Name: potato virus x-based microrna silencing (vbms) in potato.
Uploaded: 2020-05-11T13:30:00
Description: Watch this Scientific Journal Video about Potato Virus X-Based microRNA Silencing VbMS In Potato. at JoVE.com

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.

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		<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 ...

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