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.
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.
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 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 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 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.
1. Grow Potato Plants.
2. Construct the VbMS vectors.
3. Perform PVX-based VbMS assay in potato plants.
4. Perform expression analysis.
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 determine the function of specific miRNAs or miRNA families.
Figure 3 shows the PVX-STTM165/166 potato plants (Russet Burbank) with ectopic leaf tissue growth from the abaxial side of the leaf lamina along the veins. These results show that the PVX-VbMS system could be applied to other potato species, including a major potato cultivar.
Figure 1: Schematic diagram of PVX-based VbMS vectors and the PVX-STTM165/166 structure. LB = T-DNA left border; RB = T-DNA right border; 35S = cauliflower mosaic virus 35S promoter; NOST = nopaline synthase terminator; RdRP = RNA-dependent RNA polymerase; TGB1 = triple gene block protein 1; TGB2 = triple gene block protein 2; TGB3 = triple gene block protein 3; sgP = PVX subgenomic RNA promoter; CP = coat protein; LIC Cassette = ligation-independent cloning cassette; 48 nt = 48 nucleotide imperfect stem-loop linker. STTM165/166 consists of tandem TM sequences of miR165/166 separated by a 48-nt imperfect stem-loop linker sequence. The green arrowhead in the PVX-LIC vector indicates the start site of the PVX subgenomic RNA harboring the STTM sequence. The triple minus hyphens in miRNA sequences indicate the cleavage sites. Please click here to view a larger version of this figure.
Figure 2: VbMS of miR165/166 in the tetraploid potato variety Katahdin. Phenotypes of the potato plants (Katahdin) expressing the PVX vector control or PVX-STTM165/166. Magenta arrows denote ectopically generated leaf structures in the leaf veins. The orange arrowhead denotes trumpet-like leaf structures. Bars = 1 cm. Please click here to view a larger version of this figure.
Figure 3: VbMS of miR165/166 in the tetraploid potato variety Russet Burbank. Phenotypes of the potato plants (Russet Burbank) expressing PVX vector as control or PVX-STTM165/166. Magenta arrows denote ectopically generated leaf structures in the leaf veins. Bars = 1 cm. Please click here to view a larger version of this figure.
Supplemental Figure 1: Schematic diagrams of stem-loop RT-PCR analysis of miRNAs and real-time PCR primer design. (A) Stem-loop RT-PCR analysis of miRNAs. During reverse transcription, the binding of the stem-loop primer to the 3’ miRNA initiated the reverse transcription and cDNA was synthesized. PCR products were amplified with a specific forward primer of the miRNA of interest and the universal reverse primer. (B) Real-time PCR primer design. The forward and reverse primers for Stu-miR160 and Stu-miR165/166 are shown. The forward primer was designed based on the miRNA sequence but did not include sequences that overlapped with the designed stem-loop reverse transcription primer. A 3–7-nt extension was added to the 5’ of the forward primer to adjust the length, melting temperature, and GC content. Please click here to download this file.
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 complementation sequence that results in sequestration of targeted miRNA and arrest of its activity22,35,78,79. The pairing between TM molecules and the target miRNAs blocks the function of the miRNAs by knocking down the levels of a specific miRNA or a miRNA family, which leads to upregulation of the target mRNAs. Several TM technologies have been developed for silencing of miRNAs, including endogenous miRNA target mimicry (eTM)79,80, eTM-based miRNA mimics (MIMs)35,78, short tandem target mimics (STTMs)22,53, a miRNA decoy approach with TMs integrated into the 3' UTR of protein-coding transcripts81, and miRNA SPONGEs containing miRNA binding sites with two central mismatches to target miRNAs (cmSPs)82. STTM consists of two miRNA binding sites with a 3-nt mismatch bulge, linked by a 48-nt spacer that was empirically optimized. STTM triggers efficient inhibition of target miRNAs22,53. The STTM technology has recently been successfully applied to a large-scale functional analysis of miRNAs from the model plant Arabidopsis and major crops such as rice and maize. This led to the discovery of unprecedented roles of several endogenous miRNAs involved in yield and hormone control, which holds great promise in improving crop breeding47. Based on these advantages of STTM design, we chose STTM and integrated it into the PVX vector for functional characterization of miRNAs in potato. It is worth noting that the various designs of TM molecules, such as cmSPs, MIMs, and STTMs, have variable efficacies in blocking the function of different miRNAs82. Therefore, using various TM design strategies may help to achieve more effective miRNA suppression. The length and sequence context of the unmatched bulge as well as the nucleotide alterations adjacent to the miRNA binding sites may also need to be optimized for a specific miRNA silencing outcome22,36,38,78,83. Furthermore, design of TM molecules under guidance of computational predictions together with experimental analysis will probably lead to more reliable inhibition of miRNAs84.
It was shown that the PVX-based VIGS system is effective in triggering RNA silencing in both diploid and cultivated tetraploid Solanum species. The PVX-based systemic silencing is induced and maintained throughout the foliar tissues on in vitro propagated potato plants for several cycles and on in vitro generated microtubers85. We have recently reported that the PVX-based VIGS system can silence genes of interest in several tetraploid potato cultivars, such as Ancilla, Arran Pilot, Marius Bard, and Serrana86. It remains to be determined whether the PVX-based VbMS effect can be transmitted and sustained for several generations through vegetative propagation in potato. Transgenic approaches to introduce TM molecules stably into potato plants are still recommended when the silencing effects of miRNAs of interest need to be maintained in the subsequent generations.
Numerous miRNAs involved in potato growth and development have been identified. RNA-seq, genome sequencing, and bioinformatic prediction have greatly facilitated identification of miRNAs and their targets17,19,20,21. So far, three potato genomes have been sequenced, including a doubled monoploid S. tuberosum Group Phureja clone DM1-3, a wild diploid species S. commersonii, and a diploid inbred clone M6 of S. chacoense87,88,89. Up to date, only a limited number of potato miRNAs have been functionally characterized, in most cases using TM technology. For example, the FLOWERING LOCUS T (FT) homolog SP6A acts as a mobile signal to control tuberization in potato and is targeted by a miRNA, suppressing expression of SP6A (SES), which mediates heat-induced cleavage of the SP6A transcript90,91. STTM-mediated overexpression of SES blocks the activity of SES miRNA and facilitates tuberization even under continuous heat conditions91. Knockdown of miR160, a miRNA involved in immune response, by the eTM approach showed that miR160 is required in both local and systemic acquired resistance against Phytophthora infestans in potato92.
Using a bioinformatic approach, eight unique families of miRNAs that target nucleotide binding site leucine-rich repeat (NLR) immune receptors in potato and tomato were identified93. One of the miRNA families, miR482/2118, targets several NLRs that confer resistance to various pathogens and suppression of the miR482/2118 family miRNAs mediated by transgenic expression of STTM constructs leads to enhanced resistance in tomato against P. infestans and Pseudomonas syringae13. Increasing evidence suggests that small RNAs produced in pathogens and hosts can travel between the two organisms and suppress each other’s gene expression mediated by cross-kingdom RNA interference94,95,96,97. For example, target mimics of an oomycete pathogen-derived sRNAs can scavenge these invading sRNAs and reduce pathogen infection61. It would be interesting to examine whether the present VbMS system can be employed to target pathogen-derived sRNAs to improve resistance in plants.
In summary, virus-based miRNA silencing system is rapid and cost-effective and can be performed in a high-throughput format. The PVX-based VbMS system provides an efficient and robust genetic tool to determine the function of specific miRNAs or miRNA families and the target genes.
The authors have nothing to disclose.
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&M AgriLife Research and the Hatch Project TEX0-1-9675 from USDA National Institute of Food and Agriculture to JS.
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 |