Robertson LAB

Department of Plant Biology , North Carolina State University

     
 

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ChlI-silenced Nicotiana benthamiana
 

Last updated September, 2007

We are interested in gene silencing mediated by geminiviruses, and how we can use this silencing as a tool for functional genomics. Two species of the Geminiviridae family, Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus (CaLCuV), have been used to induce gene silencing in Nicotiana benthamiana and Arabidopsis, respectively. We recently adapted a third geminivirus, Cotton leaf crumple virus, as a vector for silencing in the cotton plant; our first crop plant (Tuttle et al., 2008). This work was a collaboration between our group and Candace Haigler and Judy Brown's group and was supported by Cotton Inc.

We also focus on geminivirus-induced silencing mechanisms and how geminiviruses escape silencing in plants. We know that SGS3 and RDR6 are needed for gemini-induced gene silencing, which places them in a category closer to transgenes than to RNA viruses, at least in terms of how the host responds to them (Muangsan et al., 2004). Although 24 nt siRNAs are abundant in CaLCuV:CHLI-infected plants, only mutations in DCL4 affect VIGS of CHLI (Blevins et al., 2006).

Geminivirus vectors are especially good for silencing meristematic genes and we have silenced both PCNA and RBR in both N. benthamiana (Peele et al., 2001, Jordan et al, 2007) and Arabidopsis (Bernacki, unpublished). In both species, we found that RBR was needed for cell viability at maturity, but not during development. Development was abnormal, as described by Jordan et al., (2007).

We have also studied the cell biology of geminivirus infection in N. benthamiana. We found that TGMV causes cell-autonomous induction of DNA replication that includes replication of host DNA (Nagar et al., 2002). Steve Nagar also found that some cells had condensed chromatin, suggesting an arrest in mitosis at prophase. In collaboration with Hank Bass, we imaged through some of these cells using deconvolution microscopy (Bass et al., 2000, see photo and movie on the home page). For unknown reasons, host DNA is always marginalized. TGMV DNA replication sites can vary from one primary site to several distributed throughout the nucleus. A successful TGMV infection causes an increase in the size of the nucleus, sometimes to over 500% of its original size (Bass et al., 2000). This work was a collaboration with Hank Bass and Linda Hanley-Bowdoin.