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ARS Home » Midwest Area » Wooster, Ohio » Corn, Soybean and Wheat Quality Research » Research » Research Project #427429

Research Project: Developing Reverse Genetics Systems for Functional Analysis of Monocot Virus and Host Genes

Location: Corn, Soybean and Wheat Quality Research

Project Number: 5082-22000-001-01-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 22, 2014
End Date: Sep 21, 2019

Developing infectious cDNA clones of plant-infecting RNA viruses allows further important basic research on both the virus and its plant hosts. Viruses that are amenable to modification to carry heterologous sequences have been developed as gene silencing and gene expression vectors, allowing functional genomics in plants by targeting genes of interest for silencing or overexpression. Research is needed to develop a high-throughput system for virus-induced gene silencing (VIGS) or expression in monocotyledonous plants, which include the grass crops maize and wheat. Development of reverse genetics systems allows discovery of functions of virus genes and sequence elements by targeted mutagenesis of infectious virus cDNA, and research is needed to develop systems for major U.S. maize viruses. This project aims to develop potential virus vectors for gene silencing in monocots, and to develop a reverse genetics system for the U.S. maize virus Maize chlorotic dwarf virus (MCDV). Prioritized specific objectives are: 1. Develop infectious cDNA clones and a reverse genetics system for Maize chlorotic dwarf virus (MCDV), a major U.S. maize virus. MCDV sequences are recalcitant to propagation in Eschericia coli, a common problem for virus sequences. So far, no infectious clones of full-length MCDV or truncated replicons have been developed. Improved current cloning technologies make success more probable now. Research is needed to develop infectious clones of MCDV and to deliver cloned virus into maize directly, or using a protoplast or local host intermediate to increase inoculum. 2. Complete research examining the utility of Maize necrotic streak virus (MNeSV) as a VIGS vector in maize. MNeSV is one of very few viruses being considered for vector development for maize because of its high infectivity and relatedness to the dicot virus used for highly effective VIGS, Tomato bushy stunt virus. However, unlike related tombusviruses, MNeSV is not readily mechanically transmissible. The recalcitrance to mechanical transmission make it useful at this time for laboratory research but not to manipulate plant phenotypes in the field. Final tests of MNeSV and a report of our work developing MNeSV as a VIGS vector, including its utility and limitations for functional genomics applications are anticipated. 3. Develop Soilborne wheat mosaic virus (SBWMV) as a virus vectors. SBWMV shows promise as a monocot VIGS vector, but is still in very early stages of development for gene expression and VIGS in wheat. We will complete experiments determining the feasibility of using SBWMV for gene silencing in monocots. 4. Test Barley stripe mosaic virus (BSMV) for high-throughput functional genomics on cold-tolerance genes in wheat and barley. BSMV is one of the best currently available monocot VIGS vectors, and will be tested for utility by attempting silencing of known cold tolerance pathway genes and testing for transcriptional changes, cold tolerance, and flowering time phenotypes.

1. MCDV full length clones will be developed into low-copy plasmids with a 35S promoter, terminator, and ribozyme cassette for expression of the viral cDNA. Several clones will be developed and tested. Insertion of intron sequences will be attempted to stabilize clones. Once stable clones are developed, they will be tested by agroinfiltration to Nicotiana benthamiana followed by vascular puncture inoculation to maize. Other potential local hosts will also be tested, and protoplasts as a last option for scale up of infectious material for delivery to maize. When infectious clones and a delivery system are developed, mutagenesis will be performed and clones tested for infectivity without ability to express putative small open reading frame products. 2. Complete experiments and draft manuscripts describing results from MNeSV vector development. 3. Test alternate insertion sites and VIGS in wheat for SBWMV. Re-test SBWMV phytoene desaturase (PDS) silencing in wheat using coat protein read-through insertion site, testing SBWMV phytoene desaturase (PDS) insertion clones upstream of the 3’UTR of RNA2, and complete SBWMV RNA1 movement protein based deletion clone evaluation. 4. Test BSMV vector utility in maize and : The differential susceptibility of maize cultivars to SBWMV and BSMV is not known. The vascular puncture inoculation (VPI) technique was used to test for infectivity of both viruses in maize cultivar 'Spirit'. In first experiments, this cultivar showed infection by BSMV but not SBWMV. To more thoroughly test for the utility of these viral vectors we will carry out VPI inoculations on five maize cultivars with broad virus susceptibility.