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

Summary

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.

Abstract

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.

Introduction

Plant microRNAs (miRNAs) are characterized as 20–24 nucleotide-long, nuclear-encoded regulatory RNAs¹ and play fundamental roles in almost every aspect of plant biological processes, including growth and development^2,3, photosynthesis and metabolism^4,5,6,7, hormone synthesis and signaling^8,9, biotic and abiotic responses^10,11,<....

Protocol

1. Grow Potato Plants.

1. Propagate in vitro potato plants in culture tubes (25 x 150 mm) with Murashige and Skoog (MS) media plus Gamborg’s vitamin (MS basal salt mixture, Gamborg’s vitamin, 30 g/L sucrose, 3.5 g/L agar, pH = 5.7). Place the tubes in the growth room under 20–22 °C, 16 h light/8 h dark photoperiod, and light intensity 120 µmol/m²∙s¹.
   NOTE: New shoots and roots normally develop in 1–2 weeks from plants. Propagate plants wit.......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
100 µM dATP and 100 µM dTTP	Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA	TQAC136
3 M Sodium acetate, pH 4.0.	Teknova, Hollister, CA 95023, USA	#S0297
Acetosyringone	TCI America, Portland, OR 97203, USA	D2666-25G
Agrobacterium tumefaciens strains: GV3101, GV2260 or EHA105.
Chloroform	VWR Corporate, Radnor, PA 19087-8660, USA	VWRV0757-950ML
Dimethyl sulfoxide, DMSO	TCI America, Portland, OR 97203, USA	D0798-25G
DTT	VWR Corporate, Radnor, PA 19087-8660, USA	VWRV0281-25G
E. coli DB3.1			for maintenance of PVX-LIC and pTRV2e containing the ccdB gene
E. coli DH5α			for the destination constructs generated by LIC cloning
Fertilizer: Peters Peat Lite Special 15-0-15 Dark Weather Feed	ICL Specialty Fertilizers, Summerville, SC 29483, USA	G99260
High fidelity PCR reagents: KAPA HiFi DNA Polymerase with dNTPs	Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA, USA	7958960001
Isoamyl alcohol	VWR Corporate, Radnor, PA 19087-8660, USA	VWRV0944-1L
Koptec Pure Ethanol – 200 Proof	Decon Labs, King of Prussia, PA 19406 , USA	V1005M
MES	TCI America, Portland, OR 97203, USA	M0606-250G
MgCl2	ThermoFisher, Waltham, MA 02451, USA	MFCD00149781
M-MuLV Reverse Transcriptase	New England BioLabs, Ipswich, MA 01938-2723 USA	M0253L
Nano-drop spectrometer: NanoDrop OneC Microvolume UV-Vis Spectrophotometer with Wi-Fi	ThermoFisher, Waltham, MA 02451, USA	ND-ONEC-W
PCR machine: Bio-Rad MyCycler PCR System	Bio-Rad, Hercules, California 94547, USA	170-9703
PCR machine: Eppendorf Mastercycler pro	Eppendorf, Hauppauge, NY 11788, USA	950030010
pH meter	Sper Scientific, Scottsdale, AZ 85260, USA	Benchtop pH / mV Meter - 860031
Phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7/8.0.	VWR Corporate, Radnor, PA 19087-8660, USA	VWRV0883-400ML
Phytagel: Gellan Gum	Alfa Aesar, Tewksbury, MA 01876, USA	J63423-A1
PVX VIGS vector: PVX-LIC			Zhao et al., 2016
Real-time PCR machine: QuantStudio 6 Flex Real-Time PCR System	ThermoFisher, Waltham, MA 02451, USA	4485697
Real-time PCR reagent: KAPA SYBR® FAST qPCR Master Mix (2x) Kit	Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA 01887, USA	7959389001
Restriction enzyme: SmaI	New England BioLabs, Ipswich, MA 01938-2723 USA	R0141S
Reverse transcription reagents: qScript cDNA SuperMix	Quanta BioSciences, Gaithersburg, MD 20877 , USA	95107-100
RNA extraction Kit: E.Z.N.A. Plant RNA Kit	Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA	SKU: D3485-01
RNase Inhibitor Murine	New England BioLabs, Ipswich, MA 01938-2723 USA	M0314L
RNAzol RT	Sigma-Aldrich, St. Louis, MO 63103, USA	R4533
Soil: Metro-Mix 360	Sun Gro Horticulture, Agawam, MA 01001-2907, USA	Metro-Mix 360
T4 DNA polymerase and buffer	New England BioLabs, Ipswich, MA 01938-2723 USA	M0203S