Name: plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean
Uploaded: 2017-12-23T12:30:00.000+00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean at JoVE.com

This work was partially supported by the Direcci&#243;n General de Asuntos del Personal Acad&#233;mico, DGAPA/PAPIIT-UNAM (grant no. IN219916 to M.L and IA205117 to M-K.A) and the Consejo Nacional de Ciencia y Tecnol&#242;gia (CONACYT grant no. 240614 to M.L.).

1. Identification, Isolation, and Cloning of P. Vulgaris NIN Promoter
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C for 5 min; 30 cycles (95 &#176;C for 30 s, 55 &#176;C for 30 s, 72 &#176;C for 1 min); and 72 &#176;C for 5 min.</li>
	<li>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&#39;s instructions.</li>
	<li>Transform 2 &#181;L of the reaction into competent Escherichia coli cells using the manufacturer&#39;s instructions and plate 150 &#181;L of the transformation mixture on lysogeny brother (LB) supplemented with 50 mg/L kanamycin (Kan50). Grow the plated cells at 37 &#176;C overnight.</li>
	<li>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.</li>
	<li>Use the following PCR amplification conditions; 95 &#176;C for 3 min; 30 cycles (95 &#176;C for 30 s, 58 &#176;C for 30 s, 72 &#176;C for 75 s); and 72 &#176;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).</li>
	<li>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&#39;s instructions.</li>
	<li>Transform 2 &#181;L of LR reaction into competent E. coli cells using standard techniques as described in the manufacturer&#39;s instructions and plate 150 &#181;L of transformation mixture on LB supplemented with 100 mg/L of spectinomycin (Spe100). Grow the plated cells at 37 &#176;C overnight.</li>
	<li>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.</li>
	<li>Use the PCR conditions in step 1.3. Visualize the PCR products on a 1% agarose gel to confirm amplification. Amplicons are 700 bp.</li>
	<li>Sequence the positive plasmid pBGWFS7.0/PvNIN::GUS-GFP (Figure 1D) with the promoter-specific oligos to verify the authenticity of the insert.</li>
	<li>Transform the plasmids into A. rhizogenes strain K599 (however, any other strains of A. rhizogenes can be used). Add 2 &#181;L of the plasmid prep to 50 &#181;L of electrocompetent cells and electroporate in a 1 mm cuvette with the following settings: 1.8 kV, 25 &#181;F, and 200 &#937;.</li>
	<li>Recover cells in ~ 500 &#181;L of SOC medium and shake at 28 &#176;C for 2 h. Plate on LB-Spe100 and grow at 28 &#176;C for 2 days.</li>
	<li>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 &#176;C.</li>
	<li>Use A. rhizogenes culture harboring empty pBGWFS7.0 vector as the negative control.</li>
</ol>
2. A. Rhizogenes Transformed Hairy Roots of the Common Bean
<ol>
	<li>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.</li>
	<li>Germinate the sterilized seeds on sterile filter papers moistened with Broughton &#38; Dilworth (B&#38;D) solution22 (Table 1) for 2 days in the dark at 28 &#176;C.</li>
	<li>The day before transformation prepare the following:
	<ol>
		<li>Streak out 150 &#181;L of A. rhizogenes culture harboring the binary vector (prepared in steps 1.13 and 1.14) on solid LB Spe100 and grow at 28 &#176;C overnight.</li>
		<li>Fill a 15 mL tube with B&#38;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&#38;D solution (Figure 2C) and autoclave it.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Use a bent 200 &#181;L 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.
	<ol>
		<li>Mix the cells gently to resuspend. Do not vortex.</li>
	</ol>
	</li>
	<li>Make a 3 mm hole on the aluminum foil of the tubes from step 2.3.2 using a sterile pipette tip.</li>
	<li>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 &#181;L) drops of the cells on the wound at different positions around the hypocotyl.</li>
	<li>Carefully insert the roots of the injected seedlings into the 3 mm hole of the 15 mL tube containing B&#38;D solution. Return the entire setup into the 22 mL glass tubes and seal them to maintain humidity.</li>
	<li>Incubate the tubes in a growth chamber maintained at 28 &#176;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.</li>
	<li>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&#38;D solution constantly in both plastic and glass tubes.</li>
	<li>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 &#176;C) and irrigate with B&#38;D solution.</li>
</ol>
3. Promoter Analysis: NIN Promoter Expression in Rhizobial Nodules of the Common Bean
<ol>
	<li>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&#8722;1 nalidixic acid. Incubate at 30 &#176;C for 24 h with shaking at 300 rpm. 
	Note: Any specific antibiotics could be used in the case of transgenic Rhizobium.</li>
	<li>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.</li>
	<li>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&#38;D solution with limited nitrogen (2 mM KNO3) to promote nodulation.</li>
	<li>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 &#62; 28 dpi.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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 &#946;-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.

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 (&#8722;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 (&#8722;161 to &#8722;48 bp) of the nodule-specific promoter leghemoglobin GLB3 with the heterologous truncated promoters &#948;-p35S and &#948;-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.

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

plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean

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 &#946;-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 &#8805;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.

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 &#8805; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56140/56140fig1.jpg" /> 
Figure 1: Outline of P. vulgaris NIN gene structure and cloning. (A) A schematic representation of the NIN gene structure showing 5&#39;UTR (green), 4 exons, 3 introns, and 3&#39;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. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56140/56140fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56140/56140fig2.jpg" /> 
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 &#38; 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. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56140/56140fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56140/56140fig3.jpg" /> 
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. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56140/56140fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean at JoVE.com

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

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 ...

plant-promoter-analysis-identification-characterization-root-nodule

Universidad San Sebastian

Research

JoVE Journal

Biology

11.7K Views.  Escuela Nacional de Estudios Superiores Unidad León- Universidad Nacional Autónoma de México (UNAM). 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.

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

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Environment

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Genetics

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Polysome Purification from Soybean Symbiotic Nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

High-Efficiency Production of Transgenic Soybean Hairy Roots

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

Author Spotlight: Soybean Hairy Root Transformation for the Analysis of Gene Function  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

Bioengineering

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

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

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Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates