Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

Podsumowanie

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

Streszczenie

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter β-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates ≥2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Wprowadzenie

Promoters are important molecular biological tools which play a crucial role in understanding the regulation of the expression of genes of interest. Promoters are DNA sequences located upstream of the translation initiation codon of gene sequences and they carry the central regulatory information of genes; therefore, their correct annotation and characterization are vital to understanding gene function. Depending on the expression patterns, plant promoters are classified as constitutive, tissue-specific, or development-stage-specific and inducible¹. Advances in transcriptomic technologies, improvements in computer modeling, and the availability of increasing numbers of genome sequences for different plant species have facilitated the large-scale prediction of promoter sequences².

On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. Unlike the other model plants, the functional characterization of common bean legume (P. vulgaris) genes is impeded chiefly due to the recalcitrant nature of Phaseolus sp. for stable genetic transformation. Transient transformation systems serve as an alternative for rapid gene functional characterization studies³. In legume symbiosis research, the interaction between the legume host plant and rhizobial bacterium is one of the most tractable model systems for the functional analysis of nodule-specific genes and promoter studies. So far, several legume promoters related to these symbioses have been characterized, viz., Medicago truncatula PT4⁴, SWEET11⁵, Lotus japonicus Cyclops, UBQ⁶, VAG1⁷, Glycine max PT5⁸, Exo70J⁹, P. vulgaris RbohB^10,11,12, TRE1¹³, PI3K¹⁴, TOR¹⁵, etc.Cis elements directly influence gene regulation. The transcription factor ENBP1A binds to a Cis regulatory region (−692 bp) of early nodulin VfENOD12, and this facilitates the expression of a reporter gene in nodule primordia of Vicia faba¹⁶. Replacement of Cis regulatory regions (−161 to −48 bp) of the nodule-specific promoter leghemoglobin GLB3 with the heterologous truncated promoters δ-p35S and δ-pNOS, resulted in a loss of nodule specificity and reduced promoter activity¹⁷.

Previous reports show that the transcription factor NIN is required for the initiation of rhizobial infection in the root hair cells and is also essential for nodule organogenesis in L. japonicus¹⁸. In the present study, we describe a protocol for identification, isolation, cloning, and characterization of nodule-specific promoter in the common bean hairy roots. To achieve this, we selected a rhizobial symbiosis-specific NIN promoter of P. vulgaris and cloned in a transcriptional fusion to the chimeric reporter GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using A. rhizogenes induced hairy roots. This system generates hairy roots in less than 2 weeks after transformation. Finally, we assessed the spatiotemporal expression of NIN promoter in rhizobia colonized root nodules by GUS staining.

The procedure described here may be useful not only for the study of nodulation and mycorrhization¹¹ of legume plants, but also for the study of promoter expression patterns in roots¹⁹. Moreover, this protocol is easy to use in non-specialized laboratories.

Protokół

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.

Wyniki

The objective of this study was to assess the spatiotemporal expression pattern of nodule-specific P. vulgaris NIN. To do this, a 700 bp region upstream of the translation initiation codon of the NIN gene was selected and a set of oligos was designed as depicted in Figure 1A. Using a high-fidelity polymerase, the NIN promoter fragment was amplified, isolated (Figure 1B), and cloned into the destination binary ve...

Dyskusje

During functional analysis of genes, the study of gene expression patterns plays a crucial role in understanding the spatial and temporal regulation of the genes in vivo. A well-known method to study gene expression patterns is to clone the gene promoter region of interest, upstream to reporter genes such as fluorescent marker genes (GFP, RFP, etc.) or β-glucuronidase. Herein, we have selected a promoter region of the root nodule symbiosis (RNS) specific gene, NIN, to study the spatio-temporal expr...

Materiały

Name	Company	Catalog Number	Comments
Primers for qRT-PCR assay
pNIN Forward	CACC ATA GCT CCC CAA AAT GGT AT
pNIN Reverse	CAT CTT CCT TCC ACT AAC TAA C
M13 Forward	GTA AAA CGA CGG CCA G
M13 Reverse	CAG GAA ACA GCT ATG AC
Name	Company	Catalog Number	Comments
REAGENTS
pENTR/D-TOPO Cloning Kit	Invitrogen	K243520
Gateway LR Clonase II Enzyme Mix	Invitrogen	11791100
pBGWFS7.0	Plant systems biology	https://gateway.psb.ugent.be/vector/show/pBGWFS7/search/index/
Platinum Taq DNA Polymerase	ThermoFisher Scientific	10966018
DNeasy Plant Mini Kit	Qiagen	69104
PureLink Quick Gel Extraction Kit	ThermoFisher Scientific	K210012
Platinum Pfx DNA Polymerase	Invitrogen	11708013
Certified Molecular Biology Agarose	Bio-Rad	1613102
One Shot TOP10 Chemically Competent E. coli	Invitrogen	C404006
Nacl	Sigma-Aldrich	S7653
Tryptone	Sigma-Aldrich	T7293-250G
Yeast extract	Sigma-Aldrich	Y1625-250G
Bacteriological agar	Sigma-Aldrich	A5306-1KG
Kanamycin sulfate	Sigma-Aldrich	60615-25G
Spectinomycin sulfate	Sigma-Aldrich	PHR1441
Ethyl alcohol	Sigma-Aldrich	E7023
Bacteriological peptone	Sigma-Aldrich	P0556
Calcium chloride	Sigma-Aldrich	C1016
Nalidixic acid	Sigma-Aldrich	N8878
Magnesium sulfate	Sigma-Aldrich	M7506
Gel loading solution	Sigma-Aldrich	G7654
Name	Company	Catalog Number	Comments
EQUIPMENT
Thermocycler	Veriti Thermal Cycler	4375786
Centrifuge	Sigma	Sigma 1-14K
Gel documentation unit	Carestream	Gel Logic 212 PRO
MaxQ SHKE6000 6000 Shaking Incubator - 115VAC	Thermo scientific	EW-51708-70
Plant growth chamber	MRC	PGI-550RH
Horizantal laminarair flow cabinate	Lumistell	LH-120
Fluorescent microscope	Leica	DM4500 B
Petridish	sym laboratorios	90X15
Scalpel Blade	Fisher scientific	53223
Falcon 15mL Conical Centrifuge Tubes	Fisher scientific	14-959-53A
22 mL glass tubes	Thomas scientific	45048-16150