Virus Induced Gene Silencing (VIGS) - to study gene function in plants

A	B	C
D	E	F
G	H	I
Silencing of phytoene desaturase (PDS) gene in Nicotiana benthamiana  (A and B,) Microtom (C) and VF36 (D) tomato and Arabidopsis thaliana Col-0 (D and E) using TRV-VIGS system. Silencing of CTR1 gene in tomato (F,) CH42 gene in Arabidopsis (G) and GFP transgene in Arabidopsis (H). GFP expressing Arabidopsis (I).
MicroTom	VF36 CTR1	VF36 PDS
Silencing of phytoene desaturase (PDS) and CTR1 genes in tomato plants using TRV-VIGS system.

Virus induced gene silencing (VIGS)

RNA viruses carrying sequences homologous to a transgene or an endogenous gene can be both triggers and targets of PTGS (Baulcombe, 1999). When a virus vector is engineered to carry host-derived sequences it can be used to silence the function of those endogenous genes. PTGS triggered by the recombinant viral vectors will target both the viral vector and the host sequences corresponding to insert. The phenotype of the plant silenced by VIGS for a particular gene mimics the phenotype of loss of function mutant.

Many VIGS vectors have been described to study gene function in plants (Burch-Smith et al., 2004). We have developed a second generation TRV (Tobacco Rattle Virus)-based VIGS vector.

TRV is a bipartite positive sense RNA virus. RNA-1 can multiply and spread in the absence of the RNA2 (MacFarlane, 1999). We have constructed cDNA clones of RNA1 and RNA2 of TRV in a T-DNA expression cassette. The multiple cloning site (MCS) included in RNA2 allows the cloning of target gene sequences for VIGS. When Agrobacterium tumefaciens cultures containing TRV RNA1 and RNA2 constructs are mixed and infiltrated into the leaves of Nicotiana benthamiana viral RNA gets synthesized from the T-DNA. These RNA transcripts then serve as templates for the further replication of viral RNA by the RNA dependent RNA polymerase encoded by RNA1. Systemic infections by the virus then bring about VIGS of the targeted plant host sequences.

Our TRV-based VIGS vector can be very effectively used in plants:

• Nicotiana benthamiana (Liu et al., 2002b) (Fig. 1)
• tomato ( Ekengren et al., 2003; Liu et al., 2002a) (Fig. 2)
• petunia (Chen et al., 2004)
• potato (Brigneti et al., 2004)
• poppy (Hileman et al., 2005)
• Arabidopsis (S.P. D-K., unpublished) to identify gene function.

Our TRV-based VIGS system can also be used to silence:

• flower specific genes (Chen et al., 2004; Hileman et al., 2005; Liu et al., 2004)
• and fruit specific genes (Fu et al., 2005).

References

• Baulcombe, D. (2004). RNA silencing in plants. Nature 431, 356-363.
• Baulcombe, D. C. (1999). Fast forward genetics based on virus-induced gene silencing. Curr Op Plant Biol 2, 109-113.
• Brigneti, G., Martin-Hernandez, A. M., Jin, H., Chen, J., Baulcombe, D. C., Baker, B., and Jones, J. D. (2004). Virus-induced gene silencing in Solanum species. Plant J 39, 264-272.
• Burch-Smith, T. M., Anderson, J. C., Martin, G. B., and Dinesh-Kumar, S. P. (2004). Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39, 734-746.
• Chen, J. C., Jiang, C. Z., Gookin, T. E., Hunter, D. A., Clark, D. G., and Reid, M. S. (2004). Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence. Plant Mol Biol 55, 521-530.
• Ekengren, S. K., Liu, Y., Schiff, M., Dinesh-Kumar, S. P., and Martin, G. B. (2003). Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato. Plant J 36, 905-917.
• Fu, D. Q., Zhu, B. Z., Zhu, H. L., Jiang, W. B., and Luo, Y. B. (2005). Virus-induced gene silencing in tomato fruit. Plant J 43, 299-308.
• Hileman, L., Drea, S., de Martino, G., Litt, A., and Irish, V. F. (2005). Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy). Plant J Online publication date: 26-Aug-2005.
• Liu, Y., Nakayama, N., Schiff, M., Litt, A., Irish, V. F., and Dinesh-Kumar, S. P. (2004). Virus induced gene silencing of a DEFICIENS ortholog in Nicotiana benthamiana. Plant Mol Biol 54, 701-711.
• Liu, Y., Schiff, M., and Dinesh-Kumar, S. P. (2002a). Virus-induced gene silencing in tomato. Plant J 31, 777-786.
• Liu, Y., Schiff, M., Marathe, R., and Dinesh-Kumar, S. P. (2002b). Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30, 415-429.
• MacFarlane, S. A. (1999). Molcecular biology of the tobraviruses. J Gen Virol 88, 2799-2807.
• Meister, G., and Tuschl, T. (2004). Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343-349.

top