NLR immune receptors play a crucial role in mediating plant defense against various pathogens. We describe a TurboID-based proximity labeling method for the identification of interaction partners of Toll/interleukin-1 receptor domain containing NLRs such as immune receptors in Nicotiana benthamiana plants. This protocol provides an important reference for investigating protein-protein interactions in other plant species and it can be extended to any protein of interest in Nicotiana benthamiana.
The TurboID-based proximity labeling approach has a distinct advantage over the traditional approaches because it can be used to capture transient or weak protein-protein interactions in vivo. Start by growing N.benthamiana seeds in wet soil at a high density. Maintain them in a climate chamber with an 18-hour light and eight-hour dark photo period at 23 to 25 degrees Celsius.
Keep the plants in the chamber for about four weeks until they grow to a leaf stage of four to eight for subsequent agroinfiltration. Construct TurboID fusions and transform the plasmids into Agrobacterium tumefaciens competent cells as described in the text protocol. Use a one milliliter needleless syringe to infiltrate the inoculum of the abaxial epidermis of the fully mature N.benthamiana leaves.
At 36 hours post-filtration, infiltrate 200 micromolar biotin into the leaves and maintain the plant for an additional three to 12 hours before harvesting the leaf tissue. To collect the leaf sample, cut the infiltrated leaves at the base of the petiole, remove the leaf vein, and flash freeze the tissue in liquid nitrogen. Grind the leaf tissue with a pestle and mortar and store the powder in 15 or 50 milliliter Falcon tubes at minus 80 degrees Celsius.
To extract the total protein, transfer about 0.35 grams of leaf powder to a two milliliter tube. Be sure to maintain the tissue at low temperature in the liquid nitrogen during this process. Add 700 microliters of RIPA lysis buffer to the powder.
Vortex the tube for 10 minutes and leave the sample on ice for 30 minutes, mixing the contents every four to five minutes by inverting the tubes. Centrifuge the tubes at 20, 000 times g at four degrees Celsius for 10 minutes. Then collect the supernatant.
Remove the free biotin by running the sample through a desalting column. Remove the sealer at the bottom of the column and place it in a 50 milliliter tube. Then loosen the cap and centrifuge the column at 1, 000 times g and four degrees Celsius for two minutes.
Place the desalting column in a fresh 50 milliliter tube and equilibrate the column three times with five milliliters of RIPA lysis buffer. Centrifuge it at 1, 000 times g and four degrees Celsius for two minutes and discard the flow-through each time. Add 1.5 milliliters of protein extract to the top of the resin in the equilibrated column.
And when the extract enters the resin, add another 100 microliters of RIPA lysis buffer. Centrifuge the column for two minutes and leave the desalted samples on ice until further use. To quantify the desalted protein extracts, measure the OD595 using a Bio-Rad spectrophotometer.
Draw the standard curve based on the value of the gradient BSA solution and calculate the concentration of the desalted protein samples. Equilibrate the streptavidin C1 conjugated magnetic beads with one milliliter of RIPA lysis buffer for one minute at room temperature. Use the magnetic rack to absorb the beads for three minutes and gently aspirate the solution.
Then repeat the wash one more time. Transfer the protein extract to the equilibrated magnetic beads and incubate the tube at four degrees Celsius overnight on a rotator to affinity purify the proteins. On the next day, capture the beads with a magnetic rack and aspirate the supernatant.
Next, wash the beads with 1.7 milliliters of wash buffer one by adding it to the tube and incubate it on the rotor for eight minutes. Remove the supernatant as previously described and repeat the wash with 1.7 milliliters of wash buffer two and wash buffer three. Add 1.7 milliliters of 50 millimolar Tris HCL to remove the detergent.
Then place the tube on the magnetic rack and remove the supernatant. Repeat the wash with one milliliter of 50 millimolar Tris HCL and transfer the beads to a new 1.5 milliliter tube. Finally, wash the beads six times with 50 millimolar ammonium bicarbonate buffer for five minutes per wash.
Use 100 microliters of beads for immunoblot analysis to confirm the enrichment of the biotinylated proteins and flash freeze the rest of the sample to be stored at minus 80 degrees Celsius. Western blots were used to analyze protein expression and biotinylation in the agroinfiltrated N.benthamiana leaves. The biotinylated proteins in the infiltrated leaves were efficiently enriched with streptavidin C1 conjugated magnetic beads for subsequent mass spectrometry analysis.
Different proteins with varied sizes were captured and Western blot analysis of the enriched proteins showed smeared bands. When attempting this procedure, please remember to remove the sealer at the bottom of desalting column and loosen the caps before each centrifugation. This protocol is easily adaptable to other proteins of interest in Nicotiana benthamiana and can also be used to extend the TurboID PL in other plant species.
