The overall goal of the following experiment is to test whether effectors of pathogens are recognized by resistant proteins in plants. This is achieved by first infecting plant leaves with a liquid culture of agrobacterium or through PVX agro infection, which will transiently express effectors in the plant tissue. Then the plants are allowed to incubate and the plants are scored for cell death.
Results are obtained that show cell death responses on recognition of transiently expressed defectors by resistance proteins in plants. The main advantage of these techniques over existing methods like genetic studies with molecular markers is that these are functional assays. They not only show that the resistance gene is present in the plant, but also that it works and that defense responses are actively induced.
These methods can help answer key questions in molecular plant pathology, such as to rapidly discover bot resistance and RA and felons AFI genes, which availability of both genome sequences Demonstrating the procedure will be performed by du, a PhD student from a laboratory To generate potato plants. Maintain in vitro plantlets in sterile plastic jars containing more shige, scoog or MS medium in climate chambers at 18 degrees Celsius with a 16 hour, eight hour day night regime for two weeks. When plants grow, white roots transfer them into pots of sterilized soil.
In regulated greenhouse compartments for plants used in these experiments use four to five week old seed grown NCOA hamana, and four to five week old potato transplants generated in vitro. To prepare agrobacterium culture begin by filling 50 milliliter tubes with 10 milliliters of YEB medium, supplemented with one microliter of acetophenone, 100 microliters of MES and the appropriate antibiotics. Next pipette 20 microliters of glycerol stock of the desired strains of agrobacterium containing the gene of interest into the YEB.
Incubate the cultures at 28 degrees Celsius and 200 RPM for one to two days to an OD 600 of about 1.0. Harvest the cells by centrifugation at 3000 GS for 10 minutes. Then pour off the supernat and resuspend the pellet.
In freshly made MMA medium to an OD 600 of 0.3. Gently vortex the cells to resus suspend them for co infiltration of two bacterial strains. Mix the culture at a one-to-one ratio.
Allow the cultures to sit at room temperature for one to six hours before infiltrating plants. In the meantime, label the plants to be infiltrated with the strain and the date of the experiment to carry out infiltrations wearing eye protection. Use a one milliliter syringe without a needle to carefully and slowly inject agrobacterium suspensions into leaf panels.
Inject at least three plants and three leaves per plant to serve as triplicates to avoid cross-contamination. Change or sterilize gloves between infiltrations and avoid watering the plants until the day after. Inoculation approximately three days after infiltration, score the plants for cell death on a scale of zero to 100%with zero representing no symptoms.
100%indicating confluence cell death and intermediate values ranging from sclerosis to increasing levels of cell death. Carry out PVX agro infection in two to three week old and hamana seed grown plants and two to three week old potato transplants grown in vitro to three milliliters of YEB supplemented with antibiotics. Add 20 microliters of glycerol stock of agrobacterium incubate at 28 degrees Celsius and 200 RPM to an OD 600 of about 1.0.
Spread about 300 microliters of each agrobacterium strain onto LB agar plates with antibiotics and incubate at 28 degrees Celsius for one to two days. Use a spatula to collect the agrobacterium. Then to inoculate a leaf with a large amount of bacteria.
Dip a toothpick into the culture and use it to pierce the plant. Leaf inoculate each leaf in multiple locations through leaves per plant and three plants per strain. Two weeks after inoculation score macroscopically by recording each spot qualitatively as a yes or no.
Then calculate the percentage of responding sites and compare to the controls shown. Here are representative results of agro infiltration in end hamana and two potato species, Solenium JI 3 49 dash three and CV Desiree about three days after inoculation. There is confluence cell death in the leaf panel co infiltrated with the mixture of agrobacterium strain AGL one PVRG containing P bin plus R three A and PK seven WG two A VR three A while no death response occurs with the negative control PI plus R three A or PK seven WG two A VR three A in this figure PVX agro infection of n and two potato species S one Kae 3 54 dash one and S micro DOUM 360 dash one are shown about two weeks after inoculation.
There's expanding cell death at the sites inoculated with the positive control PGR 1 0 6 CRN two. That encodes a general cell death inducing gene from Phytophthora Infest and PGR 1 0 6 in one that encodes an illiciting gene from P Infest. No expanding cell death is observed at the site inoculated with the negative control PGR 1 0 6 empty Well.
Attempting this procedure is important to remember to cultivate good quality plant material. This technique paved the way to explore effect omics in potato and other plant species for researchers in molecular plant pathology and plant breeding. After watching this video, you should have a good understanding of how to perform efficient and robust high throughput functional analysis of can gene in plants.
