Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to Tomato Spotted Wilt Virus in transgenic tobacco

Abstract

Matrix attachment regions (MARs) are DNA elements that can increase and stabilize transgene expression. We investigated the effect of the RB7 MAR on transgenic virus resistance. Constructs for resistance to tomato spotted wilt virus (TSWV) with and without flanking RB7 MARs were used to transform tobacco and produce homozygous lines. The population with the MAR construct had a significantly higher percentage of TSWV resistant plants in the R1 generation than the nonMAR population. Each resistant line was advanced to the R4 generation, and significantly fewer MAR lines lost resistance over generations compared to the nonMAR population. Lines with TSWV resistance in growth chamber tests were also resistant in field trials. Two lines that were resistant in the R1 generation and susceptible in the R4 were examined in more detail in order to determine if transcriptional silencing of the transgene was occurring in the later generation. Short interfering 21-25 nt RNAs from the transgene that are characteristic of post-transcriptional gene silencing (PTGS) were present in the resistant R1 plants, but not the susceptible R4 plants, indicating that virus resistance was associated with PTGS of the transgene. Loss of resistance was accompanied by an increase in promoter methylation in both lines. In line M41, the transgene was fully silenced at the transcriptional level in the R4 as shown by nuclear run-on assays. In line NM13, transgene transcription and RNA accumulation was still present in the R4 generation, but the level of transcription was not sufficient to trigger PTGS, suggesting that this line may have partial transcriptional silencing. These results are consistent with the concept that MARs may prevent transcriptional silencing.