Virus-induced gene silencing (VIGS): a powerful plant functional genomics tool

          Virus-induced gene silencing (VIGS) is a RNA-mediated post-transcriptional gene silencing mechanism that protects plants against foreign gene invasion.  In addition to allowing a better understanding of how plants defend themselves against plant viruses, VIGS has emerged as an extremely powerful functional genomics tool for knocking out gene expression of target plant genes in some plants. We use Tobacco rattle virus (TRV) derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in Nicotiana benthamiana.  While VIGS has become very popular to perform reverse genetics, VIGS can also be used for forward genetics.

Optimization of VIGS:

          We used Tobacco rattle virus (TRV) derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants.  Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations since it is laborious for large scale screening and some plants are difficult to infiltrate.  We have developed a novel and simple method of agroinoculation, called “agrodrench”, where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors.  By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), 20S proteasome (PB7) or Mg-protoporphyrin chelatase (Chl H) genes in Nicotiana benthamiana and in economically important crops like tomato, pepper, tobacco, potato and Petunia; all belonging to the Solanaceae family.  An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration.  We also demonstrated that VIGS functioned to silence target genes in plant roots.  The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots.  Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.

Fast-forward genetics:

          A normalized N.  benthamiana Mixed Elicitor cDNA (cNbME) library was made with N. benthamiana leaf tissue treated with different abiotic elicitors at Dr. Greg Martin’s lab (Boyce Thompson Institute, Cornell University). The library was cloned via a Gateway reaction (Invitrogen Co.) into the destination vector pTRV2. The library was transformed into Agrobacterium strain GV2260 and arrayed in 96-well plates.  We have now used approximately 4,000 clones from this library to do VIGS in N. benthamiana.  Approximately 10-15% of the silenced plants showed developmentally abnormal phenotype when compared to the empty TRV vector infected plants. Many of the silenced plants showed chlorosis and in most cases this was due to silencing of a Rubisco gene. We are using this fast-forward genetics approach to identify plant gene involved in various processes like nonhost disease resistance and Agrobacterium-mediated plant transformation.

          Successful application of post-transcriptional gene silencing (PTGS) in both plants and animals depends on high target specificity and silencing efficiency. By computational analysis with genome and/or transcriptome sequences of 25 plant species, we predicted that about 50-70% of gene transcripts in plants have potential off-targets when used for PTGS that could obscure experimental results. We have developed a publicly available web based computational tool called “siRNA Scan” to identify potential off-targets during PTGS.  Some of the potential off-targets obtained from this tool were tested by measuring the amount of off-target transcripts using quantitative PCR. Up to 50% of the predicted off-target genes tested in plants was actually silenced when tested experimentally. Our results suggest that a high risk of off-target gene silencing exists during PTGS in plants. Our siRNA Scan tool is useful to design better constructs for PTGS by minimizing off-target gene silencing in both plants and animals.

Publications related to this project: