In vivo analysis of plant pre-mRNA splicing using an autonomously replicating vector

Abstract

In this paper, we demonstrate that an autonomously replicating plant expression vector can be used for analysis of pre-mRNA splicing determinants in intact dicot cells. This vector system relies on the Agrobacterium-mediated transfection of leaf discs with the A component of the geminivirus tomato golden mosaic virus (TGMV). Insertion of intron sequences between viral promoter and terminator sequences results in the production of high levels of pre-mRNA transcripts that are effectively and accurately spliced in vivo. Introns from the soybean B-conglycinin gene are spliced at greater than 95% efficiency indicating that the high expression levels of precursor RNA do not exceed the intron splicing capacity of these cells. Introns from the pea and wheat rbcS genes are spliced at 85% and 73% efficiency, respectively, indicating that tobacco leaf disc nuclei are capable of effectively and accurately processing particular dicot and monocot introns. Inclusion of a dicot intron in an engineered construct results in a five-fold enhancement of the level of mRNA stably expressed in dicot nuclei.