Gene Silencing and Complementation Assays.

Virus-induced gene silencing (VIGS) was performed in N. benthamiana as described (34). The specific DNA fragments with 300 base pairs (bp) from E3 (Niben101Scf00868g03005.1), LHP1-L (Niben101Scf00215g06004.1), GLYK (Niben101Scf18107g00014.1), STPK (Niben101Scf00850g01028.1), CYP (Niben101Scf02763g05012.1), and RLK (Niben101Scf08873g01009.1) predicted by VIGS tool (https://vigs.solgenomics.net/) were cloned into Tobacco Rattle Virus (TRV) RNA2 (pYL279) construct (SI Appendix, Table S1). A. tumefaciens strain GV3101 carry TRV2 and TRV RNA1 (pYL155) constructs were mixed in a 1:1 ratio and infiltrated into 2-wk-old N. benthamiana leaves. Two weeks post infiltration, upper leaves were used for the experiments. For hairpin RNA interference (RNAi), the chalcone synthase (CHSA) intron was amplified from plasmid pFGC5941. The PCR-amplified 3′StGLYK cDNA fragments were overlapped to the 5′ and 3′ end of CHSA intron from two directions. The overlapped fragment was inserted in p1300 vector and transformed into agrobacterium strain GV3101 before potato transformation which was done by Wuhan Double-helix Biology Science and Technology Company. For the complementation assays, the shuffled synonymous synthetic StGLYK sequences were generated by Genscript and cloned into expression constructs pICH86988 with C-terminal GFP tag. Synthetic proteins were coexpressed with AVRvnt1/Rpi-vnt1.1 during HR test assays.