Location: Corn, Soybean and Wheat Quality Research2011 Annual Report
1a. 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.
1b. 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.
3. Progress Report
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.
1. Virus resistant synthetic population of maize. ARS researchers at Wooster, 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 to multiple viruses in agronomically adapted materials is not available. Researchers developed a virus resistant maize population by crossing nine 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 resistance into their breeding lines.
2. 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 world. The diseases caused by these viruses have been effectively controlled for 30 years using resistant hybrids. However, when virus-resistant hybrids are widely used, there is always a chance that the pathogen will evolve to 'break' the resistance in the crop, and there have been recent reports that resistance breaking virus isolates in Europe corn and U.S. sweet corn. Research conducted by ARS scientists in Wooster, Ohio, with an Ohio State University Research Intern, determined the responses of virus-resistant U.S. corn lines to four different virus strains. Two MDMV strains (one from 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 the 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 combined. These results will assist corn breeders in developing resistant hybrids.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.