The virus induced gene silencing protocol begins with growing the aphasian strains. PT RV one, PT RV two pt, RV two PT S and PT RV two target gene on LB plates. Two days later, these strains are inoculated in liquid LB and grown overnight.
This culture is then used to inoculate a secondary induction medial liquid culture, which is also grown overnight. The next day, a bacterial suspension of each culture is prepared and both PT RV one and PT RV two or PT RV two target gene are mixed in a one-to-one ratio. The bacterial mixture is then infiltrated into end ana or tomato seedlings using a needleless syringe.
Hi, I'm Andre Velazquez from the laboratory of Greg Martin at the Department of Plant Pathology and Plant Microbiology at Cornell University. Today we'll show you how to do virus induced gene silencing tomato and nicotiana. We use this procedure in our lab to study the interactions between a plant pathogen and its host.
So let's get started. To begin this procedure for agrobacterium to fassan strains, each harboring PT RV one, PT RV two pt, RV two PDS or PT RV two host target gene plasmids are grown on LB grates supplemented with kanamycin and rifampicin. Kanamycin selects for the PTRV plasmid while rifampicin selects for the agrobacterium.
Incubate the plates at 30 degrees Celsius for two days when the bacterial colonies have grown on the LB plates, use each of the strains to inoculate four, two to three milliliter cultures of liquid LB containing kanamycin and rifampicin. Incubate the cultures at 30 degrees Celsius with shaking at 200 RPM for 16 to 18 hours. On the following day, inoculate a one to 25 dilution of each primary culture into five milliliters of a secondary liquid induction media culture with can mycin rifampicin and 200 micromolar acetophenone.
The IM mimics the environment that Agrobacterium encounters in the host Alast and the Acetophenone induces the bacterial VI genes which are required for TD NA transfer into the plant. Incubate the cultures at 30 degrees Celsius with shaking at 200 RPM for 20 to 24 hours. The next day harvest the cells by centrifugation for 10 minutes at 3000 gs.
After removing the S supernatant, add five milliliters of 10 millimolar magnesium chloride, 10 millimolar MES pH 5.5 to the pellet and resus suspend the cells by gentle vortexing, centrifuge the cells again for 10 minutes at 3000 Gs, then resuspend the cells in half the volume of the original culture with the magnesium chloride MES solution. Prepare a bacterial suspension with an OD 600 of 0.3 for each bacterial culture. Add acetone to a final concentration of 400 micromolar to the PT RV one culture.
Mix the cultures containing the PT RV one and the PT RV two or the PT RV two host target gene in a one-to-one ratio also include a PT RV two PDS control. The final acetone concentration is now 200 micromolar and each culture is at an OD 600 of 0.15. The cultures are now ready to be used for viral infiltration of the nicotiana, hamana and tomato plants and hamana plants used for silencing should be around two and a half weeks old when the coens and the first two to four true leaves have emerged.
Tomato plants are used seven to eight days post emergence when the true leaves have not yet appeared. Labeled the seedlings to be infiltrated with the gene to be silenced and the date of the experiment for tomato infiltrate both olean and for Ana. Infiltrate the biggest two true leaves.
Poke a hole into each leaf to be infiltrated with a needle. Use a one milliliter needleless syringe to infiltrate the bacterial suspension into the seedlings to avoid cross-contamination. Change gloves between infiltrations and do not water the plants until the day after the inoculation.
After infiltration, keep the plants at 20 to 22 degrees Celsius in a growth chamber with a 16 hour day length and 50%relative humidity for at least three and a half weeks before using them for assays. This is a representative result of an experiment with ana and tomato plants three and a half weeks after being silenced for the phyto desaturated gene. Both plants show the characteristic bleaching phenotype observed in plants with diminished amounts of carotinoids.
For, for PDS Silenced control plants, photobleaching starts to be seen as soon as one and a half weeks after infiltration. We've just shown you how to do virus inducing silencing in ana and tomato. In this procedure, it is important to remember to be careful to ab avoid cross contamination and to keep the plants at the proper environmental conditions that favor virus spread and silencing.
So that's it. Thanks for watching and good luck with your experiments.
