Saccharomyces cerevisiae strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0) was obtained from Open Biosystems (Huntsville, AL, USA). Plasmid-borne TEF1/2 TKY strains (MATa ura3-52 leu2-3, 112 trp1-D1 lys2-20 met2-1 his4-713 tef1::LEU2 tef2D pTEF2 URA3) were published before [69, 70, 71, 72] . The plasmid pESCHIS4-ADH-His33/CUP1-DI-72 expressing Cucumber necrosis virus (CNV) p33 and the TBSV replicon RNA, called DI-72, was described earlier [25] . The LYS2-based plasmid pRS317-Tet-His92, expressing CNV p92 under the control of Tetracycline-regulatable (Tet) promoter was constructed as follows: the Tet promoter sequence was obtained from pCM189-His92/Tet [73] by digestion with EcoRI and BamHI, and CNV p92 coding sequences from pGAD-His92 [7] digested with BamHI and PstI, followed by ligation into pRS317 vector treated with EcoRI and PstI. To generate mutations within TEF1 coding sequence by random mutagenesis, we constructed the TRP1-based plasmid pRS314-pTEF1-TEF1, which expressed TEF1 under the control of its native promoter. The TEF1 promoter sequence, the TEF1 coding region and the Cyc1 terminator sequences were amplified by PCR with the following primer pairs, #2764 (CCGCGAGCTCATAGCTTCAAAAT-GTTTCTAC)/#2765 (CCGCGGATCCGTAATTAAAACT-TAGATTAGATTGC), #2768 (CCGCGGATCCAAAATGGG-TAAAGAGAAGTCTC)/#1877 (CCGCCTCGAGTTATTTC-TTAGCAGCCTTTTGAGCAGC), and #2769 CCGCCTCG-AGGAGGGCCGCATCATGTAA/#2770 (CCGCGGTACCA-GCTTGCAAATTAAAGCCTTC), respectively. This was followed by cloning the PCR products into pRS314 digested with SacI and KpnI.