Effects of expression of BMV capsid protein on RNA1 or RNA2 stability. (A) The CP has only a moderate inhibitory effect on BMV RNA3 and RNA4 production. Agrobacterium expressing the replication proteins 1a and 2a were coinfiltrated into N. benthamiana along with culture expressing BMV RNA3. The RNAs were extracted 48 hpi and detected by Northern blots probed to detect the BMV 3′ UTR. The relative amounts of the RNA in this experiment were quantified by Phosphorimage analysis. The results are reproducible in two independent replicates. (B) RT-PCR to detect the level of transcribed RNA from the T-DNA vector in N. benthamiana in the presence of CP expression. Agrobacterium expressing RNA1 or RNA2 was coinfiltrated with different concentrations of culture expressing BMV CP. Total RNA was isolated 24 hpi and treated with RNase-free DNase I. After phenol/chloroform purification and precipitation, equal amounts of RNA were subjected to RT-PCR with appropriate primer sets. 18S ribosomal RNA was amplified from the same samples as a loading control. In the reaction marked “C,” the sample infiltrated with 1.0 OD595 was not subjected to reverse transcription before amplification by PCR. The absence of product in this lane demonstrates that the samples were not contaminated with DNA.
