Abstract: To identify essential host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model plant virus, we screened 800 yeast genes present in the yeast Tet promoters Hughes Collection. In total, we have identified 30 new host genes whose down-regulation either increased or decreased the accumulation of a TBSV replicon RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, protein metabolism/transport, or other cellular processes. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts/functions of p33 and p92(pol) viral replication proteins, (ii) the standard 10 to 20:1 ratio between p33 and p92(pol) in the viral replicase, (iii) the activity of the tombusvirus replicase, and (iv) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous genetic screening of yeast (Panavas et al., Proc. Natl. Acad. Sci. USA 102:7326-7331, 2005) led to the identification of 126 host genes (out of approximately 5,600 genes that represent approximately 95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA.