Location: Corn, Soybean and Wheat Quality Research2015 Annual Report
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.
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.
In FY15, we have made important progress on Objectives 1, 2 and 4, and completed publications associated with Objective 3. Work on Objective 1 included a large efforts towards the critical need of characterizing the epidemiology of the devastating Maize lethal necrosis (MLN) outbreak emerging in East Africa and surrounding areas, to improve our understanding of the disease, the viruses causing the disease and development of methods to detect them. Another large effort has been focused on disease management efforts, particularly in screening maize germplasm to identify lines that are resistant to one or more of the viruses involved. MLN is caused by the co-infection of Maize chlorotic mottle virus (MCMV) and any of a set of maize-infecting potyviruses. We have used deep sequencing and bioinformatics to identify the virus populations in MLN-diseased plants in Uganda and Kenya. Our data so far indicate that MCMV intergenic and coding regions have <3% nucleotide polymorphism across collection sites; whereas Sugarcane mosaic virus (SCMV) populations in East Africa have <15% nucleotide polymorphism in the polyprotein-coding sequence, including in the coat protein region important for serological detection/diagnostics. We have also made progress in characterizing at least two putative new (i.e. previously unreported) viruses detected in the East Africa sample set and commenced experiments to understand whether these may play a role in disease complexes. Other progress includes ongoing isolation and characterization of understudied or previously unknown viruses identified in grain crop virus surveys in the United States and beyond, including identifying complete or partial genome sequences by deep sequencing and confirming virus presence, sequence, and pathogenicity using standard plant virology techniques. More than 300 maize lines, hybrids and populations were screened for their responses to U.S. isolates MCMV, SCMV and MCMV plus SCMV. Strong resistance to SCMV was identified, as expected, and lines with strong tolerance to MCMV were found. For Objective 2, testing of agronomic and resistance responses of selected RIL from the Oh1VI population was completed, as was a study of the responses of U.S. grain and sweet corn hybrids to inoculation with potyviruses. For Objective 4, related to virus molecular characterization, we have identified a virus that can be used for research virus-induced gene silencing (VIGS) in maize seedlings, and have identified a novel virus silencing suppressor protein. We generated an infectious clone and various VIGS constructs for silencing in wheat using Soil-borne wheat mosaic virus (Ohio isolate) but although the constructs stably maintain inserted sequences and can silence an endogenous gene in the dicot host plant Nicotiana benthamiana, we do not observe VIGS in wheat or other monocots, suggesting that there is some biological difference in the silencing trigger in each host. Finally, we obtained preliminary data on RNAi targets that cause mortality of a leafhopper vector of maize viruses, Graminella nigrifrons.
1. Identifying disease-causing viruses in corn. Next generation sequencing (NGS) is an effective method to rapidly identify emerging viruses and assess existing virus populations, even for previously unknown viruses. 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 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). At least three putative new (or previously unknown) corn-infecting viruses were also discovered. One group of corn-infecting viruses, the potyviruses, are particularly diverse, which confounds conventional diagnostic techniques. Identification of these new viruses and isolate-specific virus genome sequences is critical to define disease epidemiology, ensure detection of all important virus variants, and limit disease spread.
2. Maize chlorotic dwarf virus encodes a novel silencing suppressor protein. RNAi (RNA interference) or RNA silencing is an important plant defense mechanisms that targets and destroys invading viruses in a sequence-dependent manner. In the pathogen-host molecular ‘arms race’, many plant viruses combat this plant defense mechanism by encoding virus suppressors of silencing. ARS scientists at Wooster, Ohio identified a Maize chlorotic dwarf virus (MCDV) protein with silencing suppressor activity. Silencing suppressors have not been previously identified for MCDV or any closely related virus. This discovery is a significant step in our understanding of gene function and pathogenicity of this major U.S. corn virus.
3. Identifying resistance and tolerance to Maize chlorotic mottle virus in maize. Maize is a staple crop in sub-Saharan Africa, where pest and disease outbreaks are key constraints to maize productivity and food security. Since 2011, maize lethal necrosis, caused by infection of maize with two viruses named Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV) has caused up to 100% losses for smallholder farmers. ARS researchers in Wooster, OH developed methods for screening maize for resistance to the viruses causing MLN. Together with collaborators from Kenya Agriculture and Livestock Research Organization, they identified maize lines that develop no or few symptoms when inoculated with U.S. isolates of MCMV or SCMV. Some of these lines also show reduced symptoms when inoculated with East African isolates of these viruses. These lines may form the basis for developing maize hybrids with tolerance or resistance to MLN, providing an affordable and environmentally sound management for this devastating disease.
4. What makes a leafhopper a vector. More than 75% of plant virus diseases are spread by insects, but we know very little about insect and virus interactions that are needed for an insect to be a virus vector, especially for viruses that replicate in the insect before transmission. Using next generation sequencing, ARS researchers in Wooster, OH and their Ohio State University collaborators determined that there are very few differences in gene expression in leafhoppers (Graminella nigrifrons) that transmit Maize fine streak virus, a rhabdovirus that replicates in the leafhopper, and those that can acquire but not transmit the virus. These results suggest that only one or a few factors that distinguish these two types of leafhoppers. We also found unexpectedly high transmission for one of the virus’ genes that suggests this gene could play a role in virus multiplication in the leafhopper. Further, we developed a system for knocking down the expression of specific genes in leafhoppers, which will allow us to test the effects of identified genes on virus multiplication in and transmission by the insects. These studies provide the basis for research to identify specific leafhopper and virus genes required for clues for rhabdovirus transmission.
Stewart, L.R., Teplier, R., Todd, J.C., Jones, M.W., Cassone, B.J., Wijeratne, S., Wijeratne, A., Redinbaugh, M.G. 2014. Viruses in maize and Johnsongrass in southern Ohio. Phytopathology. 104:1360-1369.
Morales-Cruz, K., Zambrano, J., Stewart, L.R. 2014. Co-infection and disease severity of Ohio Maize dwarf mosaic virus and Maize chlorotic dwarf virus strains. Plant Disease. 98:1661-1665.
Miao, H., Di, D., Lu, Y., Tian, L., Zhang, A., Stewart, L.R., Redinbaugh, M.G. 2014. Efficient inoculation of rice black-streaked dwarf virus to maize using Laodelphax striatellus Fallen. Journal of Phytopathology. DOI:10.1111/jph.12350.
Cassone, B.J., Redinbaugh, M.G., Dorrance, A.E., Michel, A.P. 2015. Shifts in Buchnera aphidicola density in soybean aphids (Aphis glycines) feeding on virus-infected soybean. Insect Molecular Biology. DOI 10.1111/imb.12170.
Chen, Y., Redinbaugh, M.G., Michel, A.P. 2015. Molecular interactions and immune responses between maize fine streak virus and the leafhopper vector G. nigrifrons through differential expression and RNA interference. Insect Molecular Biology. 24(3):391-401.
Edwards, M.C., Weiland, J.J., Todd, J., Stewart, L.R. 2015. Infectious Maize rayado fino virus from cloned cDNA. Phytopathology. 105:833-839.
Cassone, B.J., Cisneros-Carter, F.M., Michel, A.P., Stewart, L.R., Redinbaugh, M.G. 2015. Genetic insights into Graminella nigrifrons competence for Maize fine streak virus infection and transmission. PLoS One. 9(11):e113529. DOI:10.1371/journal.pone.0113529.
Mahuku, G., Lockhart, B.E., Wanjala, B., Jones, M.W., Kimunye, J.N., Stewart, L.R., Cassone, B.J., Sevgan, S., Johnson, N., Kusia, E., Kumar, L., Niblett, C.L., Wangai, A., Kiggundu, A., Asea, G., Pappu, H., Prasanna, B.M., Redinbaugh, M.G. 2015. Maize lethal necrosis (MLN), an emerging threat to maize-based food security in sub-Saharan Africa. Phytopathology. 105:956-965.