2010 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.
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.
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 6; however, it was postulated that this locus alone did not explain all of the resistance in Pa405. We compared the responses of near isogenic lines (NIL) carrying this genomic region and two other regions previously shown to be important for resistance to Wheat streak mosaic virus to inoculation 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 MDMV observed in some hybrids carrying Mdm1, and provide germplasm and molecular markers to assist maize and sweet corn breeders with development of MDMV resistant hybrids.
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 a 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 disease outbreaks.
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.