To assess the role of essential genes in BMV RNA replication, we co-transformed each of the ,900 strains with BMV expression plasmids pB12VG1 and pB3BG29 (Fig. 1A) . pB12VG1 expresses BMV RNA replication proteins 1a and 2a Pol [9] . pB3BG29 expresses an Rluc reporter-expressing BMV RNA3 cDNA derivative and, as an expression control, the Fluc reporter gene (Fig. 1A) . DNA-dependent transcription produces an initial (+)RNA3 transcript that serves as a template for 1a-and 2a Pol -dependent RNA3 replication and sgRNA4 synthesis via a (2)RNA3 intermediate (Fig. 1A ). Because sgRNA4, which encodes the coat protein, is derived from the (2)RNA3 intermediate, its synthesis depends on, and can serve as a reporter for, BMV RNA replication [20] . Accordingly, in pB3BG29 we replaced the coat protein ORF with the Rluc reporter gene, so that luciferase assays could be used as a rapid measure of RNA4 production and expression (Fig. 1A) . A similar approach was used for primary screening of the yeast non-essential gene deletion library [9] . Of the 892 doxrepressible strains transformed, 151 did not produce colonies after repeated transformation attempts (Table S1), suggesting that the transformation process or expression of the viral constructs significantly impacted the fitness of these strains.