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 expr...

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 border="0" cellpadding="0" cellspacing="0" width="1125">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Primers for qRT-PCR assay</td>
			<td style="width:253px;"></td>
			<td style="width:460px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Forward</td>
			<td>CACC ATA GCT CCC CAA AAT GGT AT</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Reverse</td>
			<td style="width:253px;">CAT CTT CCT TCC ACT AAC TAA C</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Forward</td>
			<td>GTA AAA CGA CGG CCA G</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Reverse</td>
			<td>CAG GAA ACA GCT ATG AC</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
			<td style="width:253px;">Company</td>
			<td style="width:460px;">Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">REAGENTS</td>
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			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pENTR/D-TOPO Cloning Kit</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">K243520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway LR Clonase II Enzyme Mix&#160;</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11791100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">pBGWFS7.0&#160;</td>
			<td style="width:253px;">Plant systems biology</td>
			<td style="width:460px;">https://gateway.psb.ugent.be/vector/show/pBGWFS7/search/index/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum Taq DNA Polymerase</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">10966018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNeasy Plant Mini Kit</td>
			<td style="width:253px;">Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">PureLink Quick Gel Extraction Kit</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">K210012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Platinum Pfx DNA Polymerase</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11708013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">Certified Molecular Biology Agarose</td>
			<td style="width:253px;">Bio-Rad</td>
			<td>1613102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">One Shot TOP10 Chemically Competent E. coli</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">C404006</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nacl</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">S7653</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tryptone</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">T7293-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Yeast extract</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">Y1625-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bacteriological agar</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Kanamycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">60615-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Spectinomycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td>PHR1441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethyl alcohol</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bacteriological peptone</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">P0556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Calcium chloride</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nalidixic acid</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">N8878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesium sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel loading solution</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">G7654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
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			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EQUIPMENT</td>
			<td style="width:253px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Thermocycler</td>
			<td style="width:253px;">Veriti Thermal Cycler</td>
			<td style="width:460px;">4375786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Centrifuge</td>
			<td style="width:253px;">Sigma</td>
			<td style="width:460px;">Sigma 1-14K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel documentation unit</td>
			<td style="width:253px;">Carestream&#160;</td>
			<td style="width:460px;">Gel Logic 212 PRO</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">MaxQ SHKE6000 6000 Shaking Incubator - 115VAC</td>
			<td style="width:253px;">Thermo scientific</td>
			<td style="width:460px;">EW-51708-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Plant growth chamber</td>
			<td style="width:253px;">MRC</td>
			<td>PGI-550RH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Horizantal laminarair flow cabinate</td>
			<td style="width:253px;">Lumistell</td>
			<td style="width:460px;">LH-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Fluorescent microscope</td>
			<td style="width:253px;">Leica&#160;</td>
			<td style="width:460px;">DM4500 B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Petridish</td>
			<td style="width:253px;">sym laboratorios</td>
			<td style="width:460px;">90X15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Scalpel Blade&#160;</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">53223</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">22 mL glass tubes</td>
			<td style="width:253px;">Thomas scientific</td>
			<td style="width:460px;">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 ve...

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

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

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

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 ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...