Hairy roots are used to obtain transformed roots in Phaseolus with a reporter construct. The resulting transgenic roots are used to examine the expression of the reporter gene. Promoter expression studies are crucial to understand the spatiotemporal expression patterns of genes.
Today we are going to demonstrate the methodologies for hairy root induction and to study promoter expression patterns in transgenic hairy roots under root nodule symbiotic conditions in Phaseolus. Estrada-Navarrete and colleagues have previously established Agrobacterium rhizogenes induced hairy root system in Phaseolus. The current method will help accelerate the duration of hairy root induction and thereby improving the versatility of the system.
Demonstrating the procedures will be my colleague Manoj-Kumar Arthikala and undergraduate students Alma Leticia and Brenda Mariana from my lab. To begin with, search the NIN gene sequence in Phaseolus vulgaris genome database from Phytozome, and select the required length of sequence upstream to ATG. Design the oligonucleotides following the appropriate promoter sequence analysis.
Freshly prepared Phaseolus genomic DNA will serve as the template to PCR amplify the promoter sequence. Prepare the PCR master mix with high-fidelity Taq polymerase and standard PCR components, along with the prior-designed oligonucleotides. Aliquot the master mix into 2 mL PCR tubes, and perform the promoter fragment amplification in a thermocycler and with a suitable program.
Separate the PCR product electrophoretically on a 1.2%agarose gel. Visualize the NIN fragment amplified at 700 base pairs under UV transillumination. Excise, elute, and clone the fragment into the Gateway entry vector pENTR/D-TOPO vector following the standard instructions.
Transform two microliters of the reaction mix into competent E.coli cells at 42 degrees Celsius for 50 seconds, and plate the transformed E.coli on LB supplemented with 50 milligrams per liter kanamycin. Grow the plated cells at 37 degrees Celsius overnight. Perform a PCR reaction for plasmids isolated from randomly selected E.coli colonies using M13 oligonucleotides.
The fragment at 1024 base pairs should indicate positive clones. Perform the LR reaction between the positive entry clone and destination binary vector. Transform two microliters of reaction mix into E.coli, and select the colonies on 100 milligrams per liter of spectinomycin at 37 degrees Celsius.
Amplify the plasmids with NIN-specific oligonucleotides. The positive clone should amplify at 700 base pairs. Sequence and verify the correctness of the clone fragment.
Mobilize the NIN promoter construct into electrocompetent K599 Agrobacterium rhizogenes cells. And once again, confirm the clone with promoter-specific oligonucleotides. Sulfur sterilize Phaseolus vulgaris seeds and germinate on sterile, wet filter papers for two days in dark at 28 degrees Celsius.
Spread 150 microliters of Agrobacterium rhizogenes cells with NIN construct and control vector on LB plates with spectinomycin selection. Grow the culture overnight at 28 degrees Celsius. Decoat Phaseolus seedlings, and scrape out Agrobacterium rhizogenes cells with a bent, 200-microliters tip from the plate.
And resuspend into Eppendorf Tube with one mL sterile, distilled water. Collect Agrobacterium rhizogenes cells into a three mL syringe, and place Phaseolus seedlings into previously prepared tube setup. Slightly prick the hypocotyls of germinated seeds with the needle tip, simultaneously injecting the Agrobacterium rhizogenes cells.
Care should be taken that the needle do not pass through the hypocotyl. Maintain the injected seedlings in a growth chamber at 28 degrees Celsius with 16 hours light and eight hour dark photoperiod. Callus on the wounded hypocotyls should be visible by five to seven days.
After two weeks, remove the primary root by cutting the stem two centimeters below the hairy roots, and transplant them into pots containing sterile vermiculite. Simultaneously, inoculate the plants with one ml Rhizobium tropici to induce nodulation. Make sure to dilute the Rhizobium culture to 6 at OD 600.
Maintain the plants in a growth chamber at 28 degrees Celsius, 16 hours light, and eight hours dark photoperiod, and irrigate with B and D solution. To study spatiotemporal expression patterns of NIN promoter, sampling is done at different time points. Take out the plants without damaging the roots, and wash the roots without any specks of vermiculite.
Randomly select the roots from NIN and control plants, and proceed to GUS histochemical staining by incubating the samples in GUS assay buffer at 37 degrees Celsius for 16 hours in dark. Observe the GUS-stained roots under stereo and compound microscope. This picture shows the Rhizobium-induced NIN promoter expression in the primary roots.
NIN promoter expression could also be seen in Rhizobium-infected root hair cells. Further, the GUS expression can be documented at different stages of root nodule development. No GUS expression was seen in vector control roots.
After watching this video, you should have a good understanding of designing your promoter constructs and studying the promoter expression patterns in transgenic hairy roots of Phaseolus. This system could be further extended to study the promoter expression patterns under other symbiotic and pathogen interactions in Phaseolus and also other eudicots.
