Location: Corn, Soybean and Wheat Quality Research2013 Annual Report
1a. Objectives (from AD-416):
1. Monitor and identify emerging insect-transmitted pathogens of maize and soybean, and identify management strategies. 2. Determine whether multiple virus resistance in maize inbred lines is the result of pleiotropic or closely linked genes, and develop and release virus-resistant germplasm to breeders. a) Determine whether resistance to potyviruses is pleiotropic in Pa405. b) Mapping multiple virus resistance in Oh1VI. c) Develop and release virus resistant germplasm. 3. Develop genetic and genomic information on two insect vectors, including the molecular response to feeding on virus-infected plants. 4. Identify virus components important for pathogenesis, insect transmission, and host interactions, and develop virus systems for gene discovery and functional analysis in maize. a) Assess viral protein complements, expression strategies, and functions in maize. b) Develop maize virus-based forward and reverse genetics systems. c) Characterize virus and insect factors needed for virus transmission, and develop methods to study these processes.
1b. Approach (from AD-416):
1. A sequence-independent approach (SIA) for amplification of viral genome sequences will be used for initial identification of viruses in suspected, symptomatic plants. Mollicutes will be identified using PCR with genus-specific ribosomal DNA (rDNA) primers. The identity of known pathogens will be confirmed with a combination of microscopic, serological and molecular assays. New viruses will be cultured in susceptible plants and characterized. As possible under permit conditions, we will test known vectors of maize and soybean diseases for their ability to transmit pathogens. Mechanical or vector transmission of pathogens will be used to screen maize or soybean germplasm for resistant genotypes. 2. To determine whether the Wsm1 and Wsm2 genes for WSMV resistance confer resistance to multiple potyviruses, to isolate or fine map these two genes in Pa405, and to develop germplasm to fine map or isolate Wsm3. The putative insertional mutants Wsm1µ and Wsm2µ plants identified in the current project will be tested for chromosomal deletions on chr. 6 and 3, respectively, prior to testing for pleiotropic gain of susceptibility to potyviruses. We will clone sequences flanking the insertion sites to identify candidate genes. Genes and cDNAs encoding Wsm1 and Wsm2 will be cloned, and sequences will be used in loss and gain of function assays to confirm gene identity. Because of the risk associated with identifying insertional mutations in Wsm1 and Wsm2, we will continue efforts to develop a fine map Wsm1 and Wsm2, using available recombinant plants and populations. Additional markers will be identified in SNP and microarray analyses. We will develop germplasm to identify mutator insertions and fine map Wsm3. 3. Use second-generation sequence analysis to build and analyze EST libraries for two important vectors of soybean and maize viruses: A. glycines and G. nigrifrons. The vectors will be fed on plants infected with viruses that are transmitted in a non-persistent (SMV), semi-persistent (MCDV), persistent-circulative (SbDV) or persistent-replicative (MFSV) manner. EST libraries will be made with RNA from: 1) A. glycines biotypes 1 and 2 fed on healthy, and SMV or SbDV-infected soybean, and 2) G. nigrifrons fed on healthy, and MCDV- and MFSV-infected maize. Libraries will be sequenced, assembled and annotated. Differential EST expression between different treatments will be verified with quantitative real-time RT-PCR (RT-qPCR), and sequences from A. glycines and G. nigrifrons will be compared with those of other vector genomes. 4. An in vivo protease assay will be used to determine MCDV polyprotein cleavage sites by co-expressing active viral protease with epitope-tagged MCDV polyprotein regions and determining sizes of cleavage products. Antibodies made against predicted small ORF-encoded proteins will be used to test for protein expression in infected plants. MCDV proteins will be tested for subcellular localization and virus protein-protein interactions, and MCDV and MFSV proteins will be tested for their ability to suppress gene silencing in N. benthamiana.
3. Progress Report:
Identified two viruses known to cause maize lethal necrosis, maize chlorotic mottle virus and sugarcane mosaic virus, in diseased maize from Kenya. Developed bioinformatic methods to identify virus sequences and analyzed RNA-Seq datasets to obtain genomic sequence of viruses from Kenya, Uganda and Ohio. Detected known and new viruses in wheat and maize samples from Ohio. Completed germplasm development for all ongoing projects. Identified new sources of resistance to Maize rayado fino virus and several other viruses among the maize nested association mapping founder lines. In collaboration with researchers at Dupont, genotyped maize inbred lines and a recombinant inbred line population using 760 single nucleotide polymorphism (SNP) markers, and developed genomic and genetic maps based on the results. Determined the responses of these lines to eight phylogenetically diverse maize-infecting viruses, and mapped virus resistance in the inbred line Oh1VI. Determined the transcriptome responses of the black-faced leafhopper to feeding on maize infected with two unrelated viruses, and the response of the soybean aphid to feeding on soybean infected with two unrelated viruses. Developed mutants of maize chlorotic dwarf virus (MCDV) proteins and used these to map protease cleavage sites in the virus. Generated antibodies to GST fusions of predicted MCDV small open reading frame (ORF)-encoded proteins, and are using these to detect expression of the proteins in vivo. Constructed and tested a series of vectors containing hairpin and linear reporter gene inserts using an infectious clone of maize necrotic streak virus. One construct showed silencing of the reporter gene. Continue optimization of parameters for testing MCDV proteins for the helper component activity. Evaluated synergism of maize dwarf mosaic virus and MCDV in maize by evaluating symptom development and virus titer. Developed rapid system for cloning MCDV sequences. Identified interactions between maize fine streak virus (MFSV) proteins 3 and 4, demonstrated that the regulation of MFSV transcript expression is different than that of mammal-infecting rhabdoviruses, with significantly higher expression of a gene toward the 3’ end of the genome (gene 3) than expected in both the plant and insect vector hosts.
1. Diagnosis of maize lethal necrosis in Kenya. Maize is a staple crop for subsistence farmers in East Africa. In 2012, a serious threat to the food security for these farmers emerged as they experienced 40 to 100% losses in their crops rapidly emerging disease of corn in Kenya. Based on disease symptoms and the presence of potential insect vectors in the field, a virus was suspected as the cause of the disease. ARS researchers in Wooster, OH collaborated with International Maize and Wheat Improvement Center (CIMMYT) and Kenya Agricultural Research Institute (KARI) scientists to identify two viruses, Maize chlorotic mottle virus and Sugarcane mosaic virus, in diseased maize. Together, these viruses cause maize lethal necrosis. Identification of the major pathogens involved in maize lethal necrosis allows ARS scientists and collaborators to identify disease control measures, to develop the screening protocols needed to breed disease resistant hybrids and cultivars, and to investigate the epidemiology of this rapidly spreading disease. Further, this information allows U.S. maize breeders and seed producers to stay ahead of a disease to which the $60 billion U.S. corn crop is vulnerable.
2. Maize rayado fino virus resistant lines. Viruses and other insect transmitted diseases cause significant losses in crops, including corn, world-wide. Controlling these diseases with virus-resistant cultivars and hybrids is both economically viable and environmentally sustainable. However, suitable virus-resistant plant materials must first be identified. Maize rayado fino (maize fine stripe) can cause large losses for producers in Central and South America, but maize lines with strong resistance to maize rayado fino virus (MRFV), the pathogen causing the disease, were not known. We screened 36 lines for resistance to MRFV and identified three maize lines with very strong resistance to MRFV, and another two lines with moderate resistance to infection by the virus. The discovery of novel sources of resistance in corn will facilitate the identification and mapping of genes for MRFV resistance and will assist maize breeders in developing cultivars and hybrids for environmentally and economically sustainable control of losses caused by this virus.
3. Vector response to virus-infected plants: Virus diseases that are moved from plant to plant by insect vectors are among the most common emerging diseases of crops and are among the most difficult to control. This is due, in part, to our lack of information about the mechanisms that viruses use to 'hitch a ride' with insect vectors and the responses the insects have to the virus that enhance or prevent movement. We know that viruses use a variety of strategies for transmission by insects that reflect different mechanisms for overcoming defense barriers in the insect. However, little was known about the responses of insect vectors to virus exposure, and no information was available on the responses of insects to viruses that use different transmission strategies. We investigated the responses of leafhoppers to feeding on maize plants infected with either a virus that binds to the surface of the insect gut or one that enters and infects insect cells for transmission using next generation sequencing. We found unexpected similarities in the responses of insects to these two very different viruses, as well as some clear differences. This information will be used to test the importance of genes involved in the common response for virus transmission, and may ultimately be useful for identifying insect factors that disrupt insect-mediated virus transmission to crops.
4. Multiple virus resistance in maize. Virus diseases in corn can cause severe yield reductions that threaten crop production and food supplies around the world. Genetic resistance to different viruses is the most economical and environmentally sustainable approach for controlling these diseases in corn and other crops. We discovered that a line of maize we developed several years ago with strong resistance to Maize chlorotic dwarf virus, is also resistant to at least nine other viruses in five different virus families. We developed a mapping population using this line, and used it to identify 17 genes for resistance to eight different viruses. Of these 17 genes, 15 were clustered in small regions of chromosomes 2, 3, 6, and 10. These results provide genetic materials and information for futher exploring the nature of virus resistance in maize. In addition, corn breeders can use maize lines and molecular markers identified in this study to develop corn lines and hybrids that are resistant to multiple viruses for economically and environmentally sustainable disease control.
5. Improved identification of wheat and corn viruses in Ohio. Traditional methods of identifying viruses and other plant pathogens in crops rely on prior information about that pathogen, making it easy to miss new or unusual pathogens. In addition, information on virus distribution in major Ohio field crops has not been updated for several decades. ARS scientists, with Ohio State University collaborators, used traditional diagnostics and “next-generation sequencing” technology to identify viruses in corn and wheat. Populations of the two major U.S. corn-infecting viruses, maize dwarf mosaic virus and maize chlorotic dwarf virus, limited primarily the reservoir host, Johnsongrass, and sweet corn. Wheat mosaic virus, the pathogen causing High Plains disease was identified for the first time in wheat and corn in Ohio, much farther east than previously reported. A custom plant virus identification bioinformatics pipeline was developed and applied to identify virus sequences from samples, allowing us to determine virus genome sequences for lab isolates of seven maize-infecting viruses. This pipeline will increase our ability to rapidly identify and manage pathogens, and to better predict future risks to crops.
Lin, J., Ali, A.K., Chen, P., Ghabrial, S., Finer, J., Dorrance, A., Redinbaugh, M.G., Qu, F. 2013. A stem–loop structure in the 59 untranslated region of bean pod mottle virus RNA2 is specifically required for RNA2 accumulation. Journal of General Virology. 94:1415-1420.