The overall goal here is to demonstrate the methodology using virus induced gene silencing for identifying genes involved in non hosts resistance to achieve VIGs of the endogenous plant. Target gene inoculate nicotiana hamama plants with tobacco rattle virus figs construct carrying a fragment of the target gene, prepare and inoculate the non hosts pathogen cultures expressing green fluorescent protein onto the gene silenced plant leaves. Next, assess the green fluorescent colonies of non hosts pathogen under UV light in the dark.
In order to identify gene silenced plants that compromise non hosts resistance, identify the target gene by sequencing the insert in virus vector. Overall, this methodology of VIX mediated forward genetic screening allows researchers to easily identify genes involved in non hosts resistance of plants against bacterial pathogens. In a short time period, We realized that the importance of combining virus induced gene silencing mediated forward genetics with GFP UV expressing pathogens to identify the non-ST resistance responsible genes.
When we did previously a tedious forward genetic screen by scoring for hyper response induced cell death and disease induced cell death. So nicotiana Ben seeds on a soilless potting mixture germinate the seeds in a growth chamber. After three weeks, transplant the seedlings into individual pots, grow them in a greenhouse for at least two to three days before the tobacco rattle virus.
Figs vector inoculation gradually thaw the frozen stalks of agrobacterium containing the cDNA clones. Intv two vector under sterile conditions inoculate the individual agrobacterium cultures onto Luria. Berti agro plates incubate the plates at 28 degrees Celsius for up to two days.
Grow four replicate colonies for each clone so that adequate agrobacterium inoculum is available for inoculation of two plants. Inoculate TRV one into Luria berti liquid medium. Harvest the overnight grown cultures by centrifugation, resuspend the bacteria in inoculation, buffer, and incubate for three hours at room temperature on a shaker at 50 RPM.
After harvesting the cells by centrifugation, measure optical density and resus suspend the bacterial pellets in five millimolar MES buffer pH 5.5 Next inoculate the T RV one bacterial culture having OD 600 of 0.3 into the axi side of three to four nicotiana hamama leaves using a needleless syringe at the site of T RV one inoculation. Prick the leaves with inoculum of the respective T RV two colonies using a toothpick for two weeks. Maintain the plants under low temperature with adequate nutrition because vigorous growth is important for efficient gene silencing by pigs.
As an example, pseudomonas senge PV tomato T one is used in this experiment. Pathogens should carry a plasmid that can express G-F-P-U-V grow the PGE strains in kings be liquid, medium supplemented with 10 micrograms per milliliter. Rifampicin 50 micrograms per milliliter can mycin at 28 degrees Celsius for 12 hours.
Harvest the bacterial cultures by centrifugation, then confirm the presence of green fluorescence using a long wavelength UV lamp in the dark. After two washes in sterile water, resus suspend the cells at the desired concentration of sterile water and adjust OD 600. Then inoculate the respective pathogens onto the axial side of target.
Gene silenced leaves encircles of about 1.5 centimeter diameter if required. Simultaneously test several non hosts pathogens for their growth in the target gene silenced plants also include a vector control for viewing. Implant a bacterial growth between two and five days.
Expose the inoculated leaves to a long wavelength UV light in the dark. Be sure to wear skin and eye protection. Monitor the bacterial colonies as green fluorescent spots in the axial side of the leaf in the background of red fluorescence emitted by the leaf surface.
Verify the green fluorescence emitted by the host pathogen that acts as a positive control. Observe pathogen growth daily from two to five days post inoculation. Make a list of the clones who silencing, resulted in growth of one or more non hosts pathogens.
To remove the false positives from the first screen. Repeat the VIGs for selected clones and again, test the response of gene silenced plants to non-cost pathogens. Use colony PCR on the selected clone to amplify insert using the A TTB one and a TB two primers in the T RV two vector or primers blanking the cloning site.
Run the PCR product on an aros gel and verify amplification of the single band. Then sequence the plant gene insert in the T RV two vector using either the A TTB primers or primers flanking the cloning site. Perform blast using the sequence and identify the gene details.
A generalized protocol for silencing large numbers of genes using tobacco rattle virus based figs in nicotiana. Hamana is shown in this flow chart. Some of the gene silenced plants showed various phenotypic alterations, including stunted growth, yellowing and photobleaching phenotypes.
These data show green fluorescence of pathogen exposed to UV light in the dark before plant inoculation and their growth implants three days after inoculation the same leaf exposed under white light is shown as control. This experiment tracks the growth of G-F-P-U-V expressing bacteria in plana. Three non hosts.
Pathogens were used to demonstrate the process of identification of the gene silenced plant susceptible to non hosts pathogens in the wild type nons silenced nicotiana hamama plants with immunity. These pathogens did not grow. However, at least two non hosts pathogens grew on NB sgt.
One gene silenced leaves. Quantitative data on bacterial growth demonstrates the process of identifying gene silenced plants compromised for non-ST resistance. Using this figs mediated forward genetics approach, we have silenced about 5, 000 genes in nicotiana hamana and identified plants compromising non-ST resistance over a period of about one and a half years.
As an example, non hosts pathogen growth in two gene silenced plants identified from this screen is shown here. We hope to have conveyed how to perform virus induced gene silencing for hundreds of genes, and then assess the silenced plants by observing the growth of non-cost pathogens in plana to dissect non-cost resistance. By using this virus induced gene, silencing mediated protocol, one can able to silence hundreds of genes in about two to three weeks time period, and then assess the growth of non horse pathogens labeled with GFP UV in about two to three hours a day, spanning around two to five days.
