Yeast strain TKY102 was co-transformed with constructs pESCHIS4-ADH-His33/CUP1-DI-72 and pRS317-Tet-His92 to induce TBSV repRNA replication according to standard Lithium acetate-PEG protocol [74] . The transformed yeast cultures were grown in a Synthetic Complete (SC) media with 2% glucose lacking leucine, histidine, lysine and uracil (SC-ULHK 2 ) by shaking at 29uC overnight. To completely suppress TBSV replication before induction, 1 mg/ml Doxycycline was added to the media to inhibit the expression of p92. The plasmid pool carrying the randomly mutated TEF1 gene was introduced into the yeast cells already transformed with the two virus expression plasmids by in vivo gap repair mechanism via homologous recombination (Fig. S1A) [75] . Briefly, pRS314-pTEF1-TEF1 was digested with enzymes to truncate the TEF1 coding sequence, and then the digested plasmid was recovered. The gapped plasmid (5-10 mg) was transformed together with overlapping PCR (20 mg) products carrying the TEF1 mutations created by random mutagenic PCR (see above). The transformed yeast cells were selected on SC media lacking uracil, tryptophan, leucine, histidine and lysine. The colonies were further streaked onto SC media plate lacking tryptophan, leucine, histidine and lysine (SC-TLHK 2 ) with 0.1% (w/v final) 5-Fluoroorotic Acid (5-FOA) media to select against the URA3-based wild-type TEF1 plasmid (Fig. S1A) . This selection was repeated once and the loss of URA3 plasmid was confirmed by the inability of the yeast strains to grow on uracil-minus media. The yeast cells carrying the randomly mutated TEF1 were grown at 29uC for 24 h in SC-TLHK 2 media with 50 mM CuSO 4 to induce virus replication. Total RNA extraction from yeast cells and Northern blotting and Western blotting were done as previously described [7, 25] .