2012 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.
Since the start of this project on 6/19/2012, we identified five tropical maize inbred lines with strong resistance to infection by Maize rayado fino virus. One of these inbred lines is a parent in an existing mapping population developed under the previous project (3607-22000-011-00D). By the end of FY12, we expect to complete the second year of testing for the presence of maize-infecting viruses in Ohio.
Chen, Y., Cassone, B.J., Bai, X., Redinbaugh, M.G., Michel, A. 2012. Transcriptome of the plant virus vector Graminella nigrifrons, and the molecular interactions of Maize fine streak rhabdovirus transmission. PLoS One. 7(7):e40613.