We thank Dr. Yule Liu from Tsinghua University for providing the PVX-LIC vector. This work was supported by a start-up fund from the Texas A&#38;M AgriLife Research and the Hatch Project TEX0-1-9675 from USDA National Institute of Food and Agriculture to JS.

1. Grow Potato Plants.
<ol>
	<li>Propagate in vitro potato plants in culture tubes (25 x 150 mm) with Murashige and Skoog (MS) media plus&#160;Gamborg&#8217;s vitamin (MS basal salt mixture, Gamborg&#8217;s vitamin, 30 g/L sucrose, 3.5 g/L agar, pH = 5.7). Place the tubes in the growth room under 20&#8211;22 &#176;C, 16 h light/8 h dark photoperiod, and light intensity 120 &#181;mol/m2&#8729;s1. 
	NOTE: New shoots and roots normally develop in 1&#8211;2 weeks from plants. Propagate plants wit...

We present a PVX-based miRNA silencing system to characterize the function of endogenous miRNAs in potato by integrating the STTM design into the PVX vector. The VbMS system proved to be effective in silencing miRNA165/166 in potato, a highly conserved miRNA family across plant species.
The TM approach has been developed to interfere with the expression of miRNAs based on an artificial miRNA target mimic that is designed to create a mismatch loop at the expected cleavage site within the miRNA ...

Plant microRNAs (miRNAs) are characterized as 20&#8211;24 nucleotide-long, nuclear-encoded regulatory RNAs1 and play fundamental roles in almost every aspect of plant biological processes, including growth and development2,3, photosynthesis and metabolism4,5,6,7, hormone synthesis and signaling8,9, biotic and abiotic responses10,11,12,13, and nutrient and energy regulation14,15. The regulatory roles of plant miRNAs are well-programmed and fulfilled typically at post-transcriptional levels by either cleaving or translationally repressing the target mRNAs.
Tremendous progress has been made towards&#160;identification, transcriptional profiling, and target prediction of miRNAs in potato16,17,18,19,20,21. However, the functional characterization of miRNAs in plants, including potato, has lagged behind other organisms due to the lack of efficient and high-throughput genetic approaches. It is challenging to perform functional analysis of individual miRNA by standard loss-of function analysis, because most miRNAs belong to families with considerable genetic redundancy22. In addition, a single miRNA can control multiple target genes23 and several different miRNAs can modulate the same molecular pathway collaboratively24,25. These properties make it difficult to characterize the function of a specific miRNA or a miRNA family.
Much of the functional analysis of miRNAs has relied heavily on gain-of-function approaches that have obvious limitations. The artificial miRNA (amiRNA) method exploits the endogenous primary transcripts (pri-miRNAs) to produce miRNAs at a high level, leading to inhibition of target gene expression26,27,28,29. However, activation tagging and miRNA overexpression using a strong constitutive 35S promoter often lead to heightened expression of miRNAs that are not representative of in vivo conditions and therefore may not reflect the endogenous function of miRNAs30. An alternative approach has been developed involving expression of miRNA-resistant forms of target genes that contain uncleavable mutations in the binding and/or cleavage sites31,32,33. But this approach can also potentially cause misinterpretation of the phenotype derived from the miRNA-resistant target transgene due to transgenic artifacts. Therefore, conclusions from these gain-of-function studies should be drawn with caution34. Another major limitation of the above-described approaches is that they require transformation, which is labor-intensive and time-consuming. Furthermore, the transgene-dependent approaches are hardly applicable for transform-recalcitrant plant species. Therefore, it is essential to develop a fast and efficient loss-of-function approach to unravel the function&#160;of miRNAs.
To bypass the prerequisite of the transformation procedure, virus-based microRNA silencing (VbMS) has been established by combining the target mimic (TM) strategies with virus-derived vectors. In the VbMS system, artificially designed TM molecules are transiently expressed from a virus backbone, offering a powerful, high-throughput, and time-saving tool to dissect the function of plant endogenous miRNAs35,36. VbMS was initially developed in N. benthamiana and tomato with the tobacco rattle virus (TRV)35,36,37 and has been extended to Arabidopsis, cotton, wheat, and maize using various other virus expression systems, including potato virus X (PVX)38, cotton leaf crumple virus (ClCrV)39, cucumber mosaic virus (CMV)40,41,42, Chinese wheat mosaic virus (CWMV)43, and barley stripe mosaic virus (BSMV)44,45.
Potato (Solanum tuberosum) is the fourth most important food crop and the most widely grown noncereal crop in the world primarily because of its high nutritional value, high energy production, and relatively low input requirements46. Several features of potato make it an attractive dicotyledonous model plant. It is a vegetatively propagated polyploid crop with high outcrossing rate, heterozygosity, and genetic diversity. However, to date, there is no report characterizing the function of miRNAs in potato using VbMS. Here, we present a ligation-independent cloning (LIC)-adapted potato PVX-based VbMS approach to evaluate the function of miRNAs in potato plants38. We selected the miR165/166 family to illustrate the VbMS assay because the miR165/166 family and their target mRNAs and Class III homeodomain/Leu zipper (HD-ZIP III) transcription factors have been extensively characterized22,47,48. The HD-ZIP III genes are key regulators of meristem development and organ polarity, and suppression of miR165/166 function leads to increased expression of the HD-ZIP III genes, resulting in pleotropic developmental defects such as reduced apical dominance and aberrant patterns of leaf polarity22,35,38,41. The readily scorable developmental phenotypes correlated with silencing of miRNA165/166 enable accurate evaluation of the effectiveness of the PVX-based VbMS assay.
In this study, we demonstrate that the PVX-based VbMS system can effectively block the function of miRNAs in potato. Because the PVX-based virus-induced gene silencing (VIGS) system has been established in a number of potato varieties49,50,51,52, this PVX-based VbMS approach can be likely applied to a broad range of diploid and tetraploid potato species.

<table border="0" cellpadding="0" cellspacing="0" style="width:691px;" width="691">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">100 &#181;M dATP and 100 &#181;M dTTP</td>
			<td style="width:188px;">Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td>
			<td style="width:120px;">TQAC136</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">3 M Sodium acetate, pH 4.0.</td>
			<td style="width:188px;">Teknova, Hollister, CA 95023, USA</td>
			<td style="width:120px;">#S0297</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acetosyringone</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">D2666-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Agrobacterium tumefaciens strains: GV3101, GV2260 or EHA105.</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chloroform</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0757-950ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dimethyl sulfoxide, DMSO</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">D0798-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DTT</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0281-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">E. coli DB3.1</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td>for maintenance of PVX-LIC and pTRV2e containing the ccdB gene</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">E. coli DH5&#945;</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td>for the destination constructs generated by LIC cloning</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Fertilizer: Peters Peat Lite Special 15-0-15 Dark Weather Feed</td>
			<td>ICL Specialty Fertilizers, Summerville, SC 29483, USA</td>
			<td style="width:120px;">G99260</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">High fidelity PCR reagents: KAPA HiFi DNA Polymerase with dNTPs</td>
			<td style="width:188px;">Roche Sequencing and Life Science, Kapa Biosystems, 
			Wilmington, MA, USA</td>
			<td>7958960001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isoamyl alcohol</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0944-1L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Koptec Pure Ethanol &#8211; 200 Proof</td>
			<td style="width:188px;">Decon Labs, King of Prussia, PA 19406 , USA</td>
			<td style="width:120px;">V1005M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MES</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">M0606-250G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MgCl2</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">MFCD00149781</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">M-MuLV Reverse Transcriptase</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0253L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;">Nano-drop spectrometer: NanoDrop OneC Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">ND-ONEC-W</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR machine: Bio-Rad MyCycler PCR System</td>
			<td style="width:188px;">Bio-Rad, Hercules, California 94547, USA</td>
			<td style="width:120px;">170-9703</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR machine: Eppendorf Mastercycler pro</td>
			<td style="width:188px;">Eppendorf, Hauppauge, NY 11788, USA</td>
			<td style="width:120px;">950030010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">pH meter</td>
			<td style="width:188px;">Sper Scientific, Scottsdale, AZ 85260, USA</td>
			<td style="width:120px;">Benchtop pH / mV Meter - 860031</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7/8.0.</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0883-400ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phytagel: Gellan Gum</td>
			<td>Alfa Aesar, Tewksbury, MA 01876, USA</td>
			<td style="width:120px;">J63423-A1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVX VIGS vector: PVX-LIC</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td style="width:167px;">Zhao et al., 2016</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Real-time PCR machine: QuantStudio 6 Flex Real-Time PCR System</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">4485697</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Real-time PCR reagent: KAPA SYBR&#174; FAST qPCR Master Mix (2x) Kit</td>
			<td style="width:188px;">Roche Sequencing and Life Science, Kapa Biosystems, 
			Wilmington, MA 01887, USA</td>
			<td style="width:120px;">7959389001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Restriction enzyme: SmaI</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td>R0141S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Reverse transcription reagents: qScript cDNA SuperMix</td>
			<td>Quanta BioSciences, Gaithersburg, MD 20877 , USA</td>
			<td style="width:120px;">95107-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">RNA extraction Kit: E.Z.N.A. Plant RNA Kit</td>
			<td style="width:188px;">Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td>
			<td style="width:120px;">SKU: D3485-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">RNase Inhibitor Murine</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0314L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">RNAzol RT</td>
			<td style="width:188px;">Sigma-Aldrich, St. Louis, MO 63103, USA</td>
			<td style="width:120px;">R4533</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Soil: Metro-Mix 360</td>
			<td>Sun Gro Horticulture, Agawam, MA 01001-2907, USA</td>
			<td style="width:120px;">Metro-Mix 360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">T4 DNA polymerase and buffer</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0203S</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

potato virus x-based microrna silencing (vbms) in potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Figure 2 shows the PVX-STTM165/166 potato plants (Katahdin) with ectopic growth of leaf tissues from the abaxial side of leaf lamina along the veins. More severe phenotypes such as trumpet-shaped leaf formation have also been observed. In contrast, no phenotypic abnormality was observed in the PVX control plants. These results show that the VbMS system was effective in suppressing endogenous miRNA function in tetraploid potato plants and the PVX-VbMS system was a robust genetic tool to deter...

Watch this Scientific Journal Video about Potato Virus X-Based microRNA Silencing VbMS In Potato. at JoVE.com

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

The traditional approach to analyzing the function of microRNAs in plants primarily relies on the generation of stable knockdown plants. This virus-based microRNA silencing system does not require tedious and time-consuming transformation. Virus-based microRNA silencing is a rapid and efficient tool for functional characterization of microRNAs in plants that can be applied to a broad range of diploid and tetraploid potato species.Demonstrating the procedure will be Jinping Zhao, a post-doctoral research associate from my laboratory. To design the target mimic module, insert a mismatch sequence into the reverse complement sequence of the microRNA of interest at the site corresponding to the 10th to 11th nucleotides of the microRNA. To amplify the short tandem target mimic fragment, set up a PCR reaction with each primer of interest, a 48 nucleotide space oligo, 10 times PCR buffer, a dNTP mixture, high fidelity PCR DNA polymerase, and double distilled water to a final volume of 50 microliters.Then amplify the microRNA by standard PCR using the parameters as indicated. To purify the short tandem target mimics, add 2.5 volumes of ethanol and a 1/10th volume of three molar sodium acetate to the PCR product. Mix vigorously before centrifuging the reaction at 14, 000 times G for 10 minutes.After removing the supernatant, rinse the pellet with one milliliter of 70%ethanol and allow the pellet to dry, then dissolve the pellet in 20 microliters of double distilled water. To set up a T4 DNA polymerase reaction, mix the purified short tandem target mimic PCR product with 10 times T4 DNA polymerase buffer, one molar dithiothreitol, 100 millimolar dATP, T4 DNA polymerase, and double distilled water to a final volume of five microliters on ice. Then incubate the mixture for 15 minutes at 37 degrees Celsius, followed by a 20-minute incubation at 75 degrees Celsius to inactivate the T4 DNA polymerase.To prepare the PVX-based VBMS construct, digest five micrograms of PVX ligation independent cloning plasmids with 2.5 microliters of SMA1 in a total volume of 100 microliters. Add an equal volume of phenol to chloroform to isoamyl alcohol, add a 25 to 24 to one ratio to the digested PVX ligation independent cloning products with vigorous mixing and collect the product by centrifugation. Add an equal volume of chloroform to isoamyl alcohol at a 24 to one ratio to the supernatant with vortexing before subjecting the tube contents to a second centrifugation.At the end of the spin, transfer the supernatant to a new centrifuge tube and add 2.5 volumes of ethanol and a 1/10th volume of three molar sodium acetate with vigorous mixing. Centrifuge the tube and rinse the pellet with one milliliter of 70%ethanol and vigorous vortexing. Centrifuge the tube again and remove the supernatant to allow the pellet to air dry.Then dissolve the digested PVX ligation independent cloning plasmid pellet in 100 microliters of double distilled water. To set up the T4 DNA polymerase reaction, mix the digested PVX ligation independent cloning vector DNA with 10 times T4 DNA polymerase buffer, one molar dithiothreitol, 100 millimolar dTTP, and T4 DNA polymerase to a final volume of five microliters on ice. Then incubate the mixture at 37 degrees Celsius for 15 minutes, followed by a 20-minute incubation at 75 degrees Celsius to inactivate the T4 DNA polymerase.To clone the short tandem target mimic sequence into the PVX ligation independent cloning vector, mix five microliters of the T4 DNA polymerase-treated short tandem target mimic PCR product with five microliters of the T4 DNA polymerase-treated PVX ligation independent cloning plasmid and incubate the mixture for five minutes at 70 degrees Celsius. At the end of the incubation, cool the tube contents to 22 degrees Celsius in a PCR machine at a rate of 0.1 degrees Celsius per second, leaving the tube in the thermocycler for 30 minutes once the target temperature is reached. At the end of the incubation, transform five microliters of ligation independent cloning reaction product into E.coli DH5 alpha using standard transformation protocols and grow the transformed bacteria on an LB plate containing 50 micrograms per milliliter kanamycin.After verifying the sequences of the short tandem target mimic fragments by terminator cycle sequencing, isolate the PVX VBMS plasmids from the validated clones and transform them into the appropriate Agrobacterium strains. To inoculate four-week-old in vitro potato plants with Agrobacterium containing the PVX VBMS plasmids, pick positive transformants containing PVX VBMS vectors and inoculate the transformants in 50 milliliters of liquid LB containing 50 micrograms per milliliter of kanamycin and 50 micrograms per milliliter of rifampicin for 16 hours at 28 degrees Celsius and 220 revolutions per minute. During the incubation, streak positive Agrobacterium colonies onto at least two new LB plates containing kanamycin and rifampicin and grow the cultures at 28 degrees Celsius for one day.When the optical density at 600 nanometers reaches 1.0, collect the Agrobacterium liquid culture by centrifugation and resuspend the pellet in a sufficient volume of infiltration buffer to adjust the optical density at 600 nanometers to 1.0. After a six-hour incubation at room temperature, load a one milliliter needleless syringe with the Agrobacterium suspension and use one hand to flip and hold an expanded leaf from one potato plant. Using one finger, support the leaf lamina from the adaxial side at the desired site of infiltration.And holding the syringe vertical to the leaf surface with the other hand, infiltrate 100 to 200 microliters of Agrobacterium culture into the abaxial side of the lamina. Next, use a toothpick to collect colonies from the Agrobacterium culture plates and to scratch the stem surface of the first one or two internodes of the infiltrated potato plants and the epidermis of the stem, then grow the infiltrated plants in the greenhouse for two to four weeks. When phenotypes appear, use scissors to collect samples with phenotypes from the VBMS and control plants.Isolate the total RNA from the collected tissues and use reverse transcriptase PCR to analyze the microRNA and target mRNA expression in each sample. In these images, PVX short tandem target mimic 165/166 potato plants with ectopic growth of leaf tissues from the abaxial side of leaf lamina along the veins can be observed. More severe phenotypes such as trumpet-shaped leaf formation have also been observed.In contrast, no phenotypical abnormalities are observed in PVX control plants, demonstrating that the VBSM system is effective in suppressing endogenous microRNA function in tetraploid potato plants. PVX short tandem target mimic 165/166 potato plants exhibit ectopic leaf to tissue growth from the abaxial side of the leaf lamina along the lamina veins, demonstrating that the PVX VBMS system can be applied to other potato varieties, including major potato cultivars. It is essential to simultaneously infiltrate the leafs and to inoculate the stem of the same plant with the same Agrobacterium culture.Transgenic approaches to stably introducing target mimic molecules into potato plants are recommended when the silencing state of the targeted microRNAs needs to be maintained in the offspring generations.

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3.0K 次观看.  Texas A&M University System. The traditional approach to analyzing the function of microRNAs in plants primarily relies on the generation of stable knockdown plants. This virus-based microRNA silencing system does not require tedious and time-consuming transformation. Virus-based microRNA silencing is a rapid and efficient tool for functional characterization of microRNAs in plants that can be applied to a broad range of diploid and tetraploid potato species.Demonstrating the procedure will be Jinping Zhao, a post...