1. Identification, Isolation, and Cloning of P. Vulgaris NIN Promoter

1. Identify the promoter sequence for the gene of interest. There are several genome databases and analysis tools available for plants such as Phytozome, Ensembl Plants, NCBI, etc. A legume promoter P. vulgaris NIN (PvNIN; Phvul.009G115800) was used in this study²⁰.
2. Design oligos either for Gateway or traditional restriction enzyme cloning depending on the vector system used (Figure 1A).
   NOTE: Standard (25-35 bp), desalted primers work fine. Reverse oligo sequences that flank between the promoter and gene are advisable.
3. Amplify the PvNIN promoter region (or promoter region of the gene of interest) from freshly isolated P. vulgaris genomic DNA using the promoter-specific oligo set. Use a high-fidelity polymerase with the following PCR conditions: 95 °C for 5 min; 30 cycles (95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min); and 72 °C for 5 min.
4. Run the amplified fragment on 1% agarose gel at 60 V on a 7 x 10 cm gel tray, and elute the corresponding amplicon (700 bp, Figure 1B) and clone into the pENTR/D-TOPO vector according to the manufacturer's instructions.
5. Transform 2 µL of the reaction into competent Escherichia coli cells using the manufacturer's instructions and plate 150 µL of the transformation mixture on lysogeny brother (LB) supplemented with 50 mg/L kanamycin (Kan50). Grow the plated cells at 37 °C overnight.
6. Pick 3-6 colonies and culture them overnight in LB medium containing Kan50. Isolate plasmid DNA using the method of choice and analyze the plasmid by PCR using the vector specific M13 oligos. For PCR, use 100 ng of plasmid, 0.3 pm of oligos, 10X PCR buffer, 0.5 U Taq DNA polymerase, and 10 mM dNTPs.
7. Use the following PCR amplification conditions; 95 °C for 3 min; 30 cycles (95 °C for 30 s, 58 °C for 30 s, 72 °C for 75 s); and 72 °C for 7 min. Visualize the PCR products on a 1% agarose gel. Ensure that the correct insertions have a band at 1,024 bp and vectors without inserts will have a 324 bp band (Figure 1C).
8. Perform the LR reaction between an entry vector (pENTR/D-TOPO-PvNIN) and destination vector pBGWFS7.0²¹ using a commercial enzyme mix according to the manufacturer's instructions.
9. Transform 2 µL of LR reaction into competent E. coli cells using standard techniques as described in the manufacturer's instructions and plate 150 µL of transformation mixture on LB supplemented with 100 mg/L of spectinomycin (Spe100). Grow the plated cells at 37 °C overnight.
10. Pick approximately 5 colonies and culture them overnight in LB medium containing Spe100. Isolate the plasmid DNA using a method of choice and analyze the plasmid by PCR using the promoter-specific oligos.
11. Use the PCR conditions in step 1.3. Visualize the PCR products on a 1% agarose gel to confirm amplification. Amplicons are 700 bp.
12. Sequence the positive plasmid pBGWFS7.0/PvNIN::GUS-GFP (Figure 1D) with the promoter-specific oligos to verify the authenticity of the insert.
13. Transform the plasmids into A. rhizogenes strain K599 (however, any other strains of A. rhizogenes can be used). Add 2 µL of the plasmid prep to 50 µL of electrocompetent cells and electroporate in a 1 mm cuvette with the following settings: 1.8 kV, 25 µF, and 200 Ω.
14. Recover cells in ~ 500 µL of SOC medium and shake at 28 °C for 2 h. Plate on LB-Spe100 and grow at 28 °C for 2 days.
15. Perform colony screening as mentioned in step 1.7 and use the PCR conditions as in step 1.3. Make glycerol stocks from positive clones and store them in -80 °C.
16. Use A. rhizogenes culture harboring empty pBGWFS7.0 vector as the negative control.

2. A. Rhizogenes Transformed Hairy Roots of the Common Bean

1. Sterilize bean (P. vulgaris cv. Negro Jamapa) seeds (approximately 20) in 50 mL of 96% ethanol for 1 min and discard the ethanol; then add 50 mL of 20% bleach (sodium hypochlorite) for 10 min, with constant mixing in a laminar flow hood. Remove the bleach and wash in excess sterile water 3 times.
   NOTE: Other P. vulgaris cultivars can be used for hairy root induction.
2. Germinate the sterilized seeds on sterile filter papers moistened with Broughton & Dilworth (B&D) solution²² (Table 1) for 2 days in the dark at 28 °C.
3. The day before transformation prepare the following:
   1. Streak out 150 µL of A. rhizogenes culture harboring the binary vector (prepared in steps 1.13 and 1.14) on solid LB Spe100 and grow at 28 °C overnight.
   2. Fill a 15 mL tube with B&D solution and replace the screw cap with double layered aluminum foil. Assemble this into a 22 cm glass tube containing 10 mL of B&D solution (Figure 2C) and autoclave it.
4. On the day of transformation, assemble the following sterile materials in a laminar flow hood: germinated seeds from step 2.2, culture plate from step 2.3.1, tubes from step 2.3.2, sterile double distilled water (10 mL), pipettes and pipette tips, sterile forceps, and 3 mL syringes.
5. Use a bent 200 µL tip to scrape A. rhizogenes cells from the plate into a microcentrifuge tube and resuspend in 1 mL of the double sterile water. Similarly, prepare vector control cells separately.
   1. Mix the cells gently to resuspend. Do not vortex.
6. Make a 3 mm hole on the aluminum foil of the tubes from step 2.3.2 using a sterile pipette tip.
7. Collect the cells (either promoter or vector control) from step 2.5 with the syringe and slightly prick the hypocotyls of the germinated seeds with the needle tip (Figure 2G).
   NOTE: Make sure the needle penetrates (4-6 times) the vascular tissue and inject small (~ 5 µL) drops of the cells on the wound at different positions around the hypocotyl.
8. Carefully insert the roots of the injected seedlings into the 3 mm hole of the 15 mL tube containing B&D solution. Return the entire setup into the 22 mL glass tubes and seal them to maintain humidity.
9. Incubate the tubes in a growth chamber maintained at 28 °C with 16 h light:8 h dark photoperiod.
   Notes: 3-4 days after incubation, the plants grow up to the rim of the 22 mL glass tubes. At this stage, remove the caps and seal the rim with parafilm around the stem as shown in Figure 2I.
10. Observe the callus at the wounding sites at 5-7 days, and find ~ 2 cm hairy roots by 10-12 days after transformation.
    NOTE: It is important to maintain the B&D solution constantly in both plastic and glass tubes.
11. After 2 weeks, remove the primary root by cutting the stem 2 cm below the hairy roots and transplant the primary roots into pots containing sterile vermiculite. Maintain the plants in a growth chamber (16 h light:8 h dark at 28 °C) and irrigate with B&D solution.

3. Promoter Analysis: NIN Promoter Expression in Rhizobial Nodules of the Common Bean

1. For induction of root nodules, inoculate Rhizobium tropici or Rhizobium etli (that are compatible to the common bean) in 100 mL of PY medium (0.5 g peptone, 0.3 g yeast extract) supplemented with 700 mM CaCl₂ and 20 mg mL⁻¹ nalidixic acid. Incubate at 30 °C for 24 h with shaking at 300 rpm.
   Note: Any specific antibiotics could be used in the case of transgenic Rhizobium.
2. Pellet the cells in a centrifuge for 3 min at 2,800 x g at room temperature and resuspend in 10 mL of 10 mM MgSO₄. Adjust the OD of cells to 0.6 at OD₆₀₀ by dilution with 10 mM MgSO₄.
3. Inoculate 1-2 mL of culture (prepared from step 3.2) per plant (both promoter and vector control) to induce nodule growth on the hairy roots. Irrigate the plants with B&D solution with limited nitrogen (2 mM KNO₃) to promote nodulation.
4. Depending on the experimental need, collect the hairy roots at different time points. Collect samples for studying infection threads between 3-5 days post inoculation (dpi).
   Note: Primordial nodule and young nodules can be studied at 6-9 dpi and 10-12 days dpi, respectively. Finally, mature and functional nodules can be studied at 14-21 dpi, and senesced nodules at > 28 dpi.
5. Process the root/nodules for the activity of reporter genes (GUS or GFP). Analyze GUS activity according to the protocol described by Jefferson²³. Observe GFP under a fluorescence microscope. Excite GFP fluorescence with a blue argon ion laser (488 nm), and collect the emitted fluorescence from 510 to 540 nm.