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 ' part of the transcribed region of the MP transgene direct elimination of recombinant PVX genomes. Remarkabty, successive portions of this 3' part, which can be as small as 60 nucleotides; all tag PVX genomes for degradation. These observations suggest that the entire 3' 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.

Resistance in plants transformed with the P1 or P3 gene of tobacco vein mottling potyvirus.

“…For the tospovirus tomato spotted wilt virus, only the expression of sequences of the N or NSm genes resulted in resistance (Prins et al, 1997). Evidence exists for a minimal length of the transgene needed (Sijen et al, 1996) but clearly further effort is necessary to identify other features of the RNA sequence that may influence its ability to trigger gene silencing. In the task of designing transgenes for potyvirus resistance, and in view of our results, it seems important to select for regions of near 90 % identity between strains to obtain a wider resistance.…”

“…When a plant is transformed with a viral sequence, PTGS will suppress accumulation of the transgene mRNA and of any viral RNA with sequence similarity with the transgene, leading to protection (English et al, 1996). PTGS probably occurs by cytoplasmic degradation of the targeted RNAs (Lindbo et al, 1993 ;Sijen et al, 1996 ;Tanzer et al, 1997) and a similar phenomenon has been described recently in nontransgenic, virus-infected plants (Ratcliff et al, 1997).…”

Pathogen‐derived resistance targeted against the negative‐strand RNA of tobacco mosaic virus: RNA strand‐specific gene silencing?

“…Virus resistance associated with post-transcriptional gene silencing has also been described in transgenic plants expressing virus-derived sequences from regions other than the replicase open-reading frame. In all of these examples the resistance is dependent upon extreme sequence similarity between the sense RNA products of the transgene and the inoculated virus (de Haan et al, 1992;English et al, 1996;Guo and García, 1977;Lindbo et al, 1993;Mueller et al, 1995;Pang et al, 1996;Prins et al, 1996;Rubino and Russo, 1995;Sijen et al, 1996aSijen et al, , 1996bSmith et al, 1994;Tenllado et al, 1995Tenllado et al, , 1996van der Vlugt et al, 1992). In this paper we use the term 'homology-dependent resistance' when referring to the examples in which the mechanism is related to gene silencing.…”

“…To further elucidate the resistance mechanism in the TMV 54K transgenic plants, we have carried out an analysis, described here, using approaches that have been previously exploited in the characterisation of homologydependent virus resistance and gene silencing (English et al, 1996(English et al, , 1997Hobbs et al, 1993;Sijen et al, 1996b). These analyses show that the target of the resistance mechanism is the negative-strand RNA of the 54K RNA.…”

Induction of RNA-directed DNA methylation and heritable transcriptional gene silencing as a tool to engineer novel traits in plants

“…Posttranscriptional gene silencing (PTGS) was first discovered in transgenic petunia plants whose flower color pattern changed when the gene that encodes the key enzyme for anthocyanin biosynthesis was overexpressed (Napoli et al 1990, van der Krol et al 1990). Similar phenomena have also been reported for other transgenic plants including tomato and tobacco plants transformed with a construct transcribing a gene involved in a metabolic pathway (de Carvalho et al 1992;Goring et al 1991;Smith et al 1990;Vaucheret 1993) or tobacco plants that acquired resistance against viruses as a consequence of transcribing genes or gene segments derived from the viruses Lindbo et al 1993;Mueller et al 1995;Sijen et al 1996;Smith et al 1994). The mechanism of PTGS is also identical to similar gene silencing phenomena in other organisms such as quelling in Neurospora crassa (Cogoni and Macino 1997) and RNA interference in Caenorhabditis elegans (Fire et al 1998).…”