Progress 03/14/07 to 03/13/12
Outputs
Progress Report Objectives (from AD-416): Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non- structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV. 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. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the ability to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done. Yeast two hybrid methodology will be used to determine if potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC- Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant- negative interference effects on WSMV infection. This project has been approved by the University of Nebraska IBC on March 23, 2007 at Biosafety level 2 (BL-2). This is the final report for 5440-22000-023-00D, which expired in FY 2012 and was replaced by 5440-22000-024-00D. Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are the most important viral pathogens of wheat in the U.S. Great Plains region, and the latter virus was found to be prevalent in the 2008 growing season. Both these viruses are spread from plant to plant by the wheat curl mite. Research was undertaken to understand the etiology of TriMV, and interactions of WSMV and TriMV with wheat and their mite vector. The sequence of TriMV genome was determined and identified as a distinct virus from WSMV. The viral coat protein of TriMV was expressed in bacteria and used to produce a high quality antiserum, providing a much- needed method for high-throughput detection method for the management of TriMV and for germplasm screening in wheat breeding programs. This antiserum also can be used to develop TriMV diagnostic kits by biotech companies. A new wheat variety, Mace, was developed in collaboration with an ARS plant geneticist researcher at Lincoln, NE which provides significant genetic resistance to WSMV and TriMV. Since both viruses are transmitted by the same vector, mixed infections by WSMV and TriMV are expected to be commonplace. In co-infections, WSMV and TriMV synergistically interact with each other resulting in increased virus concentrations in wheat plants which causes severe effects on wheat growth and yield in susceptible cultivars compared to the resistant cultivar Mace. Therefore, cultivar selection will be an important management strategy for the control of the WSMV/TriMV disease complex in wheat. The double virus resistance of Mace should encourage other wheat breeders to incorporate its virus resistance locus in their breeding programs as well. The gene for green fluorescent protein (GFP) was inserted into WSMV genome to form bright fluorescent aggregates in infected cells. The availability of GFP-tagged virus has facilitated rapid monitoring of virus presence/spread within a wheat plant during virus infection. GFP- tagged virus can be used as a non-destructive screening tool in the development of new WSMV-resistant wheat varieties. The GFP-tagged virus will also facilitate examining virus-host and virus-vector interactions to better understand the virus life cycle for possible clues to develop new disease management strategies by breaking the disease cycle. In addition to genetic resistance, plants are known to have innate defenses against invading viruses. In turn, viruses have evolved with proteins to overcome host defense mechanisms. The P1 protein of WSMV was identified as a suppressor of RNA silencing. The coat protein and HC-Pro genes of WSMV were shown to play critical roles in transmission of WSMV by the wheat curl mite. The outcome of this research provides a new WSMV/TriMV disease management strategy to wheat growers, a new tool for breeders to screen for WSMV resistance, a new diagnostic method for TriMV for extension Plant Pathologists, and advances the understanding of WSMV biology.
Impacts
(N/A)
Publications
- Young, B.A., Stenger, D.C., Ou, F., Morris, T., Tatineni, S., French, R.C. 2012. Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms. Virus Research. 163: 672-677.
- Tatineni, S., Robertson, C.J., Garnsey, S.M., Dawson, W.O. 2011. A plant virus evolved by acquiring multiple non-conserved genes to extend its host range. Proceedings of the National Academy of Sciences. 108:42:17366-17371.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non- structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV. 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. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the abilty to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done Yeast two hybrid methodology will be used to determine if Potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC- Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant- negative interference effects on WSMV infection. This project has been approved by the University of Nebraska IBC on March 23, 2007 at Biosafety level 2 (BL-2). Wheat streak mosaic virus (WSMV) is transmitted by the wheat curl mite. One viral gene, Helper component-proteinase, is required for mite transmission. Six mutations were individually introduced into the carboxyl-terminus of the coat protein of WSMV to replace acidic amino acid residues with alanine. This region of the coat protein was chosen because it is thought to be close to the surface of virus particles. Mutated virus were inoculated to wheat and then tested for mite transmission. Transmission rates of an aspartic acid replacement at position 289 and a similar substitution at position 326 were reduced to 15% compared to 55% for wild type virus. The ability of WSMV and triticum mosaic virus (TriMV) to alter disease symptoms in mixed infections with two other cereal viruses was tested. Wheat infected with brome mosaic virus in combination with either WSMV or TriMV had much more severe symptoms than singly infected plants. Similar results were obtained with barley stripe mosaic virus. This suggests that TriMV is able to suppress RNA silencing in wheat to a similar degree as WSMV. Three populations of wheat curl mite were tested for their abilities to transmit TriMV. These mite populations, designated as Nebraska, Montana and South Dakota biotypes can be distinguished by mitochondrial DNA cytochrome oxidase subunit 1 gene sequences. All three biotypes are efficient vectors of WSMV as was shown by single mite transfers from infected source plants to test plants. However, only the Nebraska biotype was able to transmit TriMV using single mites. Transmission of TriMV by Montana and South Dakota biotypes required the transfer of hundreds of mites from infected source plants to test plants. Accomplishments 01 Only one strain of wheat curl mite is an efficient vector of triticum mosaic virus. Triticum mosaic virus is an emergent virus infecting whea in the US. It, like wheat streak mosaic virus, is transmitted by the wheat curl mite. Previous studies determined that there are genetically distinct strains of the wheat curl mite present in the region. Researchers in Lincoln, NE, in cooperation with the University of Nebras investigated whether all strains of the wheat curl mite are equally competent to transmit triticum mosaic virus. They demonstrated that one mite strain was much more efficient in transmitting the virus than other strains, while all mite strains are equally capable of transmitting whea streak mosaic virus. Consequently the prevalence of triticum mosaic vir is expected to vary from place to place and from year to year depending the strain composition of wheat curl mite populations. This information will be useful to extension plant pathologists for understanding why the incidence of triticum mosaic virus may differ from that of wheat streak mosaic virus. Ultimately, the information should assist wheat growers better manage the crop. 02 Wheat streak mosaic virus coat protein is a mite transmission determinan Wheat streak mosaic is the most economically important virus in the Gre Plains region of the US and is transmitted by wheat curl mites. Previously, the helper component-proteinase of Wheat streak mosaic virus was identified as a determinant of wheat curl mite transmission. ARS scientist in Lincoln, NE, in collaboration with scientists at the University of Nebraska-Lincoln identified the coat protein of Wheat stre mosaic virus as an additional viral determinant involved in efficient mi transmission. Mutations in the coat protein affected the mite transmission efficiencies by 2-3-fold compared to that of wild-type viru Identification of helper component-proteinase and coat protein as mite transmission determinants will provide scientists with information on 'candidate genes' to produce virus resistant transgenic wheat by disrupting the virus life cycle.
Impacts
(N/A)
Publications
- Tatineni, S., Mcmechan, A., Hein, G., French, R.C. 2011. Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking. Virology. 410 (2011) 268-281. doi:10.1016/j.virol.2010.10.043.
- Tatineni, S., Van Winkle, D.H., French, R.C. 2011. The N-terminal region of Wheat streak mosaic virus coat protein is a host- and strain-specific long-distance transport factor. Journal of Virology. 85: 1718-1731.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non- structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV. 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. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the abilty to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done Yeast two hybrid methodology will be used to determine if Potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC- Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant- negative interference effects on WSMV infection. The ability of the wheat curl mite to simultaneously transmit both wheat streak mosaic virus and the newly emergent wheat virus, triticum mosaic virus was experimentally confirmed. We found that the virus content of both Wheat streak mosaic virus and Triticum mosaic virus was two to seven fold higher in mixed infections compared to single infections with each virus in susceptible wheat varieties. On the other hand, virus content of both viruses was much lower in infected plants of the Wheat streak mosaic virus�resistant variety Mace. Therefore, the risk of spread of both viruses by the wheat curl mite may be increased in susceptible wheat varieties. The gene for green fluorescent protein (GFP) was inserted into an infectious clone of Wheat streak mosaic virus (WSMV). Expression of GFP requires it to be cleaved from the WSMV polyprotein by viral proteinases. We examined a range proteinase cleavage sites for release of GFP from the viral polyprotein. Expression of GFP was stable for at least seven serial passages and for 120 days after inoculation. This provides a valuable tool to study transient gene expression in plants and for screening for virus resistance. A stable, infectious cDNA clone of the genome of triticum mosaic virus was developed. RNA transcribed in vitro was highly infectious to wheat, barley, and triticale, demonstrating that this clone has biological properties identical to native virus. This will greatly facilitate studies of viral gene structure and function. Improved understanding of virus replication and transmission may lead to new ways for controlling the disease and reducing yield losses. Resistance of the maize line SDp2 to the type strain of wheat streak mosaic virus was shown to be due to a block in systemic infection but not to a lack of replication in inoculated leaves. By making gene exchanges between the type strain and a resistance breaking strain, Sidney 81, and by site-directed mutagenesis, we have identified four specific amino acid residues in the viral coat protein that enable the virus to overcome resistance to infection exhibited by SDp2. These differences in coat protein sequences likely correspond to resistance/susceptibility genes in the host and may impact studies designed to identify resistance genes in corn. Accomplishments 01 Mixed infections of wheat streak mosaic virus (WSMV) and the newly emergent triticum mosaic virus (TriMV) multiply infection in susceptible wheats. As WSMV and, TriMV, are transmitted by the same mite vector, mix infections by the two viruses are expected to be commonplace. ARS scientists in Lincoln, NE examined the effects of mixed infections in tw wheat varieties susceptible to WSMV, and one variety, Mace, that is resistant to WSMV. There, research indicated that there was increased virus content in susceptible varieties infected by both viruses, which could enhance their natural spread by mites. Thus, it is very important that growers use resistant varieties where available and follow current cultural disease control practices such as delayed planting of susceptib varieties in the fall. 02 Development of an improved wheat streak mosaic virus clone for tracking viral infection. ARS scientists in Lincoln, NE have constructed a clone Wheat streak mosaic virus containing a gene for green fluorescent protei (GFP). GFP gives a green fluorescence to infected tissue when viewed wit a microscope under blue light. The GFP gene was modified so that it produces bright green aggregates in infected cells. This has greatly improved the ability to trace spread of the virus in infected plant tissues and will lead to a better understanding of virus-host interactio It additionally provides a valuable tool for screening plants for disea resistance. 03 Development of infectious clones of Triticum mosaic virus. Triticum mosa virus is the type member of the recently established Poacevirus genus of the potyvirus family. Little is known about the biology of these viruses and no molecular studies have been done. ARS scientists in Lincoln, NE have developed the first infectious clones for genus of viruses and established their biological activity. This will greatly facilitate studies of viral gene structure and function and may lead to new ways fo controlling the disease and reducing yield losses.
Impacts
(N/A)
Publications
- Albiach-Marti, M.R., Robertson, C., Gowda, S., Tatineni, S., Belliure, B., Garnsey, S.M., Folimonova, S.Y., Moreno, P., Dawson, W.O. 2010. The Pathogenicity Determinant of Citrus Tristeza Virus Causing the Seedling Yellows Syndrome is Located at the 3�-Terminal Region of the Viral Genome. Molecular Plant Pathology, Volume 11 (1), pages 55-67.
- Tatineni, S., Graybosch, R.A., Hein, G.L., Wegulo, S.N., French, R.C. 2010. Wheat Cultivar-Specific Disease Synergism and Alteration of Virus Accumulation During Co-Infection with Wheat Streak Mosaic Virus and Triticum Mosaic Virus. Phytopathology, volume 100, pages 230-238.
- Tatineni, S., Ziems, A.D., Wegulo, S.N., French, R.C. 2009. Triticum Mosaic Virus: A Distinct Member of the Family Potyviridae with an Unusually Long Leader Sequence. Phytopathology. 99:943-950.
- Tatineni, S., Gowda, S., Dawson, W.O. 2010. Heterologous Minor Coat Proteins of Citrus Tristeza Virus Strains Affect Encapsidation, but the Coexpression of HSP70h and p61 Restores Encapsidation to Wild-Type Levels. Virology. Volume 402:262-270.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non- structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV. 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. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the abilty to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done Yeast two hybrid methodology will be used to determine if Potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC- Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant- negative interference effects on WSMV infection. Significant Activities that Support Special Target Populations This research involves the interaction of streak mosaic virus (WSMV) with both plant hosts as well as with its mite vector. Experiments were designed identify and characterize WSMV determinant(s) of pathogenicity enhancement and suppression of the host defense RNA silencing pathway; identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite; and develop and evaluate transgenic wheat expressing WSMV non-structural proteins. An infectious clone of the type strain of WSMV was constructed for experiments designed to determine WSMV host range determinants. By exchanging genes between the sidney isolate (pathogenic on SDp2 corn) and the type isolate (nonpathogenic on SDp2 corn) we determined that the viral coat protein (CP) was the major determinant of host range. WSMV HCPro, NIa, and CP have been cloned in yeast new yeast two hybrid vectors for investigating protein-protein interactions. The first four amino acids of HCPro were found to be dispensable for mite transmission but deletion of eight amino acids abolished vector transmission. The reporter gene green fluorescent protein (GFP) was cloned downstream of the HCPro and NIa genes of WSMV and transferred into binary vectors for wheat transformation. Ten independent transgenic wheat lines expressing WSMV coat protein were developed. Antisera were made to all WSMV nonstructural proteins (P1, HCPro, P3, CI, NIa, and NIb) which will aid in screening transgenic wheat for gene and protein expression levels. cDNA of a new wheat virus, Triticum mosaic virus, was cloned and the complete genome sequence was determined. Phylogenetic analysis revealed that Triticum mosaic virus should be placed in a new genus in the plant virus family Potyviridae and this taxonomic proposal was approved by the Potyviridae Study Group of the International Committee on Taxonomy of Viruses. We found that wheat infected both Triticum mosaic virus and WSMV exhibited more severe symptoms than single infections of wheat with either virus. Single mite transmission assays revealed that Triticum mosaic virus is effieciently transmitted by the Nebraska biotype of the wheat curl mite. Technology Transfer Number of New/Active MTAs(providing only): 1
Impacts
(N/A)
Publications
- Tatineni, S., Afunian, M.R., Gowda, S., Hilf, M.E., Bar-Joseph, M., Dawson, W.O. 2009. Characterization of the 5�- and 3�-terminal subgenomic RNAs produced by a capillovirus: evidence for a CP subgenomic RNA. Virology 385 (2009) 521�528.
- Stenger, D.C., French, R.C. 2009. Wheat streak mosaic virus genotypes introduced to Argentina are closely related to isolates from the American Pacific Northwest and Australia. Archives of Virology: 154:331-336.
- Tatineni, S., Afunian, M.R., Hilf, M.E., Gowda, S., Dawson, W.O., Garnsey, S.M. 2009. Molecular Characterization of Citrus tatter leaf virus Historically Associated with Meyer Lemon Trees: Complete Genome Sequence and Development of Biologically Active In Vitro Transcripts. Phytopathology Volume 99, Pages 423-431.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non- structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV. 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. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the abilty to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done Yeast two hybrid methodology will be used to determine if Potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC- Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant- negative interference effects on WSMV infection. Significant Activities that Support Special Target Populations Experiments were designed identify and characterize wheat streak mosaic virus (WSMV) determinant(s) of pathogenicity enhancement and suppression of the host defense RNA silencing pathway; identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite; and develop and evaluate transgenic wheat expressing WSMV non- structural proteins. Twelve deletion mutants of the P1 protein of WSMV were constructed in expression vector plasmids and evaluated in a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. This revealed that the first 17 amino acid residues of P1 are dispensable for suppression of host plant RNA silencing. Several of these deletion mutants have been cloned into a full length infectious clone of WSMV and are being evaluated for effects on virus viability and symptom development. The complete genome sequence was determined for the Nebraska maize chlorotic mottle virus isolate used in disease synergism assays with WSMV. A GUS reporter gene was fused to the amino-terminus of HC-Pro in an infectious clone of WSMV, inoculated to wheat, and exposed to wheat curl mites. Histochemical staining of recovered mites revealed the presence of GUS-HC-Pro fusion protein in mite mouth parts. A study underway will examine whether mites retain the HC-Pro fusion protein following a molt. The P3 and NIa genes of WSMV have been cloned in binary vectors for wheat transformation. Clones of His-tagged HC-Pro and His- tagged P1 have been constructed to aid the study of potential protein- protein interactions and antisera is being produced for nonstructural proteins of WSMV using synthetic peptides. An infectious clone of the type strain of WSMV is being constructed for experiments designed to determine WSMV host range determinants. A new wheat virus, Triticum mosaic virus, was identified in the spring and fall of 2008. A portion of the viral genome has been cloned using PCR and potyvirid-specific primes, and will be sequenced shortly. A span of 4 kbp of the mitochondrial DNA from the wheat curl mite has been sequenced. This segment contains, in order the 12S and 16S ribosomal RNA genes, the gene subunit two of NADH dehydrogenase, the Met tRNA gene, and the genes for cytochrome oxidase subunits 1 and 2. This gene order is unique among mitochondrial DNA of metazoans yet reported. This research is a component of National Program 303 Component 2. Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors.
Impacts
(N/A)
Publications
- Young, B.A., Hein, G.L., French, R.C., Stenger, D.C. 2007. Substitution of conserved cysteine residues in Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite. Archives of Virology 152:2107- 2111.
- Stenger, D.C., Young, B.A., Qu, F., Morris, T., French, R.C. 2007. Wheat streak mosaic virus lacking HC-Pro is competent to produce disease synergism in double infections with maize chlorotic mottle virus. Phytopathology 97:1213-1221.
- Stenger, D.C., French, R.C. 2008. Complete nucleotide sequence of a maize chlorotic mottle virus isolate from Nebraska. Archives of Virology 153: 995-997.
- Folimonova, S.Y., Folimonov, A.S., Tatineni, S., Dawson, W.O. 2008. Citrus tristeza virus: survival at the edge of the movement continuum. Journal of Virology, Volume 82, page 6546-6556.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Objective 1. Identify and characterize WSMV determinant(s) of pathogenicity enhancement (disease synergism) and suppression of the host defense RNA silencing pathway. Objective 2. Identify and characterize WSMV determinant(s) responsible for semipersistent transmission by the wheat curl mite. Objective 3. Develop and evaluate transgenic wheat expressing WSMV non- structural proteins (P1, HC-Pro, P3, NIa) for gene complementation and pathogen-derived resistance to WSMV. 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. We will use a Agrobacterium tumefaciens/Nicotiana benthamiana system based on induced silencing of a green fluorescent protein (GFP) transgene. Individual protein coding regions of wheat streak mosaic virus (WSMV) will be cloned into a binary shuttle vector in A. tumefaciens. Each WSMV protein will be tested for the abilty to restore GFP expression in infiltrated leaves. Protein domains involved in the suppression phenotype will be identified by in vitro mutagenesis. Effects of mutants on virus pathogenicity will be tested by introducing identified mutations into an infectious WSMV cDNA clone and tested for disease synergism in mixed infections with maize chlorotic mottle virus. Experiments will be done Yeast two hybrid methodology will be used to determine if Potential interactions between WSMV structural proteins (coat protein and NIa) with a known mite transmission determinant, HC-Pro, will be evaluated using immunoprecipitation, yeast two hybrid and in vitro binding assays. Relevant protein domains will be identified by in vitro mutagenesis and evaluated for effects on mite transmission. Four WSMV proteins (P1, HC- Pro, P3, NIa) will be expressed in transgenic wheat and evaluated for trans-complementation with deletion mutants of WSMV. A lethal HC-Pro mutant will be expressed in wheat and evaluated for potential dominant- negative interference effects on WSMV infection. Accomplishments Wheat streak mosaic virus P1 protein is a suppressor of plant RNA gene silencing. Plants have developed basal defenses against viral attack by degrading viral RNA, termed RNA silencing. Aphid transmitted relatives of wheat streak mosaic virus use their HC-Pro protein to inhibit host RNA silencing. However, the HC-Pro gene of wheat streak mosaic virus can be entirely deleted with little effect on virus multiplication. Each of the wheat streak mosaic virus encoded proteins was tested for suppression of RNA silencing revealing that the P1 protein of wheat streak mosaic virus fulfills this role. This research provides new information on molecular basis of pathogenicity of wheat streak mosaic virus. 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).
Impacts
(N/A)
Publications
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