2013 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.
The objective of this project is to develop more efficient and effective methods for direct modification of soybean and other legume species (such as common bean). Although some methods exist for genetic modification of soybean, these are typically inefficient and slow, requiring close to a year of calendar time to generate a modified plant. Further, methods for transformation of other species such as common bean are even less efficient, due to genetic and developmental barriers to plant tissue culture and regeneration. Lastly, existing transformation methods for soybean and bean require introduction of foreign DNA, which makes the modified plants subject to concerns about side-effects of genetic modification.
This project therefore is focused on developing methods that can use endogenous plant enzymatic mechanisms to precisely target and cut genes of interest without introducing foreign DNA. The method under investigation is to use plant viruses to carry an engineered enzyme (a "zinc finger nuclease," or ZFN) that is capable of making targeted double-stranded cuts in DNA. Progress in the past year includes:
• Assessment of the "Soybean mosaic virus" (SMV) to be used, in soybean and common bean, as a vector to carry the DNA-modifying enzyme;
• Characterization of additional viruses, including sequencing and identification of an uncharacterized strain of Bean Pod Mottle Virus;
• Development and testing of a panel of RNA constructs to be used in SMV. These include constructs that are capable of self-cleaving out of the virus and various constructs to test capacity for expression in the target plant.
Remaining steps include the crucial testing of the SMV carried ZFN construct in the plant and testing to determine whether changes carried out in one plant can be carried into the next generation of plants via the seeds.
The impact of this project is development of methods that may allow dramatic improvements in speed and precision of plant improvement to enable progress towards plants that have desirable characteristics for producers and consumers.