E coli dh5α competent cells

Viral RNAs were extracted using a commercial kit (QIAgen, Hilden, Germany). The cDNA was obtained by reverse transcription of three virus strains using the primer Tx30 (5′- GACTAGTTCTAGATCGCGAGCGGCC-GCCC(T)₃₀-3′) and SuperScriptTM III reverse transcriptase (Life Technologies, Carlsbad, CA, USA). Then, we amplified the entire ORF2 and ORF3 by PCR, using primers ORF2-1F (sense: 5′-CTATAAATAGATCTTGGTACCATGAAGATGGCGTCCAATGACCG-3′) and Tx30 (antisense). The 2484-bp PCR products were treated with Bgl II (Merck KGaA, Darmstadt, Germany) and NotI (New England Biolabs, MA, USA) restriction enzymes, and ligated into the pVL1392 vector (Invitrogen Life Technologies, Waltham, MA, USA). The plasmid was amplified by E. coli DH5α competent cells (Thermo Fisher Scientific, Waltham, MA, USA), then transfected into Sf9 insect cells (ATCC biotechnology, Manassas, VA, USA). The supernatant (50.0 mL) from infected cells was purified. The VLPs were purified by ultracentrifugation through a 30% (wt/vol) sucrose cushion, followed by cesium chloride (CsCl; Merck KGaA, Darmstadt, Germany) density gradient. The VLPs were then banded by CsCl gradient ultra-centrifugation at 36,000 rpm for 20 h. A total of 330 μL supernatant was fractioned for 10 tubes from the top (lowest density) to the bottom (highest density).

Aptamers obtained from the last round of SELEX were amplified by PCR and were cloned into a pTG19-T vector (Vivantis, Selangor, Malaysia). The ligation reaction was transformed into E. coli DH5α competent cells (Thermo Fisher Scientific, Waltham, MA USA) and plated on LB agar containing 50 μg.ml⁻¹ ampicillin. Colonies were analyzed with colony PCR. Each positive clone was amplified with FITC-labeled primers and the fluorescence intensity was estimated by flow cytometer. Then aptamer candidates with the highest binding affinities were selected for further analysis.

To generate the CRISPR/Cas9 construct targeting the FT2 gene, we followed the procedures described by Jacobs et al. (2015) (link). The sgRNA within the first exon of the hybrid poplar FT2 gene was obtained from a predesigned SNP-free gRNA dataset.¹ The DNA fragment of the U6 promoter and scaffold were amplified using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, MA, United States) and purified from the gel using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, PA, United States). The oligonucleotides used to generate the construct are listed in Supplementary Table 1. Briefly, SwaI- and SpeI-digested p201N Cas9 plasmid (Jacobs et al., 2015 (link)) was mixed with the U6 single-strand oligonucleotide (containing the sgRNA sequence) and scaffold DNA fragments and ligated using NEBuilder^® HiFi DNA Assembly Master Mix (New England Biolabs, MA, United States). The reaction product, shown in Supplementary Figure 1A, was transferred to E. coli DH5α competent cells (Thermo Fisher Scientific, MA, United States) following procedures reported by Inoue et al. (1990) (link). Positive bacterial clones containing the CRISPR/Cas9-FT2 construct were identified by colony PCR using the primers Ubi3p218R and ISCeIR (Supplementary Table 1). The correct assembly and sequence of the CRISPR/Cas9-FT2 construct was confirmed by DNA sequencing using Ubi3p218R primer.