Robertson LAB

Department of Plant Biology , North Carolina State University

     
 

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Last updated September, 2007

We developed silencing vectors derived from two members of the Begomovirus genus of Geminiviridae: Tomato Golden Mosaic Virus (TGMV) and Cabbage Leaf Curl Virus (CaLCuV). Both viruses were engineered to facilitate insertion of plant gene sequences, 100 - 800 bp in size, without compromising viral replication or spread. Some geminiviruses require a coat protein for movement and can accept only about 100 bp of extra sequence. We showed that extensive silencing could result from such small sequences and that fragments from different genes cloned in tandem or in different genome components could produce silencing of multiple genes. These results suggest that geminiviruses, which infect many economically valuable plants, may be a useful tool for discovering or verifying gene function.

The CaLCuV VIGS vector (shown above, right) has two components, designated A and B, which are similar in size. E. coli plasmids are used to progagate the viral vector. The two plasmids, pMTCbLCV::CH42 and pCPCbLCV.002, are mixed together before bombardment. Once inside the plant cells, the viral proteins catalyze DNA replication and movement. The common region (CR) is indicated by a light area. The CR contains the viral origin of DNA replication and is conserved between the A and B components, but varies between members of the begomoviruses. The A DNA (carried in pMTCbLCV::CH42 above) encodes genes needed for replication, gene expression, and encapsidation. AL1 is also known as AC1(required for replication), AL2 as AC2, and AL3 as AC3. The VIGS vector lacks AR1, the coat protein gene, which is replaced by silencing DNA corresponding to the gene of interest (CH42 is now called CHLI and encodes the I subunit of magnesium chelatase). The B component contains two proteins needed for viral movement, BR1 (BV1) and BL1 (BC1).

To order the CaLCuV VIGS system from Addgene, click here. For more information about the CaLCuV VIGS system, click here.