RNA-Mediated Virus Resistance: Role of Repeated Transgenes and Delineation of Targeted Regions.

Abstract

Resistance to cowpea mosaic virus (CPMV) in transgenic Nicotiana benthamiana plants is RNA mediated. In resistant CPMV movement protein (MP) gene-transformed lines, transgene steady state mRNA levels were low, whereas nuclear transcription rates were high, implying that a post-transcriptional gene-silencing mechanism is at the base of the resistance. The silencing mechanism can also affect potato virus X (PVX) RNAs when they contain CPMV MP gene sequences. In particular, sequences situated in the 3[prime] part of the transcribed region of the MP transgene direct elimination of recombinant PVX genomes. Remarkably, successive portions of this 3[prime] part, which can be as small as 60 nucleotides, all tag PVX genomes for degradation. These observations suggest that the entire 3[prime] part of the MP transgene mRNA is the initial target of the silencing mechanism. The arrangement of transgenes in the plant genome plays an important role in establishing resistance because the frequency of resistant lines increased from 20 to 60% when transformed with a transgene containing a direct repeat of MP sequences rather than a single MP transgene. Interestingly, we detected strong methylation in all of the plants containing directly repeated MP sequences. In sensitive lines, only the promoter region was found to be heavily methylated, whereas in resistant lines, only the transcribed region was strongly methylated.