Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes

Abstract

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50؇C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

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.…”

Viróides e virusóides: relíquias do mundo de RNA

“…Nas últimas décadas, diversas empresas farmacêuticas têm investido no desenvolvimento visando o controle de vírus humanos como o da (HCV) e o (HIV) (Persidis, 1997). Outras aplicações para as ribozimas em estudos de evolução (Tsang & Joyce, 1996), no controle de viróides via plantas transgênicas (Yang ., 1997) e em estudos de interação de moléculas de RNA com fatores do hospedeiro (Daròs & Flores, 2002) foram recentemente relatadas, e provavelmente continuarão sendo alvo de estudos e discussões sobre o potencial biotecnológico das ribozimas, abrindo um futuro promissor para o uso destas moléculas do passado.…”

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.…”