The plasmid p35STunos contains an infectious cDNA clone (GeneBank accession AF530055.2) corresponding to the TuMV isolate YC5 obtained from infected calla lily (Zantedeschia sp.)92 (link). The 11 TuMV cistrons (corresponding to the virus proteins P1, HC-Pro, P3, P3N-PIPO, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP) were amplified by polymerase chain reaction (PCR) from p35STunos with the Phusion High-Fidelity DNA polymerase (Thermo) by using the corresponding pairs of primers, including Gateway adapters, listed in Supplementary Data 5 .
For the Y2H system based on the GAL4 promoter36 (link), the PCR products were cloned by recombination with the In-Fusion enzyme (Clontech) into the yeast bait vector pGBKT7 (Clontech), which was digested with EcoRI and BamHI. This generated a translational fusion of the virus protein (bait protein) with the GAL4 DNA-binding domain. The construction for P3N-PIPO was done in two steps. First, part of the P3 cistron was amplified by PCR and cloned into the plasmid pGBKT7. Second, one adenine was inserted in the putative frameshift site (GGAAAAAA) by site-directed mutagenesis to express the virus protein without the need of frameshifting93 (link).
For the screening based on the sUbq (interactions occurring in the membrane)40 (link), the PCR products were cloned by recombination in vivo to obtain the CubPLV translational fusion (in the case of P3, P3N-PIPO, and 6K2). For that, the bait vector pMetYC-gate was digested with PstI and HindIII and then was co-transformed together with the PCR product into the yeast strain THY.AP4. Transformants were selected on SD/-Leu medium after incubation at 30 °C for 5 days.
For the BiFC constructs, PCR products from all TuMV genes were recombined into the plasmid pDONR207 by using the BP Clonase II Enzyme mix (Invitrogen). For cloning the different A. thaliana Col-0 genes, total RNA was extracted from plant tissues by using the Trizol reagent (Invitrogen) following the manufacturer’s recommendations and further purified by LiCl precipitation. The corresponding cDNAs were synthesized by using the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas) with a polyT+N-primer. Full-length ORFs were amplified by PCR from those cDNAs with the Phusion High-Fidelity DNA polymerase (Thermo) by using suitable primers, including Gateway adapters (Supplementary Data5 ). The constructs were then recombined into the plasmid pDONR207 by using the LR Clonase II Enzyme mix (Invitrogen). All constructed plasmids were amplified in Escherichia coli strain DH5α, purified, and verified by sequencing.
For the Y2H system based on the GAL4 promoter36 (link), the PCR products were cloned by recombination with the In-Fusion enzyme (Clontech) into the yeast bait vector pGBKT7 (Clontech), which was digested with EcoRI and BamHI. This generated a translational fusion of the virus protein (bait protein) with the GAL4 DNA-binding domain. The construction for P3N-PIPO was done in two steps. First, part of the P3 cistron was amplified by PCR and cloned into the plasmid pGBKT7. Second, one adenine was inserted in the putative frameshift site (GGAAAAAA) by site-directed mutagenesis to express the virus protein without the need of frameshifting93 (link).
For the screening based on the sUbq (interactions occurring in the membrane)40 (link), the PCR products were cloned by recombination in vivo to obtain the CubPLV translational fusion (in the case of P3, P3N-PIPO, and 6K2). For that, the bait vector pMetYC-gate was digested with PstI and HindIII and then was co-transformed together with the PCR product into the yeast strain THY.AP4. Transformants were selected on SD/-Leu medium after incubation at 30 °C for 5 days.
For the BiFC constructs, PCR products from all TuMV genes were recombined into the plasmid pDONR207 by using the BP Clonase II Enzyme mix (Invitrogen). For cloning the different A. thaliana Col-0 genes, total RNA was extracted from plant tissues by using the Trizol reagent (Invitrogen) following the manufacturer’s recommendations and further purified by LiCl precipitation. The corresponding cDNAs were synthesized by using the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas) with a polyT+N-primer. Full-length ORFs were amplified by PCR from those cDNAs with the Phusion High-Fidelity DNA polymerase (Thermo) by using suitable primers, including Gateway adapters (Supplementary Data
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