Progress 02/15/04 to 02/14/08
Outputs
OUTPUTS: We have determined the complete nucleotide sequences of over thirty genomes of Barley yellow dwarf (BYDV) and Cereal yellow dwarf viruses (CYDV). BYDV and CYDV cause similar symptoms, are phloem-limited, and are transmitted only by aphids. Here we report that, among viruses collected across the US and selected international locations, BYDV-PAV is the most common virus and its sequence is remarkably invariant across diverse geographic locations. The severe (in oats) PAV-like BYDV-PAS virus is relatively rare, but present in regions as diverse as Alaska, New York and Hungary. A new BYDV-PAV-like virus (BYDV-PAV-CN) that differs substantially from BYDV-PAV or BYDV-PAS was discovered by collaborators in China. In contrast to most BYDVs, CYDV isolates showed striking variation. The severe CYDV-RPS virus, originally isolated in Mexico, appears to be common in western North America north to Alaska, while the mild CYDV-RPV-NY virus may predominate in the east. In the Midwest, the
CYDVs consist of diverse chimeric isolates, containing a patchwork of CYDV-RPS and CYDV-RPV-NY-like sequences, as well as some entirely new sequences. We found that the major recombination sites in CYDVs coincide with the ends of the coat protein open reading frame (ORF) and also were present within the readthrough domain ORF. The proteins encoded by both ORFs are required for aphid transmission. Thus recombination that exchanges aphid vector specificity, and possibly symptom severity determinants, may occur frequently among CYD viruses. This recombination may also be a way by which CYDVs generate entirely new aphid vector specificities. We determined the first complete genome sequence of BYDV-RMV, one of the original five serotypes described by Rochow. BYDV-RMV is defined by its efficient transmission by Rhopalosiphum maidis, a maize aphid. BYDV-RMV accumulates to very low levels in plants, relative to other BYDVs and CYDVs, making it difficult to obtain enough material for
sequencing. The complete sequence revealed that it is an entirely new virus. The 5' half of its genome and coat protein gene are more closely related to dicot-infecting poleroviruses than to CYDV or BYDV. Thus, BYDV-RMV must be assigned a new name. In addition to the results and methods already published, the completed viral genome sequences have been disseminated publically by posting on Genbank. The above discoveries have been presented in relevant scientific meetings (e.g. Plant and Animal Genome, American Phytopathological Society) and two manuscripts describing the most recent discoveries are being written up for publication in 2008.
PARTICIPANTS: W. Allen Miller, PD, Iowa State University; Stewart M. Gray, co-PD, Cornell University, USDA-ARS; Joseph M. Anderson, co-PD, Purdue University, USDA-ARS; Randy Beckett, sequencing manager and technician, Iowa State University; Emily Benson, technician, Cornell University, USDA-ARS; Dawn Smith, technician, Cornell University, USDA-ARS; Laura Sweets, contact, provided YDV isolates from Missouri, University of Missouri; Robert Hunger, contact, provided YDV isolates from Oklahoma, Oklahoma State University; Roland Sigvald, contact, provided YDV isolates from Sweden, Swedish University of Agricultural Sciences, Uppsala, Sweden; Elizabeth Borer, contact, provided YDV isolates from California, Oregon, Oregon State University; Carolyn Malmstrom, contact, provided YDV isolates from California, Michigan State University; Jelena Kraft, undergraduate, learned to sequence YDV isolates, Iowa State University; Rachel Smith, undergraduate, learned to sequence YDV isolates, Iowa State
University; Rachel Sheldahl, undergraduate, learned to sequence YDV isolates, Iowa State University; Deborah Bertrand, undergraduate, learned to sequence YDV isolates, Iowa State University; Kelli Barr, undergraduate, learned to sequence YDV isolates, Iowa State University; Chris Siguenza, school teacher, learned to sequence YDV isolates, Iowa State University; Jill Hansen, high school teacher, Panama City FL, learned to sequence YDV isolates, Iowa State; Natasa Dukic, grad student, University of Belgrade, learned to sequence YDV isolates, Iowa State University
TARGET AUDIENCES: Plant pathologists, cereal breeders breeding for virus resistance, grassland ecologists, molecular plant virologists, viral bioinformaticists
PROJECT MODIFICATIONS: International components of the project were reduced owing to difficulty of shipping isolates in the post- 9/11 era. Bioinformatics and database components were reduced because the computer specialist who was a co-PD (Xiaowu Gai) resigned from Iowa State University, and no funds had been budgeted for salary for a computer specialist, as his salary had been provided by the university. More training of students in virus sequencing was performed than planned as indicated in the Participants list.
Impacts
The yellow dwarf viruses are the most economically important, ubiquitous viruses of wheat, barley and oats, worldwide. Different strains vary greatly in symptomatology, aphid vector, or ability to break resistance. They are often difficult to distinguish by serological methods which detect only viral coat protein, or by PCR methods that rely on the coat protein sequence. This focus on coat protein or the gene encoding it is inadequate for detection because yellow dwarf viruses that differ greatly in symptomatology, aphid vector, or genome organization can have very similar coat protein sequences. Sequences of the COMPLETE genomes of many isolates from around the world are providing us with an understanding of how much these viruses vary in sequence. Availability of full-length sequences allows design of specific nucleic acid-based detection methods (e.g. PCR, gene chips), and may shed light on genes that confer the different phenotypes. Researchers in China, Czech
Republic, Turkey, Ireland, North Carolina, Cornell University and Michigan State University, among others, have been - or will be - using our sequence data in their research on topics as diverse as translational recoding, identification of field isolates, and effect of viruses on grassland ecology. The freely accessible full-length sequences will be essential tools for research on understanding and controlling YDVs in rich and poor countries. Moreover this work is relevant for quarantine purposes, as isolates thought to be strains of the same virus are actually totally different viruses. Application of these data to virus identification and resistance breeding should lead to more sustainable cropping practices, increase yields and benefit the farmer and ultimately the consumer.
Publications
- Liu F, Wang X, Liu Y, Xie J, Gray SM, Zhou G, Gao B. (2007) A Chinese isolate of barley yellow dwarf virus-PAV represents a third distinct species within the PAV serotype. Arch Virol. 152, 1365-1373.
- Gray, S. M. 2008. Barley Yellow Dwarf. In: APS Compendium of Wheat Diseases. William Bockus, ed. APS Press, St. Paul MN (In press).
- Gray, S. M. 2008. Aphid transmission of plant viruses. In: Current Protocols in Microbiology. R Coico, T Kowalik, J Quarles, B Stevenson, R Taylor, eds. John Wiley & Sons, Inc. New York (In press).
- Beckett R, Miller WA (2007) Rapid full-length cloning of non-polyadenylated RNA virus genomes. In Current Protocols in Microbiology (R Coico, T Kowalik, J Quarles, B Stevenson, R Taylor eds.) John Wiley & Sons, Inc. New York. pp. 16F.3.1 - 16F.3.18.
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Progress 01/01/06 to 12/31/06
Outputs
We have determined the complete nucleotide sequences of over thirty genomes of Barley yellow dwarf (BYDV) and Cereal yellow dwarf viruses (CYDV). Both viruses cause similar symptoms, are phloem-limited, and are transmitted only by aphids. They exist in low abundance in plants and are difficult to sequence in their entirety. Here we report that, among viruses collected across the US and selected international locations, the PAV serotype of BYDV is the most common strain and its sequence is remarkably invariant among diverse geographic locations. In contrast, CYDV isolates show striking variation. The severe CYDV-RPS virus, originally isolated in Mexico, appears to be common in western North America all the way to Alaska, while the mild CYDV-RPV New York virus predominates in the east. In the Midwest, the CYDVs consist of diverse chimeric isolates, containing a patchwork of CYDV-RPS and CYDV-RPV-NY-like sequences, as well as CYDV isolates containing entirely new
sequences. Using the TOPALi (http://bioinformatics.oxfordjournals.org/cgi/reprint/20/11/1806) computer program, we found that the major recombination sites in CYDVs coincide with the ends of the coat protein open reading frame (ORF) and also were present within the readthrough domain ORF. Both ORFs are required for aphid transmission. Thus recombination that exchanges aphid vector specificity, and possibly symptom severity determinants, may occur frequently among CYD viruses. The near complete sequence of the RMV serotype reveals it to be a polerovirus like the CYDVs, but more closely related to other poleroviruses, such as Beet chlorosis virus, than to CYDVs, suggesting it is a completely different virus species that may merit a name other than CYDV or BYDV.
Impacts
The yellow dwarf viruses are the most economically important, ubiquitous viruses of wheat, barley and oats, worldwide. Different strains vary greatly in symptomatology, aphid vector, or ability to break resistance. They are often difficult to distinguish by serological methods which detect only viral coat protein. Sequences of the complete genomes of many isolates from around the world are providing us with an understanding of how much these viruses vary in sequence. Availability of full-length sequences allows design of specific PCR-based detection methods, and may shed light on genes that confer the different phenotypes. Researchers in Czech Republic, Turkey, Ireland, and Michigan State University, among others, have been -- or will be -- using our sequence data in their research on topics as diverse as translational recoding, identification of field isolates, and effect of viruses on wild grass ecology. The freely-accessible, full-length sequence database will be
an essential tool for research on understanding and controlling YDVs in rich and poor countries. This will lead to more sustainable cropping practices, increase yields and benefit the farmer and ultimately the consumer.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
PAV, MAV, RPV, and RMV serotypes of Barley yellow dwarf and Cereal yellow dwarf viruses were sequenced. Eleven near-complete BYDV-PAV sequences (minus terminal regions) from seven states (KS, MO, IA, OK, CA, AR, NY) showed a high level of similarity to each other and to previously published sequences of Illinois and Japanese isolates. Phylogenetic distance values of <0.0156 were calculated between these unknown and known isolates using the Neighbor Joining method. Sequence variation occurred primarily in the highly variable region of the 3 prime untranslated region between bases 5000-5100. A single near-complete sequence of a Swedish BYDV-MAV was not significantly different from published sequences of this serotype. Additional MAV isolates are currently in the sequencing pipeline. Three CYDV-RPV isolates from Missouri and New York showed high similarity to a published New York isolate. However, one of two Missouri isolates showed a marked similarity in the 3 prime UTR
to the severe, resistance breaking CYDV-RPS from Mexico. Significantly, these two Missouri isolates, from different counties, would be judged identical based on serotype alone. Only by complete sequencing can the spread of potentially more severe isolates be studied. The 3 prime half of a member of the maize-infecting RMV serotype was sequenced. This is the first sequence of an RMV isolate outside of the coat protein. It shows striking divergence from other BYDV and CYDV isolates, perhaps a function of its different vector (Rhopalosiphum maidis), its favored host (maize rather than Triticeae and Avena), or its extremely low titer in plants. Other isolates were characterized serologically and for aphid vector specificity. Isolates from nine states (AK, AR, CA, FL, IA, MT, NY, OR, SD) and Sweden in (or related to) serotypes RMV, PAV, RPV, and MAV were tested for transmission by five different aphid species. Representatives of these isolates will be sequenced in 2006. This should provide
information on the specific sequence determinants of their biological properties. Co-PI Gray attempted agroinfection of Nicotiana benthamiana by introducing full-length clones of CYDV and BYDV genomes, constructed in the Miller lab, into Agrobacterium expression vectors. He found that the CYDV clone, but not the BYDV clone, replicated and spread in agro-inoculated N. benthamiana plants. This should be valuable for future functional genomics studies.
Impacts
Barley yellow dwarf virus and its cousin, cereal yellow dwarf virus, are the most economically important, ubiquitous viruses of wheat, barley and oats, worldwide. Different strains vary greatly in symptomatology, aphid vector, or ability to break resistance. They are often difficult to distinguish by serological methods which detect only viral coat protein. Sequences of the complete genomes of many isolates from around the world will give us an understanding of how much these viruses vary in sequence. Availability of full-length sequences allows design of specific PCR-based detection methods, and may shed light on genes that confer the different phenotypes. Labs in Serbia and Iran have been positively impacted by this project. Natasa Dukic of Servia visited for one month, learning PCR and cloning methods. She will provide Serbian BYDV isolates to sequence. We are acknowledged by international collaborators in: Rastgou et al., 2005, Relationships of Barley yellow dwarf
virus-PAV and Cereal yellow dwarf virus-RPV from Iran with viruses of the family Luteoviridae. Eur. J. Plant Pathol. 113:321. The freely accessible full-length sequence database, YDDB, will be an essential tool for research on understanding and controlling YDVs in rich and poor countries. This will lead to more sustainable cropping practices, increase yields and benefit the farmer and ultimately the consumer.
Publications
- Henry M, Beckett R, Miller WA (2004) Cereal yellow dwarf associated with CYDV-RPS. In: Viruses and Virus Diseases of Poaceae (Graminae) H Lapierre, P-A Signoret, eds. INRA Press, Paris. pp 561-562.
- Lapierre H, Henry M, Beckett R, Miller WA (2004) Cereal yellow dwarf associated with CYDV-RPV. In: Viruses and Virus Diseases of Poaceae (Graminae) H Lapierre, P-A Signoret, eds. INRA Press, Paris. pp 563-565.
- Miller WA, Beckett R, Jacquot E, Lapierre H (2004) Genus Luteovirus. In: Viruses and Virus Diseases of Poaceae (Graminae) (H Lapierre, P-A Signoret, eds.) INRA Press, Paris. pp 370-374.
- Miller WA, Beckett R, Ordon F, Friedt W, Lapierre H (2004) Barley yellow dwarf associated with BYDV-PAV. In: Viruses and Virus Diseases of Poaceae (Graminae) H Lapierre, P-A Signoret, eds. INRA Press, Paris. pp 465-471.
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