2011 Annual Report
1a.Objectives (from AD-416)
Develop virus-induced gene silencing (VIGS) constructs to knock out putative disease resistance genes in soybean, in order to test and establish gene function. Also test and develop protocols to precisely modify genes in soybean or other legume species, using a combination of VIGS and zinc finger nuclease (ZFN) technologies.
1b.Approach (from AD-416)
The cooperator will work with the ADODR and soybean researchers in the CICGRU on two tasks that use specialized knowledge and technology related to virus-induced gene silencing (VIGS) and use of viruses for transient gene over expression. First, assemble gene constructs (genes, promoters, and appropriate vector and selection sites) to substantially decrease or knock out gene expression of targeted genes. VIGS technologies used in the cooperator lab enable gene knockdown in transient assays (assays used within a single plant). Second, test and develop protocols to extend VIGS technologies to produce persistent, multi-generation gene knockout. Specifically, develop constructs containing zinc finger nucleases (which can be designed to precisely and permanently modify DNA), and deliver these to plant germ-line cells using modified VIGS vectors. The Soybean Mosaic Virus (SMV) and Bean Pod Mosaic Virus (BPMV) have been modified in the cooperator’s lab to express payload genes in the host plant. SMV additionally has the potential to express the ZFN in the seed and embryo, and the virus can be removed in a subsequent cross. The cooperator will first test for gene expression of a SMV-carried reporter gene in germ line cells and in a second generation in soybean, and then will test for expression and function of BPMV and SMV constructs that carry ZFNs.
We have tested a wide range of soybean and bean species and relatives for naturally occurring viruses, and have tested susceptibility of these for a specific virus (soybean mosaic virus, SMV) that has also been developed (by the cooperator) as a vector for virus-induced gene silencing (VIGS). Testing for plant/virus compatibility is important for identifying virus and plant combinations that can be used in subsequent tests of gene modification. Of 20 bean species and varieties tested for naturally occurring viruses in Iowa, four were infected (hyacinth bean, winged bean, common bean, guar bean), with a total of four bean virus types (cowpea mosaic, southern bean mosaic, potyvirus group, soybean mosaic). These viruses are being evaluated as vectors for gene modification. Of varieties tested specifically with SMV, the susceptible varieties included two bean cultivars, soybean, and hyacinth bean. Progress on this project is monitored through regular face-to-face meetings.