Location: Corn, Soybean and Wheat Quality Research
2021 Annual Report
Objectives
1. Monitor and identify emerging insect-transmitted pathogens of maize and soybean using standard and bioinformatics-based approaches, and develop management strategies. Sub-objective 1.A: Identification and diagnosis of viruses and virus populations in maize. Sub-objective 1.B: Develop tools to characterize emerging maize-infecting viruses. Sub-objective 1.C: Characterize role of E. coryli in damage caused by BMSB.
2. Identify virus factors important for pathogenesis, transmission and host interactions, and develop virus systems for gene discovery and functional analysis in maize and other cereals. Sub-objective 2A: Characterize Maize chlorotic dwarf virus factors important for pathogenesis and interactions with plant hosts. Sub-objective 2B: Develop systems for working with full-length infectious cDNAs of maize viruses. Sub-objective 2C: Define insect vector interactions with plant host and viral pathogens.
3. Identify and characterize mechanisms of action of genetic loci for virus resistance in maize. Sub-objective 3A: Identify and characterize loci providing tolerance/resistance to MCMV in maize and sorghum. Sub-objective 3B: Characterize interactions among potyviruses, MCMV and virus resistance/tolerance in maize. Sub-objective 3C: Characterize and map novel soybean quantitative trait loci (QTL) for host plant resistance to brown marmorated stinkbug (BMSB).
4. Characterize pathogen vectoring relationships of and between emerging insect pests and vectors of maize pathogens using comparative genetic and genomic analyses to identify factors that can be disrupted for disease control.
Approach
Developing control strategies for insect-transmitted diseases requires knowledge about the pathogen, crop host, disease vector, and interactions among them and with the environment. Under Objective 1 we will combine standard serological and molecular approaches for diagnostics with next generation sequencing (NGS) approaches to identify and define population structures for emerging insect-vectored pathogens of maize and soybean. We will use this information to develop targeted molecular and serological diagnostics for emerging diseases, identify virus vectors and identify other factors important for disease development and spread. The identity and populations of yeast of yeast transmitted to soybean by brown marmorated stink bug (BMSB) will be defined using NGS, and traditional plant pathological approaches will be used to determine its role in damage caused by the stink bug. Under Objective 2, molecular biological and biochemical approaches will be used to virual protein structure and function for Maize chlorotic dwarf virus. Molecular biological approaches will be used to develop and improve infectious cloned cDNAs for maize infecting viruses. For Objective 3, methods we previously developed for phenotypic analysis of plant responses to Maize chlorotic mottle virus and BMSB will be used to map resistance in biparental and association mapping populations using molecular and NGS approaches for genotyping. Interactions between known maize potyvirus resistance genes and potyvirus isolates will be assessed in near isogenic lines carrying defined resistance genes and alleles using the development of symptoms and virus titer in inoculated plants. NGS genomic and transcriptomic analyses of leafhoppers feeding on healthy and virus-infected plants under different environmental conditions will be used to develop comparisons of leafhopper species.
Progress Report
Scientists in Wooster, Ohio made significant progress on all four objectives of the project in its fourth year:
Objective 1, to monitor, characterize, and develop management strategies for emerging viruses, is ongoing as maize and soybean viruses spread, emerge, and are detected across the U.S. and worldwide. Bioinformatic analysis of a 2018 maize virus survey was completed, and at least one potentially novel virus was identified in silico. Validation and characterization of novel viruses and virus variants are needed and ongoing. Synergistic interactions between maize yellow mosaic virus (MaYMV), sugarcane mosaic virus (SCMV), and/or maize chlorotic mottle virus (MCMV) were characterized and indicated that MaYMV has measurable and significant interactions with each virus. MaYMV caused significant stunting and leaf reddening symptoms under growth chamber conditions. Evaluation of more than 30 maize inbreds for MaYMV resistance was completed and ten asymptomatic lines were identified. Potyvirus samples, that were previously collected from East Africa, were successfully purified to eliminate contamination by other viruses and will facilitate downstream virulence testing. These accomplishments represent significant progress towards the completion of goals to rapidly identify and characterize pathogens of maize and soybean and to identify genetic resistance (1.A.1), survey U.S. maize for virus sequences (1.A.2), and develop tools for understanding epidemiology of maize lethal necrosis (MLN) in East Africa (1.B).
Under Objective 2, substantial progress has been made in basic virology research. Maize chlorotic dwarf virus (MCDV) constructs were built for in planta assays to evaluate putative polyprotein proteolysis sites. Eleven MCDV-S protease mutants were created and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that lose catalytic activity.
MCDV-M1 and MCDV-Severe infectious clones were created, P51 protein was swapped to evaluate their effects on disease severity in maize, and P27 protein was deleted and replaced with GFP to evaluate P27 as the helper component in MCDV vector transmission. An MDMV infectious clone was developed to modify gene expression in maize, allowing overexpression or knocking down of multiple genes simultaneously. These results support the characterization of MCDV virus factors involved in pathogenesis and host-pathogen interactions (2.A) and development of systems for working with full-length infectious clones of maize viruses (2.B).
Significant progress has been made towards the identification and characterization of loci of genetic resistance in maize as part of Objective 3. A genome wide association study (GWAS) analysis of MCMV tolerance in the maize Goodman 282 population led to the identification of several significant tolerance loci. Quantitative resistance loci were mapped in a recombinant inbred line population derived from one of the most MCMV tolerant lines identified from the Goodman population. This work led to the identification of a major MCMV resistance locus. Several maize recombinants in the MCMV tolerance regions derived from N211 were identified on chromosomes 3 and 5 and will be used to facilitate development of fine mapping populations. Evaluation of MCMV and potyvirus levels in MCMV tolerant line, N211, and susceptible OH28 were performed. Results from this experiment indicated that MCMV titer in the tolerant line was reduced by approximately 100 thousand-fold two weeks post infection. Multiplexed RT-qPCR assays for MCMV and SCMV were refined to improve accuracy and reproducibility. Development of a maize synthetic population, OhMCMV-1, was completed and evaluated for MCMV and MLN resistance. The population was developed by intermating five MCMV tolerant maize inbred lines and conducting one complete cycle of recurrent selection for MCMV resistance. The OhMCMV-1 population has significantly higher levels of MCMV and MLN resistance than found among the five parents. This population is planned for public release and will serve as a valuable breeding tool for MLN resistance. Continued progress was made towards the conversion of N211, KS23-5, and KS23-6 lines from yellow to white endosperm through marker assisted backcrossing. KS23-5 and KS23-6 have been successfully converted to white endosperm, while N211 is partially converted. Endosperm conversion will allow for more rapid deployment and development of MLN tolerant varieties in East Africa. Screenings of a population of 233 lines from the germplasm enhancement of maize (GEM) project for resistance to MDMV and SCMV were completed and submitted to the GEM database. These efforts support the identification, characterization (3.A.1, 3.B.1, 3.B.2), and fine mapping (3.A.2) of virus resistance loci in maize.
Objective 4, genetic and genomic characterization of pathogen vectoring relationships between insect vectors of maize pathogens, is nearly complete moving into the final year of the project. Leaf hopper data sets were analyzed and virus-responsive and temperature responsive differentially expressed transcripts were detected. Annotation of these datasets is ongoing. Strand specific RT-PCR assays were developed and used to demonstrate MFSV replication in non-vector leaf-hopper species Dalbulus maidis and Macrosteles quadrilineatus. Research performed as part of this objective has helped to inform pathogen vectoring relationships between important leaf-hopper pests and viruses of corn and led to identification of several potential factors that could be disrupted for disease control.
Accomplishments
1. A recently detected corn virus causes disease and interacts with co-infecting viruses that cause maize lethal necrosis (MLN). A new corn-associated polerovirus, often termed maize yellow mosaic virus (MaYMV) was recently discovered and found to be highly prevalent globally (Asia, Africa, South America, but not yet described in the U.S.) along with other corn viruses that cause a devastating disease called maize lethal necrosis (MLN) in co-infected corn plants. Whether the newly discovered polerovirus caused disease or contributed to MLN was unknown. ARS scientists at Wooster, Ohio, elucidated that MaYMV causes significant stunting, but other symptoms such as leaf reddening are often very mild. This indicated that the virus is likely to impact yield but evade facile visual detection, and that identification and breeding of genetic resistance in corn to this virus is advisable due to its high potential for yield reduction. Disease instigated by MaYMV was evaluated in single and mixed infections by the virus alone and in all possible combinations with two other corn viruses. MaYMV had measurable interactions with each of the two other viruses in mixed infections, causing significantly increased disease severity. These discoveries were published in Plant Disease and have spurred further research to identify sources of genetic resistance to MaYMV and improved information on management of MaYMV and MLN in corn, which is of great benefit to growers, breeders, and seed companies.
2. Maize dwarf mosaic virus-based tool for simultaneous gene silencing and expression in corn. Ever since the first genome sequence of corn was developed, there has been a need for research tools to examine and understand the function of genes, gene families, and genetic pathways in the plant. Historically, such tools have been very labor-intensive (generating transgenic plants or random mutagenesis), but viruses provide a shorter timescale method to target individual genes to study their function. ARS scientists at Wooster, Ohio, in collaboration with researchers at The Ohio State University (OSU), developed a new virus-based tool from maize dwarf mosaic virus (MDMV) with unique and unprecedented capability for corn. The MDMV-based tool can simultaneously overexpress and knock down expression of multiple corn genes, allowing study of either individual genes or entire genetic pathways in maize without the generation of transgenic or mutagenized plants. MDMV-based clones have already been shared under Material Transfer Agreements with two North Carolina State University and one Ohio State University laboratory, the Army Corps of Engineers via Defense Advanced Research Projects Agency, and have been requested from two international laboratories shortly after publication to enable studies of corn gene function and biology. An invention disclosure was made for this work.
Review Publications
Stewart, L.R. 2021. Sequiviruses and Waikaviruses (Secoviridae). Encyclopedia of Virology. vol. 3, pp. 703-711. Oxford: Academic Press.
Mlotshwa, S., Xu, J., Willie, K.J., Khatri, N., Marty, D., Stewart, L.R. 2020. Engineering maize rayado fino virus for virus-induced gene silencing. Plant Direct. 4(8). Article e00224. https://doi.org/10.1002/pld3.224.
Stewart, L.R., Willie, K.J. 2021. Maize yellow mosaic virus interacts with maize chlorotic mottle virus and sugarcane mosaic virus in mixed infections, but does not cause maize lethal necrosis. Plant Disease. Article 33736468. https://doi.org/10.1094/PDIS-09-20-2088-RE.
Xie, W., Marty, D., Xu, J., Khatri, N., Willie, K.J., Buckner Moraes, W., Stewart, L.R. 2021. Simultaneous gene expression and multi-gene silencing in Zea mays using maize dwarf mosaic virus. BMC Biology. 21. Article 208. https://doi.org/10.1186/s12870-021-02971-1.
