Cloning Gene Fragments into pALSV-RNA2u Vector

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.