Processing of complementary sense RNAs ofDigitariastreak virus in its host and in transgenic tobacco

Abstract

We have used a polymerase chain reaction (PCR) procedure to analyse low abundance complementary sense RNAs of Digitaria streak virus (DSV) from infected leaves of Digitaria setigera. This study has confirmed that both spliced and unspliced RNAs are synthesised by the same transcription unit. The position of the intron has been proven from sequencing cDNAs corresponding to the spliced RNA. Although the majority of cDNAs have 3' ends at coordinate 1063, downstream from a consensus polyadenylation sequence, a minor population of RNAs with heterogeneous 3' ends has also been identified. Two major RNA species with alternative splice sites or 3' ends, previously identified by nuclease S1 protection assays, could not be detected, but a cDNA species was observed with an apparent 90bp insertion at the 5' end of the intron. In transgenic tobacco containing integrated dimers of DSV DNA, the major unspliced RNA could readily be detected, but no spliced RNA was present. This may be a reason why DSV DNA did not replicate in tobacco. In addition, neither the minor population of heterogeneous RNAs nor the cDNA species with the insertion could be detected. The failure of the intron to be spliced in tobacco and its low activity in Digitaria is discussed in relation to recent studies on RNA splicing in plants and has led us to the conclusion that the geminivirus introns may be intrinsically inefficient.