Lr gateway cloning reaction

For construction of transient silencing vector of Pha13, the oligonucleotide pair Pha13-hpRNA-F/Pha13-hpRNA-R (S3 Table) was used to generate the hairpin dsDNA fragments. The hairpin dsDNA fragments were cloned into the Gateway entry vector pENTR/D-TOPO (Thermo Fisher-Scientific) following the manufacturer’s instructions to generate pENTR-Pha13-hpRNA. Then, LR Gateway cloning reaction (Thermo Fisher-Scientific) was conducted to transfer the hairpin RNA fragments from pENTR-Pha13-hpRNA into 35S promoter driven pB7GWIWG2(I) [48 (link)] to obtain phpPha13. The method for construction of transient silencing vector of PhaNPR1, PhaRdR1, and PhaGRX was similar to that described above, except the oligonucleotide pairs PhaNPR1-hpRNA-F/PhaNPR1-hpRNA-R, PhaRdR1 -hpRNA-F/PhaRdR1-hpRNA-R, and PhaGRX-hpRNA-F/ PhaGRX-hpRNA-R (S3 Table) were used to generate the hairpin dsDNA fragments.
For construction of virus-induced gene-silencing vector, plant RNA was used as a template to amplify the fragments of Pha13 by RT-PCR with the primer pair attB1-Pha13-F/attB2-Pha13-R (S3 Table). The amplified fragments were cloned into the pCambia-CymMV-Gateway vector [27 (link)] by the BP Clonase II enzyme mix (Thermo Fisher-Scientific) to generate pCambia-CymMV-Pha13.

For construction of Pha13 transient overexpression vector, plant total RNA was used as a template to amplify the N terminal FLAG tagged of Pha13 by RT-PCR with the primer pairs FLAG-Pha13ORF-F/Pha13ORF-R (S3 Table). The FLAG-Pha13 fragments were cloned into the Gateway entry vector pENTR/D-TOPO (Thermo Fisher-Scientific) following the manufacturer’s recommendations to generate pENTR-FLAG-Pha13. Then, LR Gateway cloning reaction (Thermo Fisher-Scientific) was used to transfer the FLAG-Pha13 fragments from pENTR-FLAG-Pha13 into the 35S promoter driven overexpression vector, pK2GW7 [48 (link)], to obtain pPha13-oe. For generation of A20 and/or AN1 mutant on pPha13-oe (Fig 1C and 1D), site-directed mutagenesis was conducted by QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). For A20 mutant, we substituted the conserved 3^rd and 4^th cysteine to glycine at A20 (C29G and C32G). For AN1 mutant, we substituted the conserved 3^rd cysteine and 1^st histidine to glycine at AN1 (C111G and H121G). The A20 mutated clone and AN1 mutated clone was designated pPha13A20m and pPha13AN1m, respectively. The A20 and AN1 double mutated clones was designated pPha13A20mAN1m. Primer pairs used for site directed mutagenesis are listed in S3 Table.

For construction of vectors used for subcellular localization analysis, the primer pairs, Pha13-ORF-F/Pha13-ORF-R and Pha13-ORF-F/Pha13-ORF-NONSTOP-R (S3 Table) were used to amplify 2 sets of Pha13 ORF (with or without stop codon). All the amplified ORF fragments of Pha13 were cloned into the Gateway entry vector pCR 8/GW/TOPO Gateway (Thermo Fisher-Scientific) following the manufacturer’s recommendations to generate pCR8-Pha13 and pCR8-Pha13-NONSTOP. Then, LR Gateway cloning reaction (Thermo Fisher-Scientific) was used to transfer the ORF fragment of Pha13 from pCR8-Pha13 into p2FGW7 driven by 35S promoter [48 (link)] to obtain N-terminal GFP fused clones (pG-Pha13). To obtain C-terminal GFP-fused clones (pPha13-G), we transferred pCR8-Pha13-NONSTOP into p2GWF7. Protoplast isolation and transfection were as described [27 (link)]. Transformed protoplasts were detected for florescence signals by confocal microscopy (Zeiss LSM 780, plus ELYRA S.1) with excitation at 488 nm and emission at 500 to 587 nm for GFP, and excitation at 543 nm and emission at 600 to 630 nm for mCherry.