducted under Objective 4 of the project Year 1 (FY2003) milestones (clone and sequence CP genes, initiate phylogenetic analysis) and project Year 2 (FY2004) milestones (complete phylogenetic analysis). 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and
durability of the technology products? For FY 2005, knowledge has been transferred to potential users as four peer reviewed publications, six invited book chapters, two national meeting presentations, and one invited presentation at a university. It is expected that knowledge transferred will be used primarily by other scientists, although some of the information presented in book chapters may be utilized by industry and regulatory agencies as basic reference sources.
Impacts
(N/A)
Publications
- French, R.C., Stenger, D.C. 2005. Reciprocal monophyly of the genera potyvirus and rymovirus is supported by genome sequences of agropyron mosaic virus and hordeum mosaic virus. Archives of Virology 150: 299-312.
- Stenger, D.C., Hein, G.L., Gildow, F.E., Horken, K., French, R.C. 2005. Plant virus hc-pro is a determinant of eriophyid mite transmission. Journal of Virology 79, 9054-9061.
- Stanley, J., Bisaro, D.M., Briddon, R.W., Brown, T.K., Fauquet, C.M., Harrison, B.D., Rybicki, E.P., Stenger, D.C. 2005. Family geminiviridae. In The International Committee on the Taxonomy of Viruses, 8th Report. Eds. Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., and Ball, L. A. Elsevier/Academic Press, London. pp. 301-326.
- Rabenstein, F., Schubert, J., Ehrig, F., French, R.C., Stenger, D.C. 2004. Ryegrass mosaic virus. In Viruses and Virus Diseases of the Poaceae. Eds., H. Lapierre and P.-A Signoret. INRA Editions, Paris. pp. 785-789.
- Rabenstein, F., Stenger, D.C., French, R.C. 2004. Hordeum mosaic virus. In Viruses and Virus Diseases of Poaceae. Eds., H. Lapierre and P.-A Signoret. INRA Editions, Paris. pp.474-476.
- Rabenstein, F., Stenger, D.C., French, R.C. 2004. Genus tritimovirus. In Viruses and Virus Diseases of Poaceae. Eds., H. Lapierre and P.-A Signoret. INRA Editions, Paris. pp 398-402.
- Rabenstein, F., Schubert, J., Stenger, D.C., French, R.C. 2004. Genus rymovirus. In Viruses and Virus Diseases of Poaceae. Eds., H. Lapierre and P.-A. Signoret. INRA Editions, Paris. pp. 395-398.
- French, R.C., Stenger, D.C. 2005. Patterns of temporal variation in parallel wheat streak mosaic virus lineages passaged at high mo1 suggest that genetic bottlenecks are intrinsic to serial transmission. American Society for Virology Meeting. Oral presentation, not published in journal.
- Stenger, D.C., Hein, G.L., Gildow, F.E., Horken, K., French, R.C. 2005. Wheat streak mosaic virus hc-pro is a determinant of eriophyid mite transmission. American Society for Virology Meeting. Oral presentation, not published in journal.
- Choi, I., Horken, K.M., Stenger, D.C., French, R.C. 2005. An internal rna element in the wheat streak mosaic virus p3 cistron revealed by synonymous mutations that affect both movement and replication.. Journal of General Virology. v. 86. p 2605-2614.
- Stenger, D.C., French, R.C., Gildow, F.E. 2005. Complete deletion of wheat streak mosaic virus hc-pro: a nulll mutant is viable for systemic infection. Journal of Virology. v. 79. p. 12077-12080.
- Berger, P.H., Adams, M.J., Brunt, A.A., Hammond, J., Hill, J.H., Jordan, R. L., Kashiwazalci, S., Rybicki, E., Spence, N., Stenger, D.C. 2005. Family potyviridae. Taxonomy of Viruses. In The International Committee on the Taxonomy of Viruses, 8th Report. 2005. Eds. C. M. Fauquet, Fauquet, C. M. , Mayo, M.A., Maniloff, J. Desselberger, U., and Ball, L.A. Elsevier/Academic Press, London. p 819-841.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Virus diseases of wheat are responsible for substantial and recurrent losses to American agriculture. This CRIS project represents the only ARS research project that focuses on wheat streak mosaic virus (WSMV), one of the most important viral disease agents of wheat. The research is organized into five objectives: (1) Map genetic determinants of differential host range among strains of WSMV. (2) Map determinants of mite transmission encoded by the WSMV HC-Pro gene. (3) Determine the function of a conserved sequence element in the WSMV P3 gene. (4) Define patterns of nucleotide sequence diversity within a collection of WSMV isolates and determine evolutionary forces affecting diversity in WSMV. (5) Determine the frequency of intermolecular homologous recombination among closely related genotypes of
WSMV. This research addresses Component 4 (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors) of National Program 303 (Plant Diseases). Improved understanding of the genetic basis of viral pathogenesis, gene function, vector transmission, RNA recombination, and population genetics will facilitate efforts to ameliorate the effects of viral infection in wheat and other cereal crops. A major product of this research will be scientific knowledge available in the public domain as published papers in peer reviewed journals and nucleotide sequence data deposited in public databases. Initially, primary customers of the research will be other ARS, University, and industry scientists striving to understand plant virus pathogenesis and develop new strategies for control. Ultimately, agricultural producers and consumers will benefit as this newly gained knowledge is applied to generate improved cultivars or cultural practices in which losses due to
plant virus infection are reduced. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1: Map genetic determinants of differential host range among strains of WSMV. Year 2 (FY2004), Construct chimeric WSMV genomes, initiate host range assays; Year 3 (FY2005), Construct chimeric WSMV genomes, conduct host range assays, initiate fine mapping; Year 4 (FY2006) , conduct host range assays, fine mapping; Year 5 (FY2007), conduct host range assays, fine mapping . Objective 2: Map determinants of mite transmission encoded by the WSMV HC-Pro gene. Year 2 (FY2004), construct mutants, initiate infectivity assays; Year 3 (FY2005), construct mutants, conduct infectivity assays, initiate movement and transmission assays; Year 4 (FY2006), construct mutants, conduct movement and transmission assays, initiate proteinase, replication and synergism assays; Year 5 (FY2007), complete various assays. Objective 3: Determine the function of a conserved sequence element in the
WSMV P3 gene. Year 1 (FY2003), construct mutants, initiate translation, infectivity, movement, replication assays; Year 2 (FY2004), fine mapping and complete various assays. Objective 4: Define patterns of nucleotide sequence diversity within a collection of WSMV isolates and determine evolutionary forces affecting diversity in WSMV. Year 1 (FY2003), clone and sequence CP genes, initiate phylogenetic analysis; Year 2 (FY2004), complete phylogenetic analysis. Objective 5: Determine the frequency of intermolecular homologous recombination among closely related genotypes of WSMV. Year 1 (FY2003), initiate recombination assays; Year 2 (FY2004), recombination assays, alter target length; Year 3 (FY2005), recombination assays, vary target length; Year 4 (FY2006), recombination assays. 3. Milestones: Question 3A. Objective 1: Map genetic determinants of differential host range among strains of WSMV. Year 2 (FY2004) milestone: Construct chimeric WSMV genomes, initiate host range assays. All
milestones for FY2004 have been met. Infectious clones of WSMV strains Sidney 81 (infects wheat and SDp2 corn) and EB3 (infects wheat but not SDp2 corn) have been used as sources of genes to construct a series of chimeric viral genomes using conserved restriction endonuclease sites. Additional restriction endonuclease sites have been engineered by mutagenesis into each virus strain genome to provide additional common sites for fragment exchange. Preliminary infectivity/host range assays indicate that all chimeric genomes infect wheat, but only those with the 3'-proximal two-thirds of the genome from Sidney 81 infect SDp2 corn. Objective 2: Map determinants of mite transmission encoded by the WSMV HC-Pro gene. Year 2 (FY2004) milestone: Construct mutants, initiate infectivity assays. All milestones for FY2004 have been met. Thirty-two point mutants and eleven 5'-proximal deletion mutants of Sidney 81 HC-Pro were constructed, verified by sequencing, placed within the WSMV Sidney 81
infectious clone, and evaluated for infectivity on mechanically inoculated wheat. Currently, these experiments are being repeated. The complete genomes of oat necrotic mottle virus (ONMV), Agropyron mosaic virus (AgMV), and Hordeum mosaic virus (HoMV) have been cloned, completely sequenced, and used as sources of heterologous HC-Pro genes. A manuscript describing the ONMV sequence has been written and published. A manuscript describing the AgMV and HoMV sequences has been written and submitted. Chimeric WSMV-Sidney 81 viral genomes bearing HC-Pro replacements from four strains of WSMV, and the distinct viral species ONMV, AgMV, HoMV, Turnip mosaic virus (TuMV) and Tobacco etch virus (TEV) have been constructed, verified by sequencing, and evaluated for infectivity/host range. A manuscript describing this work has been written and published. Preliminary mite transmission assays indicate that WSMV chimeric genomes having HC-Pro replacements derived from ONMV, AgMV, and TuMV have lost
the ability to be transmitted by eriophyid mites. This provides the first solid evidence that HC-Pro is a viral-encoded determinant of mite transmission. Objective 3. Determine the function of a conserved sequence element in the WSMV P3 gene. Year 2 (FY2004) milestone: fine mapping and complete various assays. All milestones for this objective have been accomplished and the objective completed on time. The P3 gene of WSMV contains a cis-acting RNA sequence element required for systemic movement and optimal replication. A draft manuscript describing this work has been written and is expected to be submitted for publication in 2004. Objective 4: Define patterns of nucleotide sequence diversity within a collection of WSMV isolates and determine evolutionary forces affecting diversity in WSMV. Year 2 (FY2004) milestone: complete phylogenetic analysis. All milestones for this objective have been accomplished and the objective completed ahead of schedule. A manuscript describing
nucleotide sequence diversity, patterns of polymorphism, and an analysis of forces affecting WSMV evolution was completed and published previously. Objective 5: Determine the frequency of intermolecular homologous recombination among closely related genotypes of WSMV. Year 2 (FY2004) milestone: recombination assays. Milestone for this objective was completed, albeit with disappointing results. WSMV-Sidney 81 constructs bearing overlapping portions of the GUS reporter gene as a recombination target have been constructed, evaluated for infectivity on wheat, and tested in co-inoculation experiments for recombination. No evidence of homologous recombination was observed (e.g. , functional GUS activity was not restored even though the co- inoculations were successful. We also attempted to generate recombinants in vivo during co-inoculations with the Sidney 81 and Type isolates of WSMV (the contingency plan for this objective) and screened mixed- infected plants for recombinants bearing
5'- and 3'- ends of the two strains by RFLP (restriction fragment length polymorphism). An alternate contingency plan has been developed to address this objective: Statistical analysis of naturally occurring recombinants in the extensive WSMV CP sequence database previously obtained in Objective 4. A plan to analyze homoplasy (shared polymorphism) present in the WSMV sequence data set has been developed which will allow partioning of shared polymorphism into that expected for recurrent (independent) mutation, with any excess shared polymorphism attributed to recombination. Question 3B. List the milestones (from the list in question #2) that you expect to address over the next three years (FY2005, FY2006, FY2007). What do you expect to accomplish year by year over the next three years under each milestone? Objective 1: Map genetic determinants of differential host range among strains of WSMV. Year 3 (FY2005) milestone: Construct chimeric WSMV genomes, conduct host range assays,
initiate fine mapping. Assay infectious clones containing engineered common restriction sites (and derivative chimeric genomes) for infectivity and host range. Initiate fine mapping of host range determinants by exchanging shorter genome fragments. Year 4 (FY2006) milestone: Conduct host range assays, fine mapping. Continue fine mapping of host range determinants by site directed mutagenesis. Year 5 (FY2007) milestone: Conduct host range assays, fine mapping. Finish host range assays. Prepare manuscript describing host range determinants encoded by WSMV. Objective 2: Map determinants of mite transmission encoded by the WSMV HC-Pro gene. Year 3 (FY2005) milestone: Construct mutants, conduct infectivity assays, initiate movement, proteinase, and transmission assays. Additional deletion mutants of WSMV HC-Pro will be constructed, sequenced, placed into the infectious clone, and tested for infectivity by mechanical inoculation wheat. Infectivity assays of point and deletion mutants will
be repeated. Initiate placement of GUS reporter gene sequences into non-infectious mutants to assess movement phenotype. Construct chimeric HC-Pro genes having portions of the gene derived from WSMV-Sidney 81 and the remainder from ONMV. Repeat mite transmission assays with WSMV Sidney 81 bearing HC-Pro gene replacements from other viruses and initiate mite transmission assays of Sidney 81/ONMV HC-Pro chimeras. Prepare manuscript describing mite transmission properties of HC-Pro replacements. Year 4 (FY2006) milestone: Construct mutants, conduct movement and transmission assays, proteinase, replication and synergism assays. Initiate mite transmission assays of relevant point and deletion mutants of Sidney 81 HC-Pro. Conduct movement assays of point and deletion mutants bearing the GUS reporter gene. Use in vitro transcription/translation system to assess proteinase activity of relevant HC-Pro mutants. Begin co-inoculations of relevant mutants with a second virus(foxtail mosaic virus
or maize chlorotic mottle virus) to assess replication and synergism assays. Initiate preparation of manuscript(s) describing infectivity, proteinase, transmission, and movement properties of point and deletion mutants. Year 5 (FY2007) milestone: Complete various assays. Complete manuscripts initiated in year 4. Initiate manuscript(s) describing synergism, replication, and properties of HC-Pro mutants. Objective 3: Determine the function of a conserved sequence element in the WSMV P3 gene. The remaining milestone to achieve is the submission and publication of a manuscript describing the completed research. It is anticipated that submission will occur prior to the end of FY2005. Objective 4: Define patterns of nucleotide sequence diversity within a collection of WSMV isolates and determine evolutionary forces affecting diversity in WSMV. Objective completed. Objective 5: Determine the frequency of intermolecular homologous recombination among closely related genotypes of WSMV. Year
3 (FY2005) revised milestone: Complete analysis of homoplasy in WSMV CP sequence data set. Year 4 (FY2006) revised milestone: Prepare and submit manuscript describing analysis of homoplasy and the relative importance of recurrent mutation and recombination with respect to shared polymorphisms. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004. Functional replacement of wheat streak mosaic virus (WSMV) HC-Pro. The HC-Pro gene of WSMV was systematically replaced with the corresponding gene from closely related strains of WSMV and from distantly related virus species. All HC-Pro replacements were functional in wheat with respect to systemic infection. The experiments demonstrate that HC-Pro genes from dissimilar sources and limited structural similarities nonetheless have conservation of function with respect to pathogenicity in wheat. Alterations in host range and symptom severity for some HC-Pro replacements
indicates that HC-Pro may encode determinants of pathogenicity. B. Other significant accomplishment. AgMV and HoMV completely sequenced. The complete nucleotide sequences of two wheat infecting viruses (e.g., Agropyron mosaic virus [AgMV] and Hordeum mosaic virus [HoMV]) were determined and deposited in the public database GenBank. Analysis of the AgMV and HoMV sequences revealed that the two viral species are most closely related to each other and are members of the genus Rymovirus. Phylogenetic analyses indicate that the genus Rymovirus should be retained as a taxonomic unit with the plant virus family Potyviridae. C. Significant activities that support special target populations. None. D. Optional Progress Report: Wheat streak mosaic virus (WSMV) HC-Pro is a determinant of eriophyid mite transmission. Preliminary experiments conducted in FY 2004 indicate that the WSMV HC- Pro gene is required for transmission by eriophyid mites. Chimeric WSMV genomes bearing HC-Pro genes
other virus species were not transmitted by the wheat curl mite vector of WSMV, even though these chimeric viral genomes were transmissible to wheat by mechanical inoculation. These data provide the first demonstration that HC-Pro of a non-aphid transmitted potyvirus mediates vector transmission. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Sequencing three complete viral genomes. The complete nucleotide sequences of oat necrotic mottle virus (ONMV; 9346 nucleotides), Agropyron mosaic virus (AgMV; 9540 nucleotides) and Hordeum mosaic virus (9463 nucleotides) have been determined and deposited in GenBank. Phylogenetic analyses indicated that ONMV is most closely related to wheat streak mosaic virus; AgMV and HoMV were most closely related to each other and are members of the genus Rymovirus. Availability of these complete nucleotide sequences will be useful to plant virologists comparing evolutionary relationships
among viruses, and the cloned viral genomes may be used as sources of distantly related genes homologous to the well characterized viruses of the genus potyvirus. The complete genome sequences also will facilitate PCR-based diagnostic assays for these viruses by permitting the design of primers to any region of the genome. Characterization of an RNA element in the Wheat streak mosaic virus (WSMV) P3 gene. An RNA element in the WSMV P3 gene was identified through functional analysis of synonymous (but not silent) mutations in the P3 gene. Because these synonymous mutations do not alter protein coding information yet dramatically affect viral movement and replication, it appears that important aspects of WSMV pathogenesis are determined by primary RNA structure. As a result, researchers concerned with potyviral pathogenesis and resistance breaking phenotypes (some of which map to the P3 gene in related viruses) may no longer assume that the observed phenotypes are due to changes at
the protein level. Thus, the research may have a major impact on experimental design and analysis of potyvirus gene structure and function with respect to pathogenesis and disease control. Functional equivalence of heterologous HC-Pro genes in wheat. HC-Pro of wheat streak mosaic virus strain Sidney 81 (WSMV-Sidney 81) was systematically replaced with the corresponding gene derived from four strains of WSMV, the tritimovirus Oat necrotic mottle virus (ONMV), the rymoviruses Agropyron mosaic virus (AgMV) and Hordeum mosaic virus (HoMV), or the potyviruses Tobacco etch virus (TEV) and Turnip mosaic virus (TuMV). These HC-Pro proteins varied in amino acid sequence identity shared with HC-Pro of WSMV-Sidney 81 from high (strains of WSMV at 86- 99%), to moderate (ONMV at 70%) to low (rymoviruses and potyviruses at 15-17%). Surprisingly, all chimeric viral genomes examined were capable of systemic infection of wheat upon inoculation with RNA transcripts produced in vitro. HC-Pro
replacements derived from tritimoviruses did not alter host range relative to WSMV-Sidney 81, as each of these chimeric viruses were able to systemically infect wheat, oat, and corn line SDp2. These results indicate that differences in host range among tritimoviruses, including the inability of ONMV to infect wheat or the inability of WSMV strains Type and El Batan 3 to infect SDp2 corn, are not determined by HC-Pro. In contrast, all chimeric viruses bearing HC- Pro replacements derived from rymoviruses or potyviruses were unable to infect SDp2 corn and oat. Collectively, these results indicate that HC- Pro from distantly related virus species of the family Potyviridae are competent to provide WSMV-Sidney 81 with all functions necessary for infection of a permissive host (wheat) and that virus-host interactions required for systemic infection of oat and SDp2 corn are more stringent. Changes in symptom severity or mechanical transmission efficiency observed for some chimeric viruses
further suggests that HC-Pro affects virulence in WSMV. This work indicates that recombination among highly divergent viruses is possible and, therefore, may impact risk assessment analyses. The work also demonstrates that highly divergent genes may be substituted while maintaining functional equivalence. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The complete nucleotide sequences of ONMV, AgMV, and HoMV, and the CP gene sequences of over 50 WSMV isolates have been deposited in GenBank. GenBank is a public-accessible data base that is used by government, university, and industry scientists. Complete nucleotide sequences of plant viral genomes are of value to other scientists concerned with plant virus evolution and classification, and to
industry scientists concerned with rapid and accurate diagnosis of viral infection in crop species. It is anticipated that the population genetics data base generated for WSMV will facilitate the emerging area of molecular epidemiology, in which the origin and biological properties of pathogens responsible for new (or recurring) epidemics may be accurately and quickly identified based on nucleotide sequence analysis of pathogen populations. Improved understanding of virus replication, movement, host range, and transmission may lead to new ways for controlling the disease and reducing yield losses. Approximately a dozen Material Transfer Agreements have been established with other scientists interested in using the WSMV infectious clone (modified as a gene expression vector), most of whom plan to use the WSMV gene vector as a tool in functional genomics projects for wheat, barley, and other cereal crops.
Impacts
(N/A)
Publications
- Stenger, D.C., French, R.C. 2004. Functional replacement of wheat streak mosaic virus hc-pro with the corresponding cistron from a diverse array of viruses in the family potyviridae. Virology 323:257-267.
- Stenger, D.C., French, R.C. 2004. Complete nucleotide sequence of oat necrotic mottle virus: a distinct tritimovirus most closel related to wheat streak mosaic virus. Archives Of Virology 149:633-640.
- Baliji, S., Black, M.C., French, R.C., Stenger, D.C., Sunter, G. 2004. Spinach curly top virus:a newly described curtovirus species from southwest texas with incongruent gene phylogenies. Phytopathology 94:772- 779.
- Hudspeth, D.S., Stenger, D.C., Hudspeth, M.E. 2003. A cox2 phylogenetic hypothesis for the downy mildews and white rusts. Fungal Diversity 13:47- 57.
- Van Winkle, D.H., French, R.C., Stenger, D.C. 2004. Quantifying symptom severity in wheat infected with wheat streak mosaic virus using both visual and digital means of assessment. American Phytopathological Society Astracts. Phytopathology 94:S99.
- Stenger, D.C., French, R.C. 2004. Systematic replacement of wheat streak mosaic virus hc-pro. American Phytopathological Society Abstracts. Phytopathology 94:S99.
- French, R.C., Stenger, D.C. 2004. Reciprocal monophyly of potyvirus and rymovirus genera, family potyviridae, is suppored by genome sequences of agropyron mosaic virus and hordeum mosaic virus. American Phytopathological Society Abstracts. Phytopathology 94: S32.
- Baliji, S., Black, M.C., French, R.C., Stenger, D.C., Sunter, G. 2004. Spinach curly top virus: a new curtovirus species revealing a history of recombination among curtoviruses. American Phytopathological Society Abstracts. Phytopathology 94:S6.
- Stenger, D.C., French, R.C. 2004. Hc-pro replacements from diverse species of the family potyviridae are tolerated by the tritimovirus wheat streak mosaic virus. American Society For Virology Meeting. (Will not be published in journal.)
- French, R.C., Stenger, D.C. 2004. Complete nucleotide sequences of agropyron mosaic virus and hordeum mosaic virus support monnophyly of the genus rymovirus, family potyviridae. American Society For Virology Meeting. (Not published in journal.)
- Surendranath, B., Black, M.C., French, R.C., Stenger, D.C., Sunter, G. 2004. Spinach curly top virus: a new curtovirus species revealing a history of recombination among curtoviruses. American Society For Virology Meeting. (Not published in a journal)
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Progress 10/01/02 to 09/30/03
Outputs
(N/A)
Impacts
(N/A)
Publications
- Stenger, D.C., Seifers, D.L., French, R.C. 2002. Patterns of polymorphism in wheat streak mosaic virus: sequence space explored by a clade of closely related genotypes rivals that between the most divergent strains. Virology 302:58-70.
- French, R.C., Stenger, D.C. 2002. Wheat streak mosaic virus (descriptions of plant viruses no. 48, revised). Description Of Plant Viruses. A.A.B. descriptions of plant viruses, No. 393.
- FRENCH, R.C., STENGER, D.C. EVOLUTION OF WHEAT STREAK MOSAIC VIRUS: DYNAMICS OF POPULATION GROWTH WITHIN PLANTS MAY EXPLAIN LIMITED VARIATION. Annual Review Of Phytopathology. 2003. Ann. Rev. Phytopathology 41:199- 214.
- Stanley, J., Bisaro, D.M., Briddon, R.W., Brown, J.K., Czosnek, J., Fauquet, C.M., Gronenborn, B., Harrison, B., Maxwell, D.P., Stenger, D.C. 2003. Revision of taxonomic criteria for species demarcation in the geminiviridae family and new updated list of geminivirus species. Archives Of Virology 148:405-421.
- Stenger, D.C., French, R.C. 2003. Population genetics of wheat streak mosaic virus provides an insight into plant virus evolution. American Phytopathological Society Annual Meeting. Phytopathology 93:(Supplement) S99.
- Seifers, D.L., French, R.C., Stenger, D.C., Martin, T.J. 2003. Biological variation among wheat streak mosaic virus isolates. American Phytopathological Society Annual Meeting. Phytopathology 93:(supplement) S78.
- Idris, A.M., Brown, T.K., French, R.C., Stenger, D.C. 2003. Variation among isolates of pepper golden mosaic virus suggests a begomovirus species complex. American Phytopathological Society Annual Meeting. Phytopathology 93 (supplement):538
- FRENCH, R.C., STENGER, D.C. USE OF COALESCENT-BASED METHODS TO INFER RATES OF INSTRASPECIFIC RECOMBINATION IN RNA PLANT VIRUSES. AMERICAN PHYTOPATHOLOGICAL SOCIETY ANNUAL MEETING. 2003. Phytopathology 93 (Supplement) S27
- FRENCH, R.C., STENGER, D.C. EVIDENCE FOR FREQUENT RECOMBINATION IN A WHEAT STREAK MOSAIC VIRUS POPULATION. American Society For Virology Meeting. 2003. (Oral Presentation)
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