User enzyme mix

PCR primers flanking predicted ORFs were designed for each of 12 selected PacBio contigs. Specific 5′ and 3′ extensions were added to primers for compatibility with the custom USER expression vectors used in this study and described previously (Witek et al., 2016). Candidate genes were PCR‐amplified from R parent genomic DNA in 25 μL PCR reactions (35 cycles with annealing at 62 °C and 7 min extension at 72 °C) using Kappa HiFI HotStart Uracil+ Fidelity Polymerase (Manufacturing, R&D Cape Town, South Africa). Purified PCR product (30 ng) was hybridized with 30 ng pICSLUS0003 vector in the presence of 1 μL USER enzyme mix (New England Biolabs, Inc., Ipswich, MA) as described previously (Witek et al., 2016). All constructs were verified by DNA sequencing. Plasmids containing candidate genes were transformed into Agrobacterium strain GV3101 for transient complementation assays or strain LBA404 for stable potato transformation using the method described previously (Mac et al., 2004). To create a construct containing Ry_sto (c630) under the control of its native regulatory elements, the whole contig (7.5 kB) assembled from PacBio reads was PCR‐amplified and introduced into USER‐vector pICSLUS0001 lacking the 35S promoter and OCS terminator. List of primers used in this study is enclosed as Table S5.

We designed pALSV-RNA2u to be able to carry a gene fragment between the movement protein and the Vp25 capsid protein upon USER cloning (Fig. 1). Target gene fragments must be cloned into pALSV-RNA2u without generating premature stop codons or altering the reading frame of the viral polyprotein. The fragments were amplified from leaf cDNA [10 (link)] with USER-compatible primers (Additional file 1: Table S1). Cloning reactions were composed of 10 µL of purified PCR fragment(s), 1 µL of PacI/Nt.BbvCI-digested pALSV-RNA2u vector (ca. 50 ng), and 1 U of USER enzyme mix (New England Biolabs). The reaction was incubated at 37 °C for 20 min and transformed into E. coli without ligation. Correct insertions were verified by Sanger sequencing and the new constructs were transformed into Agrobacterium strain AGL-1. For the single-gene silencing strategy, individual fragments of approximately 180 bp from LaPDS, LaLDC, and GFP were used. For the PDS co-silencing strategy, primers were designed to amplify and fuse together the same gene fragments used for the single-gene strategy to give a total insert size of approximately 370 bp. The coding sequences of LaPDS, LaLDC, and GFP are shown in Additional file 1: Sequences S1, S2 and S4, respectively, and the VIGS fragments are highlighted therein.

Primers used in this study are listed in Table 2. All fragments were amplified with oligomers having uracil incorporated, using the Phusion U polymerase (Thermo Scientific). The plasmids and promoter library were constructed by the uracil-specific excision reagent (USER) cloning method (Geu-Flores et al. 2007 (link); Nour-Eldin et al. 2006 (link)). In brief, 1 μl of 5× HF buffer (Thermo Scientific) and 1 U of USER™ enzyme mix (New England Biolabs, 1 U/ml) were added to 10 µl of the mixture of purified PCR products, plasmid backbone, or genes.
The reaction mixture was incubated for 25 min at 37 °C, followed by 25 min of incubation at a temperature optimized for annealing of the fragments for 25 min. 8 µl of water was added to the reactions, reaching a final volume of 20 µl. 5 µl diluted USER mixture was used to transform chemically competent E. coli TOP10 cells (Thermo Scientific) (Sambrook and Russell 2001 ).

Mutagenesis was performed as described previously [19 (link)]. Briefly, mutations were introduced into the Gateway entry vector pEntr223-rAbcc6 by uracil-specific excision reagent (USER) cloning with the primers listed in Table 2 using Phusion U PCR master mix (Thermo Scientific, Waltham, MA, USA). PCR fragments were purified using the Nucleospin gel and PCR cleanup kit (Macherey-Nagel, Düren, Germany) and assembled using the USER enzyme mix (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. Resulting circular constructs were verified by Sanger sequencing and transformed into competent E. coli DH5alpha cells. The cDNAs encoding pEnter223-rAbcc6 mutants were subsequently subcloned into a Gateway compatible pQCXIP expression vector using LR Clonase-II (Thermo Scientific, Waltham, MA, USA).

The TRIzol-based technique was used to extract total RNA (Life Technologies, CA, United States). The NanoPhotometer^® spectrophotometer was used to verify RNA purity (OD₂₆₀/OD₂₈₀; OD₂₆₀/OD₂₃₀; IMPLEN, CA, United States). ProtoScript Reverse Transcriptase (New England BioLabs, Ipswich, MA, United States) was used to synthesize primary stranded cDNA using the Purification Kit (Bacteria; Illumina, San Diego, CA, United States). Bioanalyzer 2100 was used to analyze RNA integrity (Agilent, Santa Clara, CA). The Illumina MRZB12424 Ribo-Zero rRNA was used to eradicate rRNA from 1 μl of total RNA. The secondary stranded cDNA was prepared with buffer and a combination of dUTP, dGTP, dATP, and dCTP. The USER enzyme mix was then used to disintegrate the second-strand cDNA (New England BioLabs, Ipwich, MA, United States). Finally, the raw reads were produced and sequenced on the Illumina Novaseq 6000 platform (GENE DENOVO, China; Ju et al., 2019 (link)).