Ectopic expression of the sgRNA1 promoter. (A) Map of the construct PAVSG1A that contains a duplicated subgenomic promoter region (gray box) inserted in the unique KpnI site of PAV6. The KpnI site was duplicated in the cloning process. The dashed line represents the expected artificial sgRNA1A produced from the duplicated promoter. (B) Northern blot shows viral RNAs from protoplasts (24 hpi) inoculated with PAV6 (lanes 2 and 3) and PAVSG1A which has the 314-nt region expected to contain the sgRNA1 promoter duplicated in the KpnI site (sgRNA1A, lanes 4 and 5). uninf., uninoculated protoplasts (lane 1). RNA degradation products formed a band just below the position of the 18S rRNA (rRNA “shadow”) caused by the very abundant rRNA. The probe is transcript from pSP10, complementary to the 3′-terminal 1.5 kb of the viral gRNA. (C) Northern blot analysis of RNAs from protoplasts (24 hpi) infected with full-length transcripts containing the following portions of the sgRNA1 promoter region duplicated in the KpnI site: 2SL, nt 2595 to 2692; I, nt 2611 to 2692; J, nt 2595 to 2679; K, nt 2611 to 2679.