A golden-gate compatible TRV2 virus induced gene silencing (VIGS) vector.

Virus induced gene silencing (VIGS) is a useful tool in plant biology to systemically silence a

gene by RNA interference (RNAi). Co-expression of tobacco rattle virus 1 (TRV1), which

contains the genome of the viral vector, with TRV2 containing the target sequence for RNAi,

can be achieved by Agrobacterium-mediated gene expression in Nicotiana benthamiana

(Burch-Smith et al., 2006). Existing methods of cloning TRV2 plasmids with the targeting

sequence of interest include time consuming Restriction-Ligation cloning, expensive

Gateway cloning or Ligation Independent Cloning (LIC). Whilst LIC is relatively quick and

cost-effective, it relies on having a pre-linearised vector. Therefore, Golden-Gate cloning is

preferable in that it is a 1-step cloning method. Therefore, we modified the existing TRV2

vector to be compatible with golden gate cloning. To add extra flexibility, we incorporated the

overhangs used for pRNAiGG, a vector for carrying out transient, local silencing in leaf

tissue by hairpin RNAi (Yan et al., 2012). Therefore, the resulting TRV2gg is compatible with

the same insert for pRNAiGG enabling transient, local silencing or systemic silencing.

TRV2gg contains an unwanted BsaI recognition site in the backbone, however a simple

golden gate protocol of 37˚C for 2 h or a protocol that ends with a ligation-optimised step, is

sufficient to easily clone multiple positive colonies.