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ARS Home » Midwest Area » Wooster, Ohio » Corn, Soybean and Wheat Quality Research » Research » Research Project #423065

Research Project: CONTROL OF VIRUS DISEASES IN CORN AND SOYBEAN

Location: Corn, Soybean and Wheat Quality Research

2014 Annual Report


Objectives
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.


Approach
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.


Progress Report
Surveys of viruses in Ohio wheat, maize, and an important weedy reservoir, Johnsongrass (Sorghum halepense), were completed using next generation sequencing (RNASeq). Results showed that viruses previously reported in Ohio, including Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus, and Maize chlorotic dwarf virus (MCDV) mild and type strains, are still abundant in Johnsongrass, and MDMV can be found frequently infecting sweet corn. The causal agent of High Plains disease, Wheat mosaic virus (WMoV), was discovered for the first time in Ohio in samples from four counties. Based on survey sequence data, RT-PCR is being used to determine the complete genomic sequence of WMoV. We demonstrated that maize co-infected with MDMV and MCDV, the two most abundant maize viruses in the U.S., delayed symptom recovery in co-infected plants but did not cause strong symptom synergy. Transcriptomic approaches were utilized to better understand the differential responses of susceptible and resistant maize to seed inoculation with MDMV and identify candidate genes involved in the resistance response. A comparison of transcriptome responses of soybean aphid to feeding on plants infected with Soybean mosaic virus (SMV) and Bean pod mottle virus (BPMV) showed that the aphid responded not only to the virus it vectors (SMV), but also had a significantly greater transcriptome response and reduced fecundity when fed on soybean infected with a virus it does not vector (BPMV). Ongoing work to characterize MCDV gene function and identify the putative helper component is underway: yeast-two-hybrid assays for virus protein-protein interactions have been completed, tests to optimize virion yield for helper component assays are ongoing, and efforts to identify small ORFs have been carried out without detection of these predicted proteins in infected plants. Proteolysis assays using a wheat germ in vitro transcription/translation system have shown in vitro viral protease activity but have not yet identified the cleavage sites. An improved system to use Maize necrotic streak virus (MNeSV) for virus-induced gene silencing was developed, but attempts to deliver the virus vector directly to maize by agroinoculation have not yet been successful. Several insertion sites and lengths were tested, and the most successful MNeSV vectors show patchy silencing in maize and do not retain insert sequences, problems encountered with many virus VIGS vectors. We are in the process of completing studies to identify genes for resistance to MNeSV, and to investigate the effect of temperature on the expression of resistance to potyviruses in maize. A large, ongoing effort to identify and characterize resistance to Maize chlorotic mottle virus (MCMV), which is currently causing serious problems with maize lethal necrosis (MLN) in East Africa, is underway. We are also completing a survey of viruses associated with MLN and their genomic sequences in collaboration with Kenyan, Ugandan and U.S. partners. A study to identify differential expression of insect and virus genes in vectors, hosts and non-host Graminella nigrifrons, the leafhopper that transmits Maize fine streak virus is nearly complete.


Accomplishments
1. Identification and characterization of new and emerging maize viruses. Viruses are continuously emerging and changing in distribu tion in response to environmental changes and movement by humans. Next generation sequencing (NGS) is an effective method to rapidly identify emerging viruses and assess existing virus populations, even for previously unknown viruses. Virus populations in U.S. maize and wheat have not been surveyed in Ohio for several decades, and a rapid and high-impact outbreak of maize lethal necrosis (MLN) in East Africa demands rapid pathogen assessment and response. ARS researchers at Wooster, Ohio surveyed and used NGS to obtain genome sequence data from maize and wheat viruses in the United States and east Africa, working with Ohio State University bioinformaticists to develop an in-house plant virus NGS identification pipeline. Partial and complete genome sequence data for maize viruses were obtained, including African isolates of Maize chlorotic mottle virus and Sugarcane mosaic virus and Ohio isolates of the major U.S. maize viruses Maize chlorotic dwarf virus (MCDV) and Maize dwarf mosaic virus (MDMV), as well as new viruses including a maize luteovirus in Africa and the first appearance of Wheat mosaic virus (causal agent of High Plains disease) in Ohio. Identification of these viruses and the specific isolate sequences is an important step to determine which viruses are causing disease and develop control measures.

2. Co-infection by the two major U.S. maize viruses prolongs disease symptoms. Two major U.S. maize viruses, Maize chlorotic dwarf virus (MCDV) and Maize dwarf mosaic virus (MDMV), overlap in distribution and frequently occur in co-infections. However, the impact, if any, of co-infection by the two viruses on maize disease was unknown. ARS researchers in Wooster, Ohio tested co-infection of these two viruses. Whereas young plants infected with a single virus showed some reduction in symptom severity over time, plants co-infected with the severe strain of MCDV (MCDV-S) and MDMV continued to show severe symptoms over a month after infection. Unlike some pairs of co-infecting viruses that cause extremely severe disease, the combination of the two major U.S. maize viruses may have implications for disease losses but does not result in plant death under the test conditions. These results indicate that interactions in co-infected plants depends on the virus species involved.

3. Reduced reproduction of soybean aphid feeding on virus-infected plants. Aphids are among the most important insects that transmit virus diseases to crops. To develop an understanding of virus transmission by soybean aphids, a recently introduced and important pest of soybeans, we investigated the effects of aphids feeding on virus-infected plants on gene expression. We discovered that gene expression in soybean aphids was affected dramatically when they fed on plants infected with Bean pod mottle virus (BPMV), a virus the aphids cannot transmit. A much smaller effect was seen when the aphids were fed on plants infected with Soybean mosaic virus (SMV), a virus the aphid does transmit. Surprisingly, we also found that soybean aphids fed on BPMV-infected soybeans reproduced at a lower rate than those fed on healthy or SMV-infected soybean. We also discovered that aphids fed on soybean infected with either virus prevented reproduction of Buchnera aphidicola, a bacterium that lives inside the aphids and is required for the aphids to meet their nutritional needs. This research demonstrates that interactions among insects, plants, and plant pathogens influence aphid reproductive capacity, and may provide aphid or bacterial targets for controlling this pest.

4. Identification of a gene for resistance to Maize rayado fino virus. Growing resistant hybrids and cultivars is the most economical and environmentally sustainable way to control virus diseases in the corn crop, but genes for resistance to Maize rayado fino virus (MRFV), which causes severe yield losses from the southern U.S. to South America, were not known. We mapped genes for MRFV resistance in an inbred corn line called Oh1VI that is resistant to at least ten different viruses and in an unrelated, but also highly resistant line called K11. We found a major gene for MRFV resistance in the same region of chromosome 10 in both lines. The fact that the MRFV resistance genes are in the same position in different lines suggests that the virus resistance will be effective if it is bred into hybrids and cultivars that are used by farmers.

5. Roles of three viruses in Maize lethal necrosis in Kenya and Uganda. Maize lethal necrosis (MLN) is a serious emerging disease of maize and a significant threat to maize production in Kenya, Tanzania, Uganda, and other parts of Sub-Saharan Africa. The diseases is caused by two viruses, Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), but the roles of these two viruses and Maize streak virus (MSV) in MLN were not well understood. The results of a survey of symptomatic maize plants from eastern Kenya and Uganda carried out with collaborators from Uganda, Kenya and the U.S., demonstrated that all three viruses were present, but MCMV was predominant in the collected samples. SCMV was present at high levels, but was detected mostly in plants co-infected with MCMV. MSV was found at a low level. Our results provide preliminary evidence that Napier grass, sorghum and sugarcane can serve as hosts of MCMV in East Africa. Differences detected among different antisera raised to MCMV and SCMV indicate a need for developing antisera to the East African MCMV and SCMV isolates. Our results provide information on the viruses causing MLN and their diversity, which will be important for testing virus-resistant maize that is under development.


Review Publications
Cassone, B.J., Michel, A.P., Stewart, L.R., Bansal, R., Mian, R.M., Redinbaugh, M.G. 2014. Reduction in fecundity and shifts in cellular processes by a native virus on an invasive insect. Genome Biology and Evolution. 6(4):873-885.
Zambrano, J.L., Jones, M.W., Brenner, E., Francis, D.M., Tomas, A., Redinbaugh, M.G. 2014. Genetic analysis of resistance to six virus diseases in a multiple virus-resistant maize inbred line. Theoretical and Applied Genetics. 127(4):867-880.
Cassone, B.J., Wijertine, S., Michel, A., Stewart, L.R., Chen, Y., Yan, P., Redinbaugh, M.G. 2014. Virus-independent and common transcriptome responses of leafhopper vectors feeding on maize infected with semi-persistently and persistent propagatively transmitted viruses. Biomed Central (BMC) Genomics. 15:133.
Lin, J., Gio, J., Finer, J., Dorrance, A., Redinbaugh, M.G., Qu, F. 2014. The Bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation. Journal of Virology. 88(6):3213-3222.
Stewart, L.R., Paul, P., Qu, F., Redinbaugh, M.G., Miao, H., Todd, J.C., Jones, M.W. 2013. Wheat mosaic virus (WMoV), the causal agent of High Plains disease, is present in Ohio wheat fields. Plant Disease. 97:1125.
Cassone, B.J., Chen, Z., Chiera, J., Stewart, L.R., Redinbaugh, M.G. 2014. Responses of highly resistant and susceptible maize to vascular puncture inoculation with Maize dwarf mosaic virus. Physiological and Molecular Plant Pathology. 86:19-27.
Zambrano, J.L., Jones, M.W., Francis, D.M., Tomas, A., Redinbaugh, M.G. 2014. Quantitative trait loci for resistance to Maize rayado fino virus. Molecular Breeding. DOI:10.1007/S11032-014-0091-6.
Ammar, E., Correa, V.R., Hogenhout, S.A., Redinbaugh, M.G. 2014. Immunofluorescence localization and ultrastructure of Stewart’s wilt disease bacterium Pantoea stewartii in maize leaves and in its flea beetle vector Chaetocnema pulicaria (Coleoptera: Chrysomelidae). Journal of Microscopy and Ultrastructure. 2:28-33.