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.
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.
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 RbohB10,11,12, 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.
1. Identification, Isolation, and Cloning of P. Vulgaris NIN Promoter
2. A. Rhizogenes Transformed Hairy Roots of the Common Bean
3. Promoter Analysis: NIN Promoter Expression in Rhizobial Nodules of the Common Bean
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 vector pBGWFS7.0 (Figure 1C) through a Gateway approach to generate a transcriptional fusion to the chimeric reporter GUS-enhanced GFP (pBGWFS7.0/PvNIN::GUS-GFP). The pBGWFS7.0/PvNIN::GUS-GFP vector construct typically generates hundreds of independent clones. Colony screening by PCR with gene-specific oligos easily identifies the correct clones (Figure 2D). The PCR positive plasmid was Sanger sequenced with the promoter-specific oligos to verify the authenticity of the insert.
Maintaining high humidity throughout the experiment is necessary for the induction of callus and hairy roots. At the site of wounding, calli are usually observed at 5 to 7 days (Figure 2J) and ≥ 2 cm hairy roots are observed at 10 to 12 days after transformation. The majority of A. rhizogenes transformed plants successfully to produce hairy roots by 2 weeks (Figure 2K). However, the number and length of hairy roots can be variable. Therefore, select the most vigorously growing roots for analysis and further experiments. Excise the primary root by cutting the stem 2 cm below the hairy roots and transplant them into pots containing sterile vermiculite (Figure 2L) or in a hydroponics system.
To study the spatiotemporal expression of the nodule-specific NIN promoter, hairy roots were inoculated with R. tropici, and GUS activity was observed at periodic intervals post-inoculation depending on the experimental need. Inoculation with R. tropici induced a strong GUS expression in the transgenic roots, indicating that rhizobia induces the NIN expression in the common bean (Figure 3A). Further, sampling at 6-9 dpi showed GUS expression in Rhizobium infected root hair cells (Figure 3B). Phaseolus nodule primordium, and young and mature nodules also demonstrated NIN expression (Figure 3C). At all stages of nodulation, no such GUS expression was seen in the vector control roots (Figure 3D, 3E, 3F).
Figure 1: Outline of P. vulgaris NIN gene structure and cloning. (A) A schematic representation of the NIN gene structure showing 5'UTR (green), 4 exons, 3 introns, and 3'UTR (red) on chromosome number 9 of P. vulgaris. Upstream to the NIN translation initiation codon ATG, shows a promoter region on which oligos were designed. FO, forward oligo; RO, reverse oligo; ATG, start codon; TAA, stop codon. (B) PvNIN promoter region was amplified from freshly isolated P. vulgaris genomic DNA using the promoter-specific oligo set. M, 1 Kb ladder; 1, PCR reaction with gDNA showing amplicon size of 700 bp; 2, PCR reaction without gDNA (-ve control). (C) Screening of the pENTR/D-TOPO-PvNIN plasmids by PCR using the vector specific M13 oligos. Correct insertions have a band at 1,024 bp (lane 3-6) and vectors without inserts will have a 324 bp band (lane 1-2). (D) Promoter expression binary vector map used in the present study, pBGWFS7.0 vector showing PvNIN promoter in fusion with GUS and GFP. Please click here to view a larger version of this figure.
Figure 2: A. rhizogenes transformed hairy roots of the common bean. (A) The sterilized seeds of P. vulgaris cv. Negro Jamapa germinating on sterile filter paper. (B) Seeds, coat removed. (C) Tube setup for hairy root induction. (D) Scraping out A. rhizogenes culture (pNIN & control). (E) A. rhizogenes culture resuspended in sterile distilled water. (F, G) Injecting bacterial cells into hypocotyles. (H) Tube setup with Phaseolus seedlings injected with bacterial cells. (I, J) Calli formed at wounded site 5-7 dpi. (K) Hairy roots 15 dpi (L) excising the tap roots 2 cm below the site of hairy root induction. (M) Composite plants transplanted into sterile vermiculite inoculated with R. tropici. Please click here to view a larger version of this figure.
Figure 3: Promoter analysis of P. vulgaris NIN in transgenic P. vulgaris roots. Spatial and temporal pattern of NIN expression revealed by a promoterNIN::GUS-GFP construct in transgenic hairy roots incubated with GUS as a substrate. Optical microscope images of PvNIN:GUS : (A) Roots inoculated with R. tropici, (B) GUS expression in curled root hairs, and (C) young nodules. Images of control (empty vector) showing no such GUS expression in (D) roots inoculated with R. tropici, (E) curled root hairs, and (F) young nodules. Rh, root hair. Please click here to view a larger version of this figure.
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 expression patterns in P. vulgaris during RNS. The selected promoter sequence has been cloned upstream to GFP::GUS reporters using the Gateway method into a promoter expression vector.
We have described the expression of the symbiosis specific promotor in the hairy root system of P. vulgaris. The hairy root system has been widely used for functional characterization of genes, including RNAi mediated gene knock down, ectopic constitutive expression of genes, promoter expression, and protein localization in recalcitrant crops. The main advantages of the system are that it is easy, reproducible, reliable, and at the same time not labor intensive. The hairy root system has been successfully adopted in other crop legumes such as G. max24, M. truncatula25, L. japonicus26,27, tomato28, etc., with the necessary modifications. Further, the tool can be adopted in other legume and non-legume species when optimized for the suitable A. rhizogenes strain, method of inoculation, and growth conditions.
There are many critical parameters. While selecting the promoter region, care should be taken to design the oligos which would amplify the necessary promoter elements, beginning from the upstream region of the translation start sitein the protein coding genes; promoter analysis should be carried out using the available software to collect information on the transcription factors binding to the regulatory region. Take care while inducing hairy roots: inject seedlings at the appropriate stage; during injection, ensure that the needle does not pass through the hypocotyle; and maintain high humid conditions. Further, while studying promoter expression profiles in hairy roots, the hairy roots produced by A. rhizogenes are not always transformed, and hence it is important to confirm the transformed nature of the roots using GFP or GUS reporters prior to performing promoter expression studies. Suitable strains of Rhizobium should be chosen and the concentrations of Rhizobium should be adjusted as indicated in the protocol. Sampling at the appropriate stages post inoculation is crucial for documenting spatio-temporal expression of the promoter at different stages of RNS.
A. rhizogenes transformed hairy root production is one of the fastest and efficient alternative methods to generate composite plants in P. vulgaris. These composite plants cannot produce transgenic seeds, but can produce transgenic roots within a few days. In this protocol, unlike the other methods29, the closed tubes maintain constant high humidity to promote hairy roots faster at 10 days dpi.
The authors have nothing to disclose.
This work was partially supported by the Dirección General de Asuntos del Personal Académico, DGAPA/PAPIIT-UNAM (grant no. IN219916 to M.L and IA205117 to M-K.A) and the Consejo Nacional de Ciencia y Tecnològia (CONACYT grant no. 240614 to M.L.).
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 |