Fig. 4.

Mechanism of transcriptional RNAi activation involving SAGA. (A) Colony morphology of disruptants of the four genes, ubp8, gcn5, ada2, and lzf1 (Fig. S7 and Table S1). The first three genes are possible key players in SAGA, and the last one encodes an unrelated zinc-finger protein. The four genes were disrupted in DK80 to obtain Δubp8, Δgcn5, Δada2, and Δlzf1 that were infected or uninfected by CHV1-Δp69. CHV1-Δp69 was introduced into these disruptants by horizontal transfer or pretransformation with viral cDNA (Materials and Methods). (B) Northern analysis of the disruptants. Whereas DK80, Δubp8, and Δlzf1 showed up-regulation of dcl2, no such dcl2 induction was observed in the other two disruptants. (C) Requirement of DCL2 and AGL2 for virus-induced RNAi induction. The dcl2 and agl2 disruptants were retransformed with pCPXNeo-Pdcl2D2-egfp (Table S1). These two strains, together with EP155 transformant (Pdcl2D2-egfp), were fused to EP155 infected CHV1-Δp69 or MyRV1 for its horizontal transfer. Resultant fungal strains were examined for egfp induction by Northern analysis (or green fluorescence by microscopy) (Fig. S9). (D) A model for virus-induced up-regulation of dcl2 and agl2 transcription. The model is an updated version of the model previously proposed by Chiba and Suzuki (17). Virus infection is perceived by an unidentified host sensor (step 1, virus recognition), its signal is transduced and transmitted to the nucleus (step 2, SAGA recruitment) to enhance the transcription of a subset of genes including dcl2 and agl2, in which the histone acetyltransferase activities of SAGA are involved (step 3, transcriptional activation). The DUB module appears to be an independent contributor to the high transcriptional activation of only agl2. The dcl2 is induced by MyRV1, but not by CHV1-Δp69 in the absence of AGL2. This virus-specific requirement of AGL2 for the dcl2 up-regulation adds complexity to the model.