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.
- 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 with fresh MS/Gamborg’s vitamin media every month.
- Four weeks later, transplant in vitro plants into soil and grow them in a greenhouse under 20–22 °C, 16 h light/8 h dark photoperiod, and light intensity 120 µmol/m2∙s1.
NOTE: The plants with newly developed roots and leaves are suitable for transplanting. Maintain moisture for the freshly transplanted plants for the first 3–4 days.
2. Construct the VbMS vectors.
- Design and clone the short tandem TM molecules (STTM, Figure 1)22,53 for the miRNA of interest.
NOTE: Acquire miRNA sequences based on experimental data or from the miRbase database54,55,56,57,58,59. The miR166 sequence used in this study has been described previously60.
- Design the TM module by inserting a mismatch sequence, normally 5’-CTA-3’, into the reverse complement sequence of miRNA at the site corresponding to the 10th–11th nucleotides of the miRNA.
NOTE: For example, the Stu-miR160 sequence is 5’-UGCCUGGCUCCCUGUAUGCC-3’61, where the 10th–11th nucleotides is in bold. The reverse complement sequence (in deoxynucleotides) is 5’-GGCATACAGGGAGCCAGGCA-3’ and the mismatch bulge insertion site is shown in bold. The TM molecule sequence (deoxynucleotide) should be 5’-GGCATACAGG-CTA-GAGCCAGGCA-3’.
- Design primers for cloning the STTM fragment (Figure 1). Use DNA oligonucleotides with the 48-nt spacer sequence as template for PCR cloning. The forward primer consists of a LIC1 linker (5’-CgACgACAAgACCgT-3’), the forward sequence of the above designed for TM molecule, and the partial 5’ sequence of the 48-nt spacer (5’-GTTGTTGTTGTTATGGT-3’). The reverse primer consists of a LIC2 linker (5’-gAggAgAagAgCCgT-3’), the reverse complement sequence of the TM molecule, and a partial reverse complement to the 3’ sequence of the 48-nt spacer (5’-ATTCTTCTTCTTTAGACCAT-3’).
NOTE: The 48-nt spacer sequence is 5’-GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAAT-3’. For example, for STTM-miR160, the forward primer should be 5’-CgACgACAAgACCgT-GGCATACAGG-CTA-GAGCCAGGCA-GTTGTTGTTGTTATGGT-3’; the reverse primer should be 5’-gAggAgAagAgCCgT-TGCCTGGCTC-TAG-CCTGTATGCC-ATTCTTCTTCTTTAGACCAT-3’ (Figure 1).
- Amplify the STTM fragment in a volume of 50 µL by PCR using the synthesized universal 48-nt spacer as the template and a high fidelity DNA polymerase.
- Set up the PCR reaction by mixing 0.5 µL of each primer (40 µM), 0.5 µL of 48-nt spacer oligo (40 µM), 5 µL of 10x PCR buffer, 1 µL of dNTP mixture (10 mM each), 0.1 µL of high fidelity DNA polymerase (10 U/µL ), and 43 µL of ddH2O to a total volume of 50 µL. Perform standard PCR amplification as follows: 94 °C for 3 min, 32 cycles of 94 °C for 45 s, 60 °C for 45 s and 72 °C for 60 s.
- Purify the STTM fragment by ethanol precipitation. Add 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH = 4.0) to the PCR products. Mix vigorously and centrifuge at 14,000 x g for 10 min. Remove the supernatant and rinse the pellet with 1 mL of 70% ethanol. Dry the pellet and dissolve it in 20 µL of ddH2O.
NOTE: Use DNA electrophoresis to check the amplification of the STTM PCR products.
- Set up the T4 DNA polymerase reaction on ice by mixing 2.5 µL of purified STTM PCR product, 0.5 µL of 10x T4 DNA polymerase buffer, 0.05 µL of 1 M dithiothreitol (DTT), 0.25 µL of 100 mM dATP, 0.1 µL of T4 DNA polymerase (3 U/µL), and 1.6 µL of ddH2O to a total volume of 5 µL. Incubate the mixture at 37 °C for 15 min, and treat the products at 75 °C for 20 min to inactivate the T4 DNA polymerase.
- Design the TM module by inserting a mismatch sequence, normally 5’-CTA-3’, into the reverse complement sequence of miRNA at the site corresponding to the 10th–11th nucleotides of the miRNA.
- Prepare the PVX-based VbMS construct.
- Digest 5 μg of PVX-LIC plasmid38 with 2.5 µL of SmaI (20 U/µL) in a volume of 100 µL.
- Add an equal volume of phenol:chloroform:isopropanol (25:24:1, pH = 6.7/8.0) to the digested PVX-LIC products and mix vigorously. Centrifuge at 14,000 x g for 10 min and transfer the supernatant to a new centrifuge tube. Add an equal volume of chloroform:isopropanol (24:1) and vortex vigorously. Centrifuge at 14,000 x g for 10 min.
NOTE: The PVX-LIC vector harbors the LIC cassette for cloning. The LIC cassette of PVX-LIC vector contains a ccdB gene and a chloramphenicol-resistant gene and needs to be maintained/propagated in the E. coli strain DB3.1 using LB medium containing kanamycin (50 μg/L) and chloramphenicol (15 μg/L).
- Transfer the supernatant to a new centrifuge tube. Add 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH = 4.0) and mix vigorously. Centrifuge at 14,000 x g for 10 min and remove the supernatant.
- Rinse the pellet with 1 mL of 70% ethanol and vortex vigorously. Centrifuge at 14,000 x g for 10 min and remove the supernatant. Dry the pellet and dissolve the digested PVX-LIC plasmid with 100 µL of ddH2O.
- Set up the T4 DNA polymerase reaction on ice by mixing 2.5 µL of digested PVX-LIC vector DNA, 0.5 µL of 10x T4 DNA polymerase buffer, 0.05 µL of 1 M DTT, 0.25 µL of 100 mM dTTP, and 0.1 µL of T4 DNA polymerase (3 U/µL) in a total volume of 5 µL. Incubate the mixture at 37 °C for 15 min and treat the products at 75 °C for 20 min to inactivate the T4 DNA polymerase.
- Clone the STTM sequence into the PVX-LIC vector using a LIC reaction. Mix the T4 DNA polymerase-treated STTM PCR products (5 µL) and the T4 DNA polymerase-treated PVX-LIC plasmids (5 µL). Incubate at 70 °C for 5 min, cool down to 22 °C at a ramp of 0.1 °C/s, and keep at 22 °C for 30 min using a PCR machine.
NOTE: Extend the incubation time to overnight at 4 °C to achieve higher efficiency of LIC cloning.
- Transform 5 µL of the LIC reaction products into the E. coli DH5α and grow on an LB plate containing 50 μg/mL kanamycin62,63. Pick and verify positive colonies by PCR with the cloning primers and universal primer for the PVX-LIC vector followed by sequencing.
- Perform colony PCR with the forward primer for PVX-LIC (5’-GTGTTGGCTTGCAAACTAGAT-3’) in combination with the reverse primer for STTM cloning to identify positive clones. The size of the PCR band should be ~300 bp.
NOTE: Verify the sequences of STTM fragments by terminator cycle sequencing64,65.
- Perform colony PCR with the forward primer for PVX-LIC (5’-GTGTTGGCTTGCAAACTAGAT-3’) in combination with the reverse primer for STTM cloning to identify positive clones. The size of the PCR band should be ~300 bp.
- Isolate the PVX-STTM plasmids from the validated clones and transform them into Agrobacterium strains GV3101, GV2260, or EHA10562,63. Verify Agrobacterium colonies by PCR.
NOTE: Confirm Agrobacterium colonies by PCR using the forward primer for PVX-LIC and the reverse primer for STTM cloning.
3. Perform PVX-based VbMS assay in potato plants.
- Transplant 4-week-old in vitro potato plants into soil. The transplanted plants will be subjected to VbMS assay 3–4 days later.
- Inoculate potato plants with Agrobacterium containing the PVX-STTM plasmids.
NOTE: For the VbMS assay in potato, Agrobacterium-mediated infiltration and toothpick-scratching inoculation are performed simultaneously.
- Pick and inoculate positive transformants containing PVX-STTM vectors into 50 mL of liquid LB containing 50 μg/mL kanamycin and 50 μg/mL rifampicin. Grow in a 28 °C incubator at 220 rpm for 16 h until OD600 = 1.0.
- At the same time, streak the positive Agrobacterium colonies onto at least two new LB plates containing 50 μg/mL kanamycin and 50 μg/mL rifampicin and grow at 28 °C for 1 day. Include the PVX-LIC38,66 vector as a control and PVX-GFP67,68 to monitor virus spread.
- Collect the Agrobacterium liquid culture by centrifugation at 3,400 x g for 10 min at room temperature. Resuspend Agrobacterium cells with an equal volume of infiltration buffer (10 mM MgCl2, 10 mM MES, and 200 µM acetosyringone, pH = 5.6) and adjust to OD600 = 1.0. Incubate at room temperature for 6 h.
- Infiltrate the Agrobacterium culture into the abaxial side of fully expanded leaves with a 1 mL needleless syringe.
- Flip and hold a leaf with one hand, then use one finger to support the leaf lamina from the adaxial side at the infiltration site. Keep the syringe vertical to the leaf surface with the other hand and infiltrate the Agrobacterium culture into the abaxial side of lamina.
- Scrape the Agrobacterium culture from the LB plates and scratch the stem surface of the first one or two internodes of the infiltrated potato plants with a toothpick. Gently scratch the epidermis of the stem. Avoid piercing through the stem, which may cause severe damage to the plants.
- Grow the infiltrated plants at 22 °C with a 16 h light/8 h dark photoperiod and light intensity of 120 μmol/m2∙ s1 in a greenhouse.
NOTE: Phenotypes caused by miRNA silencing usually appear in 2–4 weeks postinoculation (Figure 2, Figure 3). It typically takes 10–20 days for the VbMS phenotype to appear after infiltration. The VbMS phenotype depends on the properties of the specific miRNA and target genes, growth conditions, and potato varieties.
4. Perform expression analysis.
- When phenotypes appear at 2–4 weeks postinoculation, collect tissues such as shoots, leaves, flowers, or roots with phenotypes from the VbMS plants and tissues from the control plants with scissors. Isolate total RNAs from the collected tissues.
- Check the RNA quality by electrophoresis62,63 and quantify the RNA concentration by measuring the OD260 absorbance with a spectrophotometer.
- Use stem-loop real-time reverse transcription PCR (RT-PCR) to analyze the miRNA expression.
- For specific miRNAs, design a stem-loop reverse transcription primer. Stem-loop RT primers contain a universal 5’ backbone and a 3’ 6-nt extension of a specific miRNA. Design a 5’ universal backbone (5’- GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGAC-3’) that forms a stem-loop structure. (The upper case corresponds to an inverse-repeated sequence and the lower case to the loop region).
- Part of the 5’ backbone sequence that forms a loop serves as the reverse primer for subsequent PCR amplification (bold-italic sequence in the backbone sequence). Add a 6-nt extension sequence that is reverse-complementary to the 3’ end of the miRNA of interest to the stem-loop primer to provide specificity for reverse transcription (Supplemental Figure 1A).
NOTE: Design the stem-loop reverse transcription primer as described by Chen et al.69 and Erika Varkonyi-Gasic et al.70,71,72. For example, the stem-loop reverse transcription primer for Stu-miR160 is designed as 5’-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGCATA-3’. The stem-loop reverse transcription primer for the potato miR165/166 family is designed as 5’-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGGG(A/G)A-3’. (The bold uppercase sequences provide the specificity of reverse transcription for specific miRNAs).
- Set up the reverse transcription reaction by mixing 50–200 ng of total RNA, 1 µL of the stem-loop reverse transcription primer (100 μM), 2 µL of 10x buffer, 0.2 µL of RNase inhibitor (40 U/µL), 0.25 µL of dNTPs (10 mM each), and 1 µL of reverse transcriptase (200 U/µL) on ice. Add nuclease-free ddH2O to a total volume of 20 µL.
- Perform reverse transcription using the pulsed reverse transcription procedure. Incubate the reverse transcription reaction mixture at 16 °C for 30 min, perform a temperature cycle of 30 °C for 30 s, 42 °C for 30 s and 50 °C for 1 s, for a total of 60 cycles, and inactivate the reverse transcriptase by incubation at 85 °C for 5 min.
- For real-time PCR analysis of miRNA expression, design the forward primer based on the miRNA sequence but do not include sequences that overlap with the above designed stem-loop reverse transcription primer. Add a 3–7-nt extension to the 5’ of the forward primer to optimize the length, melting temperature, and GC content (Supplemental Figure 1).
NOTE: The universal reverse primer is 5’-GTGCAGGGTCCGAGGT-3’. For example, the stem-loop reverse transcription primer for Stu-miR160 is designed as 5’-CGGCTGCCTGGCTCC-3’. The stem-loop reverse transcription primer for miR165/166 family is 5’-CGGCTCGGACCAGGCTT-3’. The bold sequences serve as 5’ extensions for primer optimization. The stem-loop reverse transcription primers and the real-time PCR primers for miRNA can be designed using the miRNA Primer Design Tool73.
- Synthesize cDNAs of the target mRNAs by standard reverse transcription PCR (RT-PCR). Incubate the reverse transcription reaction mixture at 37 °C for 60 min and inactivate the reverse transcriptase by heating to 85 °C for 5 min.
NOTE: (1) Predict target mRNAs using the psRNATarget program74 if the target mRNAs are not known. (2) The universal reverse transcription primer for mRNA is an anchored primer 5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’.
- Set up the real-time PCR reaction for both miRNA of interest and the target mRNAs. Mix 0.5 µL of template cDNA, 5 µL of 2x real-time PCR buffer with SYBR green, 0.05 µL of each forward and reverse primer (40 µM), and 4.4 µL of ddH2O in a total volume of 5 µL on ice.
- Incubate at 95 °C for 3 min, 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Perform a subsequent melting curve analysis as follows: Incubate at 95 °C for 15 s, cool down to 60 °C at a ramp of 20 °C/s, keep at 60 °C for 60 s, heat to 95 °C at a ramp of 0.2 °C/s, and keep at 95 °C for 15 s. Analyze the Ct-values using the ΔΔCt methods76,77 and plot the means with standard errors.
NOTE: (1) The real-time PCR primers for target mRNAs can be designed as described75. (2) Potato polyubiquitin 10 gene can serve as an internal control for normalization in potato. The forward primer for the potato polyubiquitin 10 gene is 5’-ATGTTGCCTTTCTTATGTGTGGTTG-3’ and the reverse primer 5’-TTATTTATTCACATAAACGACAGTTCAACC-3’. (3) For real-time PCR analysis, contamination and primer-dimer formation may generate false positive results. To monitor nonspecific amplification and increase the liability of real-time PCR analysis, it is recommended to include controls without template and reverse transcriptase for real-time PCR assays.
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.
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
|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
|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|
- Axtell, M. J., Meyers, B. C. Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data. The Plant Cell. 30, (2), 272-284 (2018).
- Chen, X. Small RNAs and Their Roles in Plant Development. Annual Review of Cell and Developmental Biology. 25, (1), 21-44 (2009).
- Rubio-Somoza, I., Weigel, D. MicroRNA networks and developmental plasticity in plants. Trends in Plant Science. 16, (5), 258-264 (2011).
- Zhang, J. -P., et al. MiR408 Regulates Grain Yield and Photosynthesis via a Phytocyanin Protein. Plant Physiology. 175, (3), 1175-1185 (2017).
- Gupta, O. P., Karkute, S. G., Banerjee, S., Meena, N. L., Dahuja, A. Contemporary Understanding of miRNA-Based Regulation of Secondary Metabolites Biosynthesis in Plants. Frontiers in Plant Science. 8, (374), (2017).
- May, P., et al. The effects of carbon dioxide and temperature on microRNA expression in Arabidopsis development. Nature Communications. 4, (1), 2145 (2013).
- Krützfeldt, J., Stoffel, M. MicroRNAs: A new class of regulatory genes affecting metabolism. Cell Metabolism. 4, (1), 9-12 (2006).
- Damodharan, S., Corem, S., Gupta, S. K., Arazi, T. Tuning of SlARF10A dosage by sly-miR160a is critical for auxin-mediated compound leaf and flower development. The Plant Journal. 96, (4), 855-868 (2018).
- Nizampatnam, N. R., Schreier, S. J., Damodaran, S., Adhikari, S., Subramanian, S. microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development. The Plant Journal. 84, (1), 140-153 (2015).
- Chinnusamy, V., Zhu, J., Zhu, J. -K. Cold stress regulation of gene expression in plants. Trends in Plant Science. 12, (10), 444-451 (2007).
- Covarrubias, A. A., Reyes, J. L. Post-transcriptional gene regulation of salinity and drought responses by plant microRNAs. Plant, Cell, Environment. 33, (4), 481-489 (2010).
- Wang, S., et al. Suppression of nbe-miR166h-p5 attenuates leaf yellowing symptoms of potato virus X on Nicotiana benthamiana and reduces virus accumulation. Molecular Plant Pathology. 19, (11), 2384-2396 (2018).
- Canto-Pastor, A., et al. Enhanced resistance to bacterial and oomycete pathogens by short tandem target mimic RNAs in tomato. Proceedings of the National Academy of Sciences. 116, (7), 2755-2760 (2019).
- Chiou, T. -J., Lin, S. -I. Signaling Network in Sensing Phosphate Availability in Plants. Annual Review of Plant Biology. 62, (1), 185-206 (2011).
- Sunkar, R., Chinnusamy, V., Zhu, J., Zhu, J. -K. Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Science. 12, (7), 301-309 (2007).
- Kwenda, S., Birch, P. R. J., Moleleki, L. N. Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection. BMC Genomics. 17, (1), 614 (2016).
- Lakhotia, N., et al. Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing. BMC Plant Biology. 14, (1), 6 (2014).
- Koc, I., Filiz, E., Tombuloglu, H. Assessment of miRNA expression profile and differential expression pattern of target genes in cold-tolerant and cold-sensitive tomato cultivars. Biotechnology, Biotechnological Equipment. 29, (5), 851-860 (2015).
- Zhang, N., et al. Identification of Novel and Conserved MicroRNAs Related to Drought Stress in Potato by Deep Sequencing. PLoS One. 9, (4), 95489 (2014).
- Xie, F., Frazier, T. P., Zhang, B. Identification, characterization and expression analysis of MicroRNAs and their targets in the potato (Solanum tuberosum). Gene. 473, (1), 8-22 (2011).
- Zhang, R., Marshall, D., Bryan, G. J., Hornyik, C. Identification and Characterization of miRNA Transcriptome in Potato by High-Throughput Sequencing. PLoS One. 8, (2), 57233 (2013).
- Yan, J., et al. Effective Small RNA Destruction by the Expression of a Short Tandem Target Mimic in Arabidopsis. The Plant Cell. 24, (2), 415-427 (2012).
- Roodbarkelari, F., Groot, E. P. Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis. New Phytologist. 213, (1), 95-104 (2017).
- Reichel, M., Millar, A. A. Specificity of plant microRNA target MIMICs: Cross-targeting of miR159 and miR319. Journal of Plant Physiology. 180, 45-48 (2015).
- Taylor, R. S., Tarver, J. E., Hiscock, S. J., Donoghue, P. C. J. Evolutionary history of plant microRNAs. Trends in Plant Science. 19, (3), 175-182 (2014).
- Schwab, R., Ossowski, S., Riester, M., Warthmann, N., Weigel, D. Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis. The Plant Cell. 18, (5), 1121-1133 (2006).
- Martin, A., et al. Graft-transmissible induction of potato tuberization by the microRNA miR172. Development. 136, (17), 2873-2881 (2009).
- Yang, L., et al. Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection. Journal of Integrative Plant Biology. 57, (12), 1078-1088 (2015).
- Tang, Y., et al. Virus-based microRNA expression for gene functional analysis in plants. Plant Physiology. 153, (2), 632-641 (2010).
- Voinnet, O. Origin, Biogenesis, and Activity of Plant MicroRNAs. Cell. 136, (4), 669-687 (2009).
- Teotia, S., Tang, G. To Bloom or Not to Bloom: Role of MicroRNAs in Plant Flowering. Molecular Plant. 8, (3), 359-377 (2015).
- Wu, G., Poethig, R. S. Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development. 133, (18), 3539-3547 (2006).
- Zhao, L., Kim, Y., Dinh, T. T., Chen, X. miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems. The Plant Journal. 51, (5), 840-849 (2007).
- Li, J., Millar, A. A. Expression of a microRNA-Resistant Target Transgene Misrepresents the Functional Significance of the Endogenous microRNA: Target Gene Relationship. Molecular Plant. 6, (2), 577-580 (2013).
- Sha, A., et al. Virus-based microRNA silencing in plants. Plant Physiology. 164, (1), 36-47 (2014).
- Zhao, J., Liu, Y. Virus-based MicroRNA Silencing. Bio-protocol. 6, (2), 1714 (2016).
- Yan, F., et al. A virus-based miRNA suppression (VbMS) system for miRNA loss-of-function analysis in plants. Biotechnology Journal. 9, (5), 702-708 (2014).
- Zhao, J., et al. An efficient Potato virus X-based microRNA silencing in Nicotiana benthamiana. Scientific Reports. 6, 20573 (2016).
- Gu, Z., Huang, C., Li, F., Zhou, X. A versatile system for functional analysis of genes and microRNAs in cotton. Plant Biotechnology Journal. 12, (5), 638-649 (2014).
- Du, Z., et al. Using a viral vector to reveal the role of microRNA159 in disease symptom induction by a severe strain of cucumber mosaic virus. Plant Physiology. 164, (3), 1378-1388 (2014).
- Liao, Q., Tu, Y., Carr, J. P., Du, Z. An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs. Scientific Reports. 5, 13178 (2015).
- Liu, X., et al. Analyses of MiRNA Functions in Maize Using a Newly Developed ZMBJ-CMV-2bN81-STTM Vector. Frontiers in Plant Science. 10, 1277 (2019).
- Yang, J., et al. Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature. Frontiers in Plant Science. 9, 1627 (2018).
- Jiao, J., Wang, Y., Selvaraj, J. N., Xing, F., Liu, Y. Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.). PLoS One. 10, (5), 0126621 (2015).
- Jian, C., et al. Virus-Based MicroRNA Silencing and Overexpressing in Common Wheat (Triticum aestivum L.). Frontiers in Plant Science. 8, 500 (2017).
- Barrell, P. J., Meiyalaghan, S., Jacobs, J. M. E., Conner, A. J. Applications of biotechnology and genomics in potato improvement. Plant Biotechnology Journal. 11, (8), 907-920 (2013).
- Peng, T., et al. A Resource for Inactivation of MicroRNAs Using Short Tandem Target Mimic Technology in Model and Crop Plants. Molecular Plant. 11, (11), 1400-1417 (2018).
- Teotia, S., Zhang, D., Tang, G. Functional Genomics: Methods and Protocols. Kaufmann, M., Klinger, C., Savelsbergh, A. Springer. New York. 337-349 (2017).
- Dommes, A. B., Herbert, D. B., Kivivirta, K. I., Gross, T., Becker, A. Virus-induced gene silencing: empowering genetics in non-model organisms. Journal of Experimental Botany. 70, (3), 757-770 (2018).
- Lacomme, C., Chapman, S. Use of Potato Virus X (PVX)-Based Vectors for Gene Expression and Virus-Induced Gene Silencing (VIGS). Current Protocols in Microbiology. 8, (1), 11-16 (2008).
- Lim, H. -S., et al. Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology. 402, (1), 149-163 (2010).
- Gleba, Y., Klimyuk, V., Marillonnet, S. Viral vectors for the expression of proteins in plants. Current Opinion in Biotechnology. 18, (2), 134-141 (2007).
- Tang, G., et al. Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs. Methods. 58, (2), 118-125 (2012).
- Kozomara, A., Griffiths-Jones, S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Research. 39, suppl 1 152-157 (2010).
- Kozomara, A., Griffiths-Jones, S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Research. 42, (1), 68-73 (2013).
- Griffiths-Jones, S. The microRNA Registry. Nucleic Acids Research. 32, suppl 1 109-111 (2004).
- Griffiths-Jones, S., Grocock, R. J., van Dongen, S., Bateman, A., Enright, A. J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research. 34, suppl 1 140-144 (2006).
- Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. miRBase: tools for microRNA genomics. Nucleic Acids Research. 36, suppl 1 154-158 (2007).
- Kozomara, A., Birgaoanu, M., Griffiths-Jones, S. miRBase: from microRNA sequences to function. Nucleic Acids Research. 47, (1), 155-162 (2018).
- Yin, K., Tang, Y., Zhao, J. Genome-wide characterization of miRNAs involved in N Gene-mediated Immunity in response to tobacco mosaic virus in Nicotiana benthamiana. Evolutionary Bioinformatics. Supplementary Files 20744 1-11 (2015).
- Dunker, F., et al. Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence. bioRxiv. 689190 (2019).
- Green, M. R., Sambrook, J. Molecular Cloning. A Laboratory Mannual 4th. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. (2014).
- Sambrook, J., Russell, D. Molecular Cloning: A Laboratory Manual. 3rd Edition. Cold spring Harbor Laboratory Press. Cold Spring Harbor, New York. (2001).
- Anderson, S., et al. Sequence and organization of the human mitochondrial genome. Nature. 290, (5806), 457-465 (1981).
- Sanger, F., Nicklen, S., Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences. 74, (12), 5463-5467 (1977).
- Qian, L., et al. Hsp90 Interacts With Tm-22 and Is Essential for Tm-22-Mediated Resistance to Tobacco mosaic virus. Frontiers in Plant Science. 9, (411), (2018).
- Voinnet, O., Baulcombe, D. C. Systemic signalling in gene silencing. Nature. 389, (6651), 553 (1997).
- Li, C., et al. A cis Element within Flowering Locus T mRNA Determines Its Mobility and Facilitates Trafficking of Heterologous Viral RNA. Journal of Virology. 83, (8), 3540-3548 (2009).
- Chen, C., et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research. 33, (20), 179 (2005).
- Varkonyi-Gasic, E., Hellens, R. P. RNAi and Plant Gene Function Analysis: Methods and Protocols. Kodama, H., Komamine, A. Humana Press. 145-157 (2011).
- Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F., Hellens, R. P. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods. 3, (1), 12 (2007).
- Varkonyi-Gasic, E. Plant Epigenetics: Methods and Protocols. Kovalchuk, I. Springer US. 163-175 (2017).
- Czimmerer, Z., et al. A Versatile Method to Design Stem-Loop Primer-Based Quantitative PCR Assays for Detecting Small Regulatory RNA Molecules. PLoS One. 8, (1), 55168 (2013).
- Dai, X., Zhuang, Z., Zhao, P. X. psRNATarget: a plant small RNA target analysis server (2017 release). Nucleic Acids Research. 46, (1), 49-54 (2018).
- Untergasser, A., et al. Primer3-new capabilities and interfaces. Nucleic Acids Research. 40, (15), 115 (2012).
- Livak, K. J., Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods. 25, (4), 402-408 (2001).
- Schmittgen, T. D., Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nature Protocols. 3, (6), 1101-1108 (2008).
- Todesco, M., Rubio-Somoza, I., Paz-Ares, J., Weigel, D. A Collection of Target Mimics for Comprehensive Analysis of MicroRNA Function in Arabidopsis thaliana. PLoS Genetics. 6, (7), 1001031 (2010).
- Franco-Zorrilla, J. M., et al. Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics. 39, (8), 1033-1037 (2007).
- Jiang, N., et al. Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato-Phytophthora infestans interaction. Horticulture Research. 6, (1), 28 (2019).
- Ivashuta, S., et al. Regulation of gene expression in plants through miRNA inactivation. PLoS One. 6, (6), 21330 (2011).
- Reichel, M., Li, Y., Li, J., Millar, A. A. Inhibiting plant microRNA activity: molecular SPONGEs, target MIMICs and STTMs all display variable efficacies against target microRNAs. Plant Biotechnology Journal. 13, (7), 915-926 (2015).
- Wong, G., Alonso-Peral, M., Li, B., Li, J., Millar, A. A. MicroRNA MIMIC binding sites: Minor flanking nucleotide alterations can strongly impact MIMIC silencing efficacy in Arabidopsis. Plant Direct. 2, (10), 00088 (2018).
- Paschoal, A. R., Lozada-Chávez, I., Domingues, D. S., Stadler, P. F. ceRNAs in plants: computational approaches and associated challenges for target mimic research. Briefings in Bioinformatics. 19, (6), 1273-1289 (2018).
- Faivre-Rampant, O., et al. Potato Virus X-Induced Gene Silencing in Leaves and Tubers of Potato. Plant Physiology. 134, (4), 1308-1316 (2004).
- Zhao, J., et al. Virus-Induced Gene Silencing in Diploid and Tetraploid Potata Species. Methods in Molecular Biology. (2019).
- Leisner, C. P., et al. Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloid-producing tuber-bearing potato species Solanum chacoense, reveals residual heterozygosity. The Plant Journal. 94, (3), 562-570 (2018).
- Aversano, R., et al. The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives. The Plant Cell. 27, (4), 954-968 (2015).
- The Potato Genome Sequencing, C. et al. Genome sequence and analysis of the tuber crop potato. Nature. 475, 189 (2011).
- Navarro, C., et al. Control of flowering and storage organ formation in potato by FLOWERING LOCUS T. Nature. 478, (7367), 119-122 (2011).
- Lehretz, G. G., Sonnewald, S., Hornyik, C., Corral, J. M., Sonnewald, U. Post-transcriptional Regulation of FLOWERING LOCUS T Modulates Heat-Dependent Source-Sink Development in Potato. Current Biology. 29, (10), 1614-1624 (2019).
- Natarajan, B., et al. MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato. Journal of Experimental Botany. 69, (8), 2023-2036 (2018).
- Li, F., et al. MicroRNA regulation of plant innate immune receptors. Proceedings of the National Academy of Sciences. 109, (5), 1790-1795 (2012).
- Weiberg, A., et al. Fungal Small RNAs Suppress Plant Immunity by Hijacking Host RNA Interference Pathways. Science. 342, (6154), 118-123 (2013).
- Huang, C. -Y., Wang, H., Hu, P., Hamby, R., Jin, H. Small RNAs - Big Players in Plant-Microbe Interactions. Cell Host, Microbe. 26, (2), 173-182 (2019).
- Shahid, S., et al. MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs. Nature. 553, (7686), 82-85 (2018).
- Weiberg, A., Jin, H. Small RNAs-the secret agents in the plant-pathogen interactions. Current Opinion in Plant Biology. 26, 87-94 (2015).