Inhibition of Viroid Infection by Antisense RNA Expression in Transgenic Plants

Abstract

Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.

Experimental Transmission ofPospiviroidPopulations to Weed Species Characteristic of Potato and Hop Fields

“…The lower detection limit of this method is about 0.03 pg per mg of fresh mass (28). For hybridization analysis of PCR products, cDNA fragments were electrophoresed in 2% agarose gels, transblotted to nylon membranes by using alkaline blotting, and hybridized to [␣- 32 P]dCTP-labeled viroid cDNAs as probes.…”

Double-stranded RNA-specific Ribonuclease Confers Tolerance against <i>Chrysanthemum Stunt Viroid</i> and <i>Tomato Spotted Wilt Virus</i> in Transgenic Chrysanthemum Plants

“…Except for the avocado sun blotch viroid group (Bonfiglioli et al 1994), viroids in general (including CSVd and PSTVd) share a similar secondary structure. Because pac1 transgenic potato and chrysanthemum exhibited tolerance to PSTVd and CSVd infection, respectively, pac1-mediated tolerance must have a potentially greater applicability to most of the viroids based on the primary structure of the genome (Matousek et al 1994, Atkins et al 1995, Yang et al 1997.…”

“…It has been difficult to control viroid disease, because factors essential for viroid survival are indispensable to the host itself. Transgenic plants expressing antisense RNA or ribozyme against viroid genome were shown to provide potential strategies for producing viroid-tolerant plants (Matousek et al 1994, Atkins et al 1995, Yang et al 1997. Because viroids-consisting of circular singlestranded RNAs-form a base-paired dsRNA-like structure in the native state as well as a dsRNA structure during rolling circle replication (Branch andDickson 1984, Ishikawa et al 1984), they might be good targets for digestion by dsRNA-specific ribonucleases.…”

Resistance to Viruses of Potato and their Vectors

“…However, there are fewer examples where such sequences of potato virus genomes have been incorporated into the potato genome and have been shown to confer resistance to those viruses. Nevertheless, this has been accomplished with sequences derived from PLRV (Barker et al, 1992;Brown et al, 1995;Ehrenfeld et al, 2004;Graham et al, 1997;Kawchuk et al, 1997;Kawchuk et al, 1990;Murray et al, 2002;Palucha et al, 1998;Rovere et al, 2001;Thomas et al, 2000), PVY (Chi et al, 2005;Hefferon et al, 1997;MakiValkama et al, 2001;Malnøe et al, 1994;Missiou et al, 2004;Okamoto et al, 1996;Racman et al, 2001;Schubert et al, 2004;Smith et al, 1995;Sokolova et al, 1994;Wefels et al, 1993), PVA (Nie et al, 2008), both PLRV and PVY (Arif et al, 2009), PVX (Doreste et al, 2002Hoekema et al, 1989;Spillane et al, 1998;Xu et al, 1995), both PVX and PVY (Lawson et al, 1990), PMTV (Germundson et al, 2002;Melander et al, 2001), TRV (Melander, 2006) and PSTVd (Matousek et al, 1994;Yang et al, 1997). Solomon-Blackburn and Barker (2001a) have suggested that transgenic resistance derived by stacking sequences from several viruses may offer to best means of obtaining multiple virus resistance, given that the sources of host-derived resistance to the various potato viruses involve different breeding lines and even different Solanum species.…”