Post-transcriptional silencing of pectin methylesterase gene in transgenic tomato fruits results from impaired pre-mRNA processing

Abstract

Transgenic tomato fruit expressing a truncated fruit pectin methylesterase (PME) sense transgene under the control of CaMV 35S promoter show developmentally regulated co-silencing of the endogenous fruit PME and sense transgene. This co-silencing is associated with the onset of fruit PME gene expression. Detectable levels of PME protein and mRNA were not present in ripening transgenic fruits. However, several smaller transgene RNA species hybridizing to the 3' end of the transgene are present in transgenic fruits, indicating degradation of sense transgene transcript. RT-PCR analyses of nuclear RNA from wild-type and transgenic fruits, primed with oligo(dT), show the presence of spliced and unspliced transcripts for the fruit PME and the transgene, indicating a post-transcriptional regulation of gene silencing. However, an accumulation of unspliced fruit PME transcript was obtained in the nuclei of transgenic fruit, using the randomly primed cDNAs in PCR amplification. Taken together, these results suggest that the ectopic expression of the fruit PME sense transgene interferes with processing of the endogenous PME pre-mRNA.