Figure 6. Replication Protein A, a ssDNA-binding protein complex, is essential for transgene-induced silencing.

A) To examine the role of RPA subunits in asexual co-suppression, an RPA70-DAmP allele was constructed to reduce the amount of RPA70 mRNA and the RPA32 gene was deleted in transgenic strain PPW22. PPW22, two independent rpa70-DAmP mutant strains, and two independent rpa32Δ mutants were 10-fold serially diluted in YPD medium, and the cell suspensions were spotted onto YPD medium with or without CsA 100 µg/ml, incubated at 30°C and 37°C for four days, and photographed. B) Expression of CPA1 and CPA2 was examined by northern blot analysis. The abundance of CPA1/2 were determined as described above. C) siRNAs were enriched from individual total RNAs and resolved in a 15% denaturing polyacrylamide TBE-urea gel. A ³²P-labeled in vitro transcribed CPA1 sense transcript was first hydrolyzed and then used to probe antisense CPA1 siRNAs.