VIGS protocol (pgR106/7)
Plasmids, strains and plant
Both pgR106 and pgR107 are binary vectors based on pGreen0000, they are basically the same vector except that the cloning site in pgR106 contains ClaI-AscI-NotI-SalI, while in pgR107 contains CalI-SmalI-SalI. AscI and NotI are rare cutting enzymes in that both of them are 8-basepair cutting enzymes and all the 8 base pairs consist of only GC, which will ensure less truncation for cDNA cloning. Therefore pgR106 is more suitable for cDNA library construction, while pgR107 is more suitable for PCR product cloning considering the SmaI site in its cloning site. The selection marker for pgR106 and pgR107 is kanamycin (50ug/ml). Both of them can replicate to high copy number in E.coli. But in agrobaterium they need helper plasmid pJIC Sa_Rep ,which carrys tetracycline as selection mark (5ug/ml), for replicating.
We recommend agrobacterium strain GV3101 to work with these two vectors because of its faster growth and high transformation efficiency, though other agrobacterium strains can also work very well with pgR106/pgR107.
Nicotianan benthamiana has been proved to be a suitable plant for easy inoculation and successful VIGS using these two vectors.
Cloning of target gene into VIGS vector
Just like normal cloning, nothing really special in this step. After transformation of E.coli., use kanamycin (50ug/ml) plate for positive colony selection. Then prepare plasmid from confirmed positive colonies and go to next step.
Transformation of agrobacterium
You can either use your constructs and helper plasmid pJIC SA_Rep together to do co-transformation of GV3101 or use your constructs only to transform GV3101 already containing pJIC SA_Rep. The transformation efficiency is much higher in the second method. Since pgR106/107 can not replicate in agrobacterium without pJIC SA_Rep, tetracycline is optional in the selective plate. But it is advisable to use both kanamycin and tetracycline as selection mark to prevent kanamycin resistant strains from contaminating.
The positive agrobacterium colony should be reconfirmed by PCR before inoculation. just to be sure the target gene insert is still there.
Toothpick inoculation of target plant
Once you get agrobacterium colonies containing VIGS constructs on the plate , pick up the colonies and stab target plant leaves on the main vein several times. Choose plants with size of about 5-6 cm in diameter for inoculation. Alternatively , you can infiltrate the agrobacterium culture into plant leaves, which normally gives stronger inoculum.