Progress 06/19/07 to 05/21/12
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop corn and soybean virus disease control strategies. � Sub-objective 1.A. Characterize the nature of host resistance to virus disease. � Sub-objective 1.B. Identify, map, and clone virus-resistance genes. � Sub-objective 1.C. Determine the effect of combining quantitative virus resistance with insect resistance on virus disease severity. � Sub-objective 1.D. Characterize insect vector/virus relationships. Objective 2: Identify and characterize emerging virus diseases in corn and soybeans. Objective 3: Develop virus-based gene vectors in corn. Soybeans and corn are the two highest value crops grown in the U.S. Although research from this laboratory and others has led to significant improvements in their management, virus diseases continue to be annual threats. Furthermore, history has shown that unanticipated, and often unknown, new virus disease problems can rapidly emerge at any time. In soybeans, the threat of virus diseases has increased even more following the introduction of the soybean aphid into the U.S. in 1999. This is the first soybean-colonizing aphid in the U.S., and the consequences for future virus disease problems in soybeans are unknown. A long-term objective of this program is to reduce corn and soybean losses attributable to virus diseases. Our strategy to do so is to identify corn and soybean viruses when they arise and to characterize their biology and epidemiology (Objective 2), and to then use this information develop practical, effective methods and strategies for minimizing crop losses (Objective 1). Our final objective (Objective 3) is to use our knowledge and expertise in maize virology to develop new tools for forward and reverse genetic analysis of maize gene function. We will develop gene silencing and expression vectors based on selected maize viruses, and use these vectors immediately to characterize the functions of candidate maize genes thought to be important for virus infection and/or resistance. Because reliable vectors are lacking for monocotyledenous plants such as corn, vectors developed under this objective would be of benefit both to corn geneticists and to those seeking to use corn for the production of non-endogenous materials. Approach (from AD-416): Our overall approach is to: 1) identify existing and emerging viruses; 2) understand their biology; and 3) develop effective disease control strategies. Known viruses will be identified using existing diagnostics. For previously uncharacterized viruses, we will culture them in healthy plants using mechanical or arthropod transmission, determine their characteristics, and develop diagnostic assays. This knowledge will be used to formulate disease control strategies, although, usually, the most effective and economic control strategy is to use virus-resistant crop varieties and cultivars. Therefore, a primary focus of this project is to identify, characterize, and map virus resistance in maize and soybean germplasm. To develop an understanding of how resistance genes work, they will be isolated and characterized, and their role in the molecular and biochemical changes associated with virus resistance will be examined. Also, factors affecting virus transmission by arthropod vectors will be characterized so that alternative disease control methods can be developed. Maize virus-based gene expression and silencing vectors will be developed to facilitate functional analysis of plant resistance genes using forward and reverse genetics. Such vectors should have broad impact as few are available for cereals. The impact of this research will be to advance our knowledge of virus diseases of corn and soybeans and provide vital information for the development of control strategies to reduce disease losses. This project terminated 6/18/12 and has been replaced with 3607-22000- 013-00D. Progress made over the first four years of the project is as follows. Objective 1: 1) mapping resistance to 7 viruses in a Oh1VI x Oh28 population; 2) developing germplasm from plants with putative mutator insertions in Wsm1 and Wsm2 and demonstrating similar responses to wheat streak mosaic virus (WSMV) and maize dwarf mosaic virus (MDMV), suggesting a pleiotropic loss of resistance; 3) mapping introgressed regions from Pa405 into Oh28 in near isogenic lines for Wsm1, Wsm2 and Wsm3, and identifying new closely linked markers for these genes; 4) identifying about 80 differentially expressed transcripts in resistant and susceptible maize after inoculation with MDMV, including ubiquitin, a small heat shock protein, a transcription factor and two lectins; 5) identifying parameters for efficient transmission of fijiviruses to maize using vascular puncture inoculation that allowed for transmission to >50% of maize seedlings; 6) identifying virus concentration as a critical factor in an vitro system for identifying maize chlorotic dwarf virus (MCDV) helper component activity; 7) demonstrating self interaction of the R78 polyprotein region and interaction of R78 with the MCDV protease. Objective 2: 1) identification of Stolbur phytoplasma transmitted by the leafhopper Reptalus panzeri as the causal agent of Maize redness (MR) in Serbia; 2) description of the roles of wheat, Johnsongrass and R. panzeri in increasing disease incidence; 3) demonstration of yield losses due to MR; 4) development of genome sequence information for stolbur phytoplasma from maize; 5) identifying the re-emergence of Maize rough dwarf virus in Spain; 4) production of antisera to Bean pod mottle virus, Wheat mosaic virus, WSMV, MDMV, and Pantoea stewartii. Objective 3: 1) demonstrating that the maize necrotic streak virus (MNeSV) coat protein, p19 and p21 are required for virus movement; 2) development of two MNeSV-based vectors with different insertion sites; 3) identification of conditions for expression of MFSV N, P and G genes, a viral replicon and T7 RNA polymerase in Drosophila S2 cells. Final year progress (FY12) includes 1) release of a virus resistant synthetic maize population; 2) identification of loci for resistance to five maize-infecting viruses in the maize inbred line Oh1VI; 3) demonstration that loci conferring resistance to MDMV and sugarcane mosaic virus (SCMV) also confer resistance to Johnsongrass mosaic virus and sorghum mosaic virus; 3) development of an infectious clone for maize dwarf mosaic virus; 4) demonstration of MDMV and maize chlorotic dwarf virus MCDV in Ohio; 5) assembly of the Graminella nigrifrons transcriptome, the leafhopper is the vector of MCDV and maize fine streak virus (MFSV); and, 6) identification of maize chlorotic mottle virus (MCMV) and SCMV as the causal agents of maize lethal necrosis in Kenya. Significant Activities that Support Special Target Populations: We made contacts with HBCU (Historically Black Colleges and Universities) Central State University and West Virginia State University, meeting with faculty and presenting seminars at CSU on 10-28-11 this year and planning a visit to WVSU in FY13. Accomplishments 01 Scientists discover maize lethal necrosis in Kenya. ARS researchers in Wooster, OH, were invited by collaborators at the Kenya Agriculture Research Institute and CIMMYT to assist with identification of suspected viral pathogens causing a serious disease in the Rift Valley of Kenya. I September 2011, a high incidence of disease symptoms were reported on maize in the Southern Rift Valley in southwestern Kenya. The disease has since spread to central Kenya. Diseased plants had symptoms characterist of virus diseases, and included leaf necrosis that caused plants to die before tasseling. Affected maize plants had small cobs with little or no grain set. Using serological and molecular diagnostics, ARS researchers identified two viruses, Maize chlorotic mottle virus (MCMV) and Sugarcan mosaic virus (SCMV), in the diseased maize plants. When these two virus infect the same maize plant, a disease known as maize lethal necrosis occurs. Although SCMV was known to be present in Kenya, this is the fir report of MCMV and of maize lethal necrosis in Kenya or anywhere in Afri The disease is a serious threat to farmers in the region, who are experiencing extensive to complete crop loss. Knowledge about the virus causing this disease provides critical information for researchers to develop control measures for farmers in Kenya. 02 Researchers develop new tool for understanding virus disease in corn. AR and Ohio State University researchers in Wooster, OH sequenced the complete genome of an Ohio isolate of Maize dwarf mosaic virus (MDMV), a developed a full-length infectious complementary DNA (cDNA) of the viral genome. MDMV is one of the most widespread and destructive corn virus pathogens in the United States and worldwide. However, tools for the molecular characterization of virus factors that affect infection of mai were lacking. The complete MDMV genome was cloned into a bacterial plasmid. This plasmid can be produced in E. coli, and then used to make infectious viral ribonucleic acid (RNA). Researchers will use the sequen information and infectious clone to identify virus sequences involved in triggering resistance or disease in corn. 03 First genome sequence information from a leafhopper. ARS and Ohio State University researchers in Wooster, OH developed genome sequence information on Graminella nigrifrons, a leafhopper vector of virus diseases. Virus diseases can cause significant losses in crops, and mos plant viruses are transmitted to new crop plants by insects. Aphids, whiteflies, planthoppers, and leafhoppers, are among the most important transmitters of viruses. Genome information on these virus transmitters, or vectors, is essential to understanding how these insects transmit viruses and for developing new ways of controlling the diseases they transmit. Some genomic information is available for aphids, whiteflies and planthoppers, but little or no genome information has been obtained for leafhoppers. The black-faced leafhopper, Graminella nigrifrons, is prevalent in the eastern U.S., and where it can transmit several differe viruses to corn. In this study, we used a high throughput sequencing approach to identify over 38,000 G. nigrifrons genes. We could tentative assign gene function to some of the genes by comparison to the genes of other insects, and identified some genes that may be involved in the insect�s innate immune system. Interestingly, five of the immunity genes were expressed at much lower levels in virus-infected leafhoppers than i uninfected leafhoppers. These results provide a sound framework for futu studies aimed at understanding the interactions between the plant virus and insect vector that are important for virus transmission.
Impacts
(N/A)
Publications
- Stewart, L.R., Bouchard, R., Redinbaugh, M.G., Meulia, T. 2012. Complete sequence and development of a full-length infectious clone of an Ohio isolate of Maize dwarf mosaic virus (MDMV). Virus Research. 165:219-224.
- Ochwo-Ssemakula, M., Aritua, V., Sengooba, T., Hakiza, J.J., Adipala, E., Ekwamu, A., Redinbaugh, M.G., Winter, S. 2012. Characterization and distribution of a Potyvirus associated with passion fruit woodiness disease in Uganda. Plant Disease. 96(5):659-665.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Objective 1: Develop corn and soybean virus disease control strategies. � Sub-objective 1.A. Characterize the nature of host resistance to virus disease. � Sub-objective 1.B. Identify, map, and clone virus-resistance genes. � Sub-objective 1.C. Determine the effect of combining quantitative virus resistance with insect resistance on virus disease severity. � Sub-objective 1.D. Characterize insect vector/virus relationships. Objective 2: Identify and characterize emerging virus diseases in corn and soybeans. Objective 3: Develop virus-based gene vectors in corn. Soybeans and corn are the two highest value crops grown in the U.S. Although research from this laboratory and others has led to significant improvements in their management, virus diseases continue to be annual threats. Furthermore, history has shown that unanticipated, and often unknown, new virus disease problems can rapidly emerge at any time. In soybeans, the threat of virus diseases has increased even more following the introduction of the soybean aphid into the U.S. in 1999. This is the first soybean-colonizing aphid in the U.S., and the consequences for future virus disease problems in soybeans are unknown. A long-term objective of this program is to reduce corn and soybean losses attributable to virus diseases. Our strategy to do so is to identify corn and soybean viruses when they arise and to characterize their biology and epidemiology (Objective 2), and to then use this information develop practical, effective methods and strategies for minimizing crop losses (Objective 1). Our final objective (Objective 3) is to use our knowledge and expertise in maize virology to develop new tools for forward and reverse genetic analysis of maize gene function. We will develop gene silencing and expression vectors based on selected maize viruses, and use these vectors immediately to characterize the functions of candidate maize genes thought to be important for virus infection and/or resistance. Because reliable vectors are lacking for monocotyledenous plants such as corn, vectors developed under this objective would be of benefit both to corn geneticists and to those seeking to use corn for the production of non-endogenous materials. Approach (from AD-416) Our overall approach is to: 1) identify existing and emerging viruses; 2) understand their biology; and 3) develop effective disease control strategies. Known viruses will be identified using existing diagnostics. For previously uncharacterized viruses, we will culture them in healthy plants using mechanical or arthropod transmission, determine their characteristics, and develop diagnostic assays. This knowledge will be used to formulate disease control strategies, although, usually, the most effective and economic control strategy is to use virus-resistant crop varieties and cultivars. Therefore, a primary focus of this project is to identify, characterize, and map virus resistance in maize and soybean germplasm. To develop an understanding of how resistance genes work, they will be isolated and characterized, and their role in the molecular and biochemical changes associated with virus resistance will be examined. Also, factors affecting virus transmission by arthropod vectors will be characterized so that alternative disease control methods can be developed. Maize virus-based gene expression and silencing vectors will be developed to facilitate functional analysis of plant resistance genes using forward and reverse genetics. Such vectors should have broad impact as few are available for cereals. The impact of this research will be to advance our knowledge of virus diseases of corn and soybeans and provide vital information for the development of control strategies to reduce disease losses. On Obj. 1 projects to characterize the nature of host virus resistance, we completed analysis of microarray experiments to test responses of virus resistant and susceptible inbred lines inoculated with maize dwarf mosaic virus, and developed quantitative (reverse transcription- polymerase chain reaction) RT-PCR assays to verify expression of identified differentially expressed genes. We published results of experiments to characterize the responses of near isogenic lines carrying Wsm1, Wsm2 and Wsm3 from Pa405 to inoculation with potyviruses. The responses of the inbred line Oh1VI and the nested association mapping population parents to Maize rayado fino virus was evaluated, and indicates we can map resistance to this virus in Oh1VI x Oh28 recombinant inbred lines (RIL). We began to evaluate RIL for responses to Maize necrotic streak virus (MNeSV) and Maize fine streak virus (MFSV). We established the sequential transmission protocol for testing for Maize chlorotic dwarf virus (MCDV) helper component and identified inefficiencies likely due to highly variable virion concentrations. We completed a yeast-two-hybrid screen for interactions among putative MCDV proteins, and have identified two MCDV protein interactions: the R78 polyprotein region self-interacts and interacts with the MCDV protease. We developed viral clones that will be used for future construction of full-length MCDV cDNAs. We did not find expression of two predicted small ORF protein products, a predicted overlapping ORFs (ORFX) and a small predicted 3� (ORF4) in the MCDV-S infected plants antibodies against ORF epitopes. For Obj. 2 projects to characterize emerging diseases in maize and soybeans, we identified Maize rough dwarf virus in symptomatic maize from southern Europe. We showed that maize redness infection causes significant yield and quality reduction in several Serbian maize hybrids, and obtained sequence data for ~90% of the maize redness phytoplasma genome. For Obj. 3 projects to develop virus-based gene vectors in corn, we used Brome mosaic virus vectors carrying phytoene desaturase (pds) sequences to determine the expected phenotypes for our similar MNeSV- based vectors. We experienced difficulties with efficient transmission of viral RNA by vascular puncture inoculation (VPI), and developed an agrobacterium-based system for expressing viral cDNA constructs in Nicotiana benthamiana. After moving constructs to the appropriate vector, we will use this system to evaluate the MNeSV vector. For the MFSV- based vector, we demonstrated simultaneous expression the viral N and P genes and the phage T7 RNA polymerase in S2 cells. Preliminary results suggest viral genes are expressed in S2 cells after incubation with virus in the presence of DEAE-dextran. Significant Activities that Support Special Target Populations Central State University (CSU) Careers in Biology seminar: Research scientists visited CSU (Historically Black College or University) at Wilberforce OH and presented careers seminar to Biology majors on USDA, the Corn and Soybean Research Unit, current research projects, and internship opportunities. Established contact with CSU biology professor to encourage internship and future colaboration. West Virginia State University research seminar: Research scientists visited WVSU (Historically Black College or University) at Institute, WV and presented current research projects and overview of Corn and Soybean Research Unit projects and objectives. Collaboration with WVSU professor has been established. Midwest Area summer internship awarded to research scientist at CSRU. Recruited candidates from CSU, OSU, and College of Wooster (COW). Hired minority student (Hispanic) from COW. Accomplishments 01 Virus resistant synthetic population of maize. ARS researchers at Woost Ohio, developed a virus resistant synthetic population of maize. Virus diseases in corn and other crops are most efficiently controlled using resistant plants, and resistance to a number of maize infecting viruses has been identified and mapped in the corn genome. However, resistance multiple viruses in agronomically adapted materials is not available. Researchers developed a virus resistant maize population by crossing nin different virus-resistant tropical and cornbelt inbred lines with the agronomically adapted line B73 and selecting virus-resistant progeny. This process resulted in increased resistance to Maize chlorotic dwarf virus, Maize dwarf mosaic virus and Sugarcane mosaic virus. The population will be useful for domestic and international maize breeders working with corn and sweet corn who need to incorporate virus resistanc into their breeding lines. 02 Resistance breaking isolates of two widespread maize-infecting viruses. The related maize-infecting viruses, Maize dwarf mosaic virus (MDMV) and Sugarcane mosaic virus (SCMV), cause disease in corn all around the worl The diseases caused by these viruses have been effectively controlled f 30 years using resistant hybrids. However, when virus-resistant hybrids are widely used, there is always a chance that the pathogen will evolve 'break' the resistance in the crop, and there have been recent reports that resistance breaking virus isolates in Europe corn and U.S. sweet co Research conducted by ARS scientists in Wooster, Ohio, with an Ohio Sta University Research Intern, determined the responses of virus-resistant S. corn lines to four different virus strains. Two MDMV strains (one fr Italy and one from Ohio) and two SCMV strains (one from Germany and one from Ohio) were tested for their ability to infect a maize line that carries a strong resistance gene called Wsm1. The MDMV from Italy and t SCMV from Ohio could infect this line, but the Ohio MDMV and the German SCMV could not. These results indicate that resistance breaking virus strains are present in the U.S. and Europe. Researchers also determined that maize with two virus resistance genes (Wsm1 plus a different gene called Wsm2) were not infected by any of the four virus strains, indicating that good resistance is achieved if the two genes are combine These results will assist corn breeders in developing resistant hybrids.
Impacts
(N/A)
Publications
- Todd, J.C., Ammar, E., Redinbaugh, M.G., Hoy, C., Hogenhout, S.A. 2010. Plant host range and leafhopper transmission of Maize fine streak virus. Phytopathology. 100(11):1138-1145.
- Jones, M.W., Boyd, E., Redinbaugh, M.G. 2011. Responses of Maize (Zea mays L.) near isogenic lines carrying Wsm1, Wsm2 and Wsm3 to three viruses in the Potyviridae. Journal of Theoretical and Applied Genetics. DOI:10. 1007/s00122-011-1622-8. 123(5):729-740.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Objective 1: Develop corn and soybean virus disease control strategies. � Sub-objective 1.A. Characterize the nature of host resistance to virus disease. � Sub-objective 1.B. Identify, map, and clone virus-resistance genes. � Sub-objective 1.C. Determine the effect of combining quantitative virus resistance with insect resistance on virus disease severity. � Sub-objective 1.D. Characterize insect vector/virus relationships. Objective 2: Identify and characterize emerging virus diseases in corn and soybeans. Objective 3: Develop virus-based gene vectors in corn. Soybeans and corn are the two highest value crops grown in the U.S. Although research from this laboratory and others has led to significant improvements in their management, virus diseases continue to be annual threats. Furthermore, history has shown that unanticipated, and often unknown, new virus disease problems can rapidly emerge at any time. In soybeans, the threat of virus diseases has increased even more following the introduction of the soybean aphid into the U.S. in 1999. This is the first soybean-colonizing aphid in the U.S., and the consequences for future virus disease problems in soybeans are unknown. A long-term objective of this program is to reduce corn and soybean losses attributable to virus diseases. Our strategy to do so is to identify corn and soybean viruses when they arise and to characterize their biology and epidemiology (Objective 2), and to then use this information develop practical, effective methods and strategies for minimizing crop losses (Objective 1). Our final objective (Objective 3) is to use our knowledge and expertise in maize virology to develop new tools for forward and reverse genetic analysis of maize gene function. We will develop gene silencing and expression vectors based on selected maize viruses, and use these vectors immediately to characterize the functions of candidate maize genes thought to be important for virus infection and/or resistance. Because reliable vectors are lacking for monocotyledenous plants such as corn, vectors developed under this objective would be of benefit both to corn geneticists and to those seeking to use corn for the production of non-endogenous materials. Approach (from AD-416) Our overall approach is to: 1) identify existing and emerging viruses; 2) understand their biology; and 3) develop effective disease control strategies. Known viruses will be identified using existing diagnostics. For previously uncharacterized viruses, we will culture them in healthy plants using mechanical or arthropod transmission, determine their characteristics, and develop diagnostic assays. This knowledge will be used to formulate disease control strategies, although, usually, the most effective and economic control strategy is to use virus-resistant crop varieties and cultivars. Therefore, a primary focus of this project is to identify, characterize, and map virus resistance in maize and soybean germplasm. To develop an understanding of how resistance genes work, they will be isolated and characterized, and their role in the molecular and biochemical changes associated with virus resistance will be examined. Also, factors affecting virus transmission by arthropod vectors will be characterized so that alternative disease control methods can be developed. Maize virus-based gene expression and silencing vectors will be developed to facilitate functional analysis of plant resistance genes using forward and reverse genetics. Such vectors should have broad impact as few are available for cereals. The impact of this research will be to advance our knowledge of virus diseases of corn and soybeans and provide vital information for the development of control strategies to reduce disease losses. On projects to characterize the nature of host virus resistance, we completed microarray hybridizations to analyze gene expression in virus resistant and susceptible inbred lines inoculated with maize dwarf mosaic virus, and are currently developing quantitative RT-PCR assays to verify expression of identified differentially expressed genes. We completed experiments to characterize the responses of near isogenic lines carrying Wsm1, Wsm2 and Wsm3 from Pa405 to inoculation with potyviruses. Resistance to Maize necrotic streak virus resistance was associated with chromosome 10 in the mutiply virus-resistant line Oh1VI. Genotyping of two recombinant inbred populations (Oh1VI x Va35 and Oh1VI x Oh28) with SSR markers is about 80% complete, and phenotypic responses of these lines to inoculation with three potyviruses is complete. Plans to screen for resistance to High Plains virus were developed. Objectives and experimental plan to characterize insect vector/virus relationships by newly hired SY were developed and approved by national programs and the area office. For projects to characterize emerging diseases in maize and soybeans, we developed plans and obtained necessary permits to identify and characterize emerging virus diseases in maize in southern Europe are in place. We are trying to obtain samples for analysis this season, although disease rates are very low this year. We completed experiments to characterize the responses of several Serbian maize hybrids to inoculation with maize redness, and are in the process of analyzing the data. For our objective to develop virus-based gene vectors in corn, we are working with Oklahoma State University collaborator to make a gene- silencing vector using Maize necrotic streak virus (MNeSV), and with OSU and John Innes Centre collaborators to develop gene expression vectors using Maize fine streak virus (MFSV) and Maize mosaic virus. We are in the process of determining phenotypes of maize inoculated with Brome mosaic virus vectors carrying phytoene desaturase (pds) sequences so that we can determine what phenotypes to expect in plants inoculated with MNeSV-based vectors designed to silence the same gene. For the MFSV- based vector, we have demonstrated low levels of L protein expression from linear constructs in Drosophila S2 cells. We also demonstrated expression of a replicon construct carrying an antisense GFP construct flanked by the viral 5� and 3� UTR. We showed that accumulation of phage T7 RNA polymerase in S2 cells was dependent on the presence of a nuclear localization signal at the N-terminus of the expressed protein. Methods were tested for visualization of cytoplasmic expression of GFP in S2 cells that are needed for further analysis of the MFSV-based vector. Accomplishments 01 Demonstrated that Maize fine streak virus (MFSV), a nucleorhabdovirus discovered in corn growing in southern Georgia several years ago, can infect wheat, oats, rye, barley, foxtail, annual ryegrass and quackgrass We established that MFSV infects and is transmitted by the leafhopper Graminella nigrifrons in a manner similar to related plant rhabdoviruses However, we also found that G. nigrifrons is not a very good vector of MFSV, because <10% of insects feeding on infected plants transmit the virus. The low rate of virus transmission by the only known vector may explain the few reports of MFSV in crops. However, the availability of perennial virus host provides a way for the virus to survive between growing seasons, and our results indicate a potential for MFSV to cause disease in several crops. Understanding how virus diseases such as MFSV spread in crops is critical for preventing economically important diseas outbreaks. 02 We identified an enhancer of Mdm1 resistance to Maize dwarf mosaic virus (MDMV) in the highly resistant maize inbred line Pa405. Mdm1 was previously identified as a dominant gene on the short arm of chromosome however, it was postulated that this locus alone did not explain all of the resistance in Pa405. We compared the responses of near isogenic lin (NIL) carrying this genomic region and two other regions previously show to be important for resistance to Wheat streak mosaic virus to inoculati with MDMV. The region on chromosome 10 alone provided no resistance to MDMV; however, it provided a significant boost to resistance in plants heterozygous for Mdm1. These results explain incomplete resistance to MD observed in some hybrids carrying Mdm1, and provide germplasm and molecular markers to assist maize and sweet corn breeders with developme of MDMV resistant hybrids.
Impacts
(N/A)
Publications
- Redinbaugh, M.G., Molineros, J., Vacha, J., Berry, S., Hammond, R.B., Madden, L.V., Dorrance, A.E. 2010. Bean Pod Mottle Virus Spread in Insect Feeding Resistant Soybeans. Plant Disease. 94(2):265-270.
- Cao, M., Ye, X., Lin, J., Zhang, X., Redinbaugh, M.G., Simon, A.E., Morris, T.J., Qu, F. 2010. The Capsid Protein of Turnip Crinkle Virus Overcomes two Separate Defense Barriers to Facilitate Viral Systemic Movement in Arabidopsis. Journal of Virology. 84(15):7793-7802.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Objective 1: Develop corn and soybean virus disease control strategies. � Sub-objective 1.A. Characterize the nature of host resistance to virus disease. � Sub-objective 1.B. Identify, map, and clone virus-resistance genes. � Sub-objective 1.C. Determine the effect of combining quantitative virus resistance with insect resistance on virus disease severity. � Sub-objective 1.D. Characterize insect vector/virus relationships. Objective 2: Identify and characterize emerging virus diseases in corn and soybeans. Objective 3: Develop virus-based gene vectors in corn. Soybeans and corn are the two highest value crops grown in the U.S. Although research from this laboratory and others has led to significant improvements in their management, virus diseases continue to be annual threats. Furthermore, history has shown that unanticipated, and often unknown, new virus disease problems can rapidly emerge at any time. In soybeans, the threat of virus diseases has increased even more following the introduction of the soybean aphid into the U.S. in 1999. This is the first soybean-colonizing aphid in the U.S., and the consequences for future virus disease problems in soybeans are unknown. A long-term objective of this program is to reduce corn and soybean losses attributable to virus diseases. Our strategy to do so is to identify corn and soybean viruses when they arise and to characterize their biology and epidemiology (Objective 2), and to then use this information develop practical, effective methods and strategies for minimizing crop losses (Objective 1). Our final objective (Objective 3) is to use our knowledge and expertise in maize virology to develop new tools for forward and reverse genetic analysis of maize gene function. We will develop gene silencing and expression vectors based on selected maize viruses, and use these vectors immediately to characterize the functions of candidate maize genes thought to be important for virus infection and/or resistance. Because reliable vectors are lacking for monocotyledenous plants such as corn, vectors developed under this objective would be of benefit both to corn geneticists and to those seeking to use corn for the production of non-endogenous materials. Approach (from AD-416) Our overall approach is to: 1) identify existing and emerging viruses; 2) understand their biology; and 3) develop effective disease control strategies. Known viruses will be identified using existing diagnostics. For previously uncharacterized viruses, we will culture them in healthy plants using mechanical or arthropod transmission, determine their characteristics, and develop diagnostic assays. This knowledge will be used to formulate disease control strategies, although, usually, the most effective and economic control strategy is to use virus-resistant crop varieties and cultivars. Therefore, a primary focus of this project is to identify, characterize, and map virus resistance in maize and soybean germplasm. To develop an understanding of how resistance genes work, they will be isolated and characterized, and their role in the molecular and biochemical changes associated with virus resistance will be examined. Also, factors affecting virus transmission by arthropod vectors will be characterized so that alternative disease control methods can be developed. Maize virus-based gene expression and silencing vectors will be developed to facilitate functional analysis of plant resistance genes using forward and reverse genetics. Such vectors should have broad impact as few are available for cereals. The impact of this research will be to advance our knowledge of virus diseases of corn and soybeans and provide vital information for the development of control strategies to reduce disease losses. Significant Activities that Support Special Target Populations The majority of our research effort was on the objective to develop corn and soybean virus disease control strategies. We showed that, BPMV incidence was lower in semi-dwarf insect-resistant soybeans than in standard insect-susceptible cultivars, but found it was also lower in a semi-dwarf, insect-susceptible cultivar. We demonstrated that contributions from �modifier genes� derived from the resistant parent Pa405 are important for resistance of maize to Maize dwarf mosaic virus (MDMV) and Sugarcane mosaic virus. We showed that Graminella nigrifrons is an inefficient vector of Maize fine streak virus (MFSV), and identified two populations of MFSV-infected insects after feeding on infected plants, insects with high virus titer that could transmit the virus and insects with low virus titer that did not transmit the virus. We are in the third year of trials to evaluate the effects of BPMV on Ohio, North Dakota and Wisconsin soybean cultivars with partial BPMV resistance; microarray analysis of the response of resistant and susceptible maize 1 and 3 days post-MDMV inoculation were done; molecular markers were used to identify cross-over events in the chromosomal regions near two WSMV resistance genes and several thousand progeny have been analyzed for these cross-overs. We completed a project with Ohio State University and Serbian collaborators to define the disease cycle for maize redness caused by stolbur phytoplasma and the life cycle of the disease vector, Reptalus panzeri. For our objective to develop virus- based gene vectors in corn, we are working with Oklahoma State University collaborator to make a gene-silencing vector using Maize necrotic streak virus (MNeSV), and with OSU and John Innes Centre collaborators to develop gene expression vectors using Maize fine streak virus (MFSV) and Maize mosaic virus. We tested constructs with maize phytoene desaturase (pds) sequences inserted into engineered cloning sites. Although some plants became infected with these constructs, none of the expected photobleaching associated with pds expression was observed. After re- examining the viral genome sequence for secondary RNA structures, a problem with disrupting the sequences near the inserted cloning sites was identified. New cloning sites were selected and constructs were made that are being tested at ARS and OkSU. Previously, we showed that the MFSV nucleoprotein and phosphoprotein can be expressed in Drosophila S2 cells using commercially available plasmids. We demonstrated expression of a replicon construct carrying an antisense GFP construct flanked by the viral 5� and 3� UTR in S2 cells. A construct for the L gene, which encodes the viral RNA dependent RNA polymerase was made and expression of these constructs in S2 cells is currently being tested. Technology Transfer Number of Other Technology Transfer: 5
Impacts
(N/A)
Publications
- Jovic, .J., Cvrkovi_, T., Mitrovi, M., Krnjanji, S., Petrovi, A., Redinbaugh, M.G., Pratt, R.C., Hogenhout, S.A., Toevski, I. 2009. Maize Redness Transmitted by Reptalus panzeri: the Disease Cycle in Serbia. Journal of Phytopathology. 99(9):1053-1069.
- Redinbaugh, M.G., Pratt, R.C. 2008. Virus Resistance. In: Hake S., Bennetzed J., Editors. Maize Handbook. 2nd edition. New York, NY: Springer- Verlag. p. 255-270.
- El-Desouky, A., Chi-Wei, T., Whitfield, A.E., Redinbaugh, M.G., Hogenhout, S.A. 2009. Cellular and Molecular Interactions of Rhabdoviruses with their Insect and Plant Hosts. Annual Review Of Entomology. 54:447-468.
- Russo, M., De Stradis, A., Boscia, D., Rubino, L., Redinbaugh, M.G., Abt, J.J., Martelli, G.P. 2008. Molecular and Ultrastructural Properties of Maize White Line Virus. Journal of Plant Pathology. 90:363-369.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Objective 1: Develop corn and soybean virus disease control strategies. � Sub-objective 1.A. Characterize the nature of host resistance to virus disease. � Sub-objective 1.B. Identify, map, and clone virus-resistance genes. � Sub-objective 1.C. Determine the effect of combining quantitative virus resistance with insect resistance on virus disease severity. � Sub-objective 1.D. Characterize insect vector/virus relationships. Objective 2: Identify and characterize emerging virus diseases in corn and soybeans. Objective 3: Develop virus-based gene vectors in corn. Soybeans and corn are the two highest value crops grown in the U.S. Although research from this laboratory and others has led to significant improvements in their management, virus diseases continue to be annual threats. Furthermore, history has shown that unanticipated, and often unknown, new virus disease problems can rapidly emerge at any time. In soybeans, the threat of virus diseases has increased even more following the introduction of the soybean aphid into the U.S. in 1999. This is the first soybean-colonizing aphid in the U.S., and the consequences for future virus disease problems in soybeans are unknown. A long-term objective of this program is to reduce corn and soybean losses attributable to virus diseases. Our strategy to do so is to identify corn and soybean viruses when they arise and to characterize their biology and epidemiology (Objective 2), and to then use this information develop practical, effective methods and strategies for minimizing crop losses (Objective 1). Our final objective (Objective 3) is to use our knowledge and expertise in maize virology to develop new tools for forward and reverse genetic analysis of maize gene function. We will develop gene silencing and expression vectors based on selected maize viruses, and use these vectors immediately to characterize the functions of candidate maize genes thought to be important for virus infection and/or resistance. Because reliable vectors are lacking for monocotyledenous plants such as corn, vectors developed under this objective would be of benefit both to corn geneticists and to those seeking to use corn for the production of non-endogenous materials. Approach (from AD-416) Our overall approach is to: 1) identify existing and emerging viruses; 2) understand their biology; and 3) develop effective disease control strategies. Known viruses will be identified using existing diagnostics. For previously uncharacterized viruses, we will culture them in healthy plants using mechanical or arthropod transmission, determine their characteristics, and develop diagnostic assays. This knowledge will be used to formulate disease control strategies, although, usually, the most effective and economic control strategy is to use virus-resistant crop varieties and cultivars. Therefore, a primary focus of this project is to identify, characterize, and map virus resistance in maize and soybean germplasm. To develop an understanding of how resistance genes work, they will be isolated and characterized, and their role in the molecular and biochemical changes associated with virus resistance will be examined. Also, factors affecting virus transmission by arthropod vectors will be characterized so that alternative disease control methods can be developed. Maize virus-based gene expression and silencing vectors will be developed to facilitate functional analysis of plant resistance genes using forward and reverse genetics. Such vectors should have broad impact as few are available for cereals. The impact of this research will be to advance our knowledge of virus diseases of corn and soybeans and provide vital information for the development of control strategies to reduce disease losses. Significant Activities that Support Special Target Populations The majority of our research effort was on the objective to develop corn and soybean virus disease control strategies. Three projects under this objective are nearing completion and publication. We showed that, although the BPMV incidence was lower in semi-dwarf insect-resistant soybeans than in standard insect-susceptible cultivars, virus incidence was also lower in a semi-dwarf, insect-susceptible cultivar. We demonstrated that contributions from �modifier genes� derived from the resistant parent Pa405 are likely to be important for complete resistance of maize to Maize dwarf mosaic virus and Sugarcane mosaic virus, since the introgression of chromosomal regions carrying Mdm1/Scmv1 and Scmv2 into a susceptible background did not by themselves provide complete virus resistance. We showed that Graminella nigrifrons is an inefficient vector of Maize fine streak virus (MFSV). Further, we demonstrated the presence of two populations of MFSV-infected insects: one with high virus titer that includes most of the vectors and one with low virus titer that does not transmit the virus. In ongoing projects under this objective: we are in the third year of trials to evaluate the effects of BPMV on Ohio, North Dakota and Wisconsin soybean cultivars with partial BPMV resistance; results from microarray analysis of the response of resistant and susceptible maize to virus inoculation indicated a small number of genes were differentially regulated by virus four days post inoculation and suggested that a slight modification of our experimental approach was warranted; molecular markers suitable for high throughput identification of cross-over events in the chromosomal regions near two WSMV resistance genes were developed and analysis of several thousand progeny for these cross-overs has begun. Under the objective to identify and characterize emerging virus diseases in corn and soybeans, we have been working with collaborators in Serbia and Ecuador to identify and characterize emerging vector-dependent diseases in maize. With Serbian collaborators we are preparing a manuscript describing the acquistion of stolbur phytoplasma from maize roots by early stage larvae of the vector Reptalus panzeri. The larvae overwinter on wheat roots and emerge as infected adults from wheat fields in late June. Wheat and Johnson grass were shown to be hosts of the stolbur phytoplasma. Preliminary experiments were carried out with an Ecuadoran collaborator to identify viruses and mollicutes assocated with an emerging disease called �cinta roja� (red stripe). For our objective to develop virus-based gene vectors in corn, we are working to make a gene-silencing vector using Maize fine streak virus (MNeSV) and gene expression vectors using Maize fine streak virus (MFSV) and Maize mosaic virus. We showed that all four MNeSV-encoded proteins are required for systemic movement of the virus, and have engineered cloning sites into the virus while maintaining infectivity. We showed that the MFSV nucleoprotein and phosphoprotein can be expressed in Drosophila S2 cells using commercially available plasmids. Technology Transfer Number of Active CRADAS: 1
Impacts
(N/A)
Publications
- Hogenhout, S.A., Ammar, E.D., Whitfield, A.E., Redinbaugh, M.G. 2008. Insect Vector Interactions with Persistently Transmitted Plant Viruses. Annual Review of Phytopathology. 46:327-359.
- De Souza, I., Schuelter, A.R., Guimaries, C., Schuster, I., De Oliveira, E. , Redinbaugh, M.G. 2008. Mapping QTL Contributing to SCMV Resistance in Tropical Maize. Hereditas. 145(4):167-173.
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