The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants

Abstract

Four ribozyme and antisense genes targeting citrus exocortis viroid (CEVd) positive-and negative-strand RNA molecules were constructed and used to transform the tomato Lycopersicon lycopersicum cv. UC82B. The tomato is a readily transformable plant and will support replication of CEVd following mechanical inoculation.The ribozyme genes contained three hammerhead catalytic motifs with long hybridizing arms and synthetic RNA transcripts were shown to cleave the target CEVd RNA molecule in vitro. Homozygous transgenic plants were produced from independent transformants expressing either ribozymes or antisense constructs. Inoculation of transgenic seedlings expressing antisense constructs targeting the negative-strand CEVd RNA molecule with CEVd resulted in a moderate reduction in the accumulation of CEVd RNA. In contrast, similarly inoculated transgenic plants expressing constructs targeting the positive-strand CEVd RNA molecule resulted in an increase in the rate of CEVd RNA accumulation. Addition of the ribozyme motifs to the antisense genes did not enhance their efficiency in the suppression of viroid replication and a moderation or elimination of the observed antisense effects was seen in plants expressing the corresponding catalytic RNAencoding genes.

Destabilization of potato spindle tuber viroid by mutations in the left terminal loop.

“…Constitutive expression of antisense RNAs targeting the (k)strand of CEVd can result in a moderate reduction in viroid accumulation in transgenic tomatoes after challenge inoculation (Atkins et al, 1995). Although RNAs containing rather large (i.e.…”

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.…”

Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes

“…Sin embargo, este resultado sólo se observó con las construcciones dirigidas contra las cadenas de polaridad (-), que se acumulan considerablemente menos que las de polaridad positiva in vivo. Además, la incorporación de un dominio catalítico (una ribozima de cabeza de martillo) al RNA antisentido no mejoró el efecto obtenido (Atkins et al, 1995). Por contra, en otros estudios similares con el PSTVd en patata no se observó apenas efecto sobre su infectividad (Matousek et al, 1994), poniendo así de manifiesto la dificultad de aplicar in vivo este tipo de estrategias.…”

“…Debido al éxito limitado de las metodologías convencionales, el interés se ha centrado en aplicar herramientas de biología molecular que previamente han resultado útiles para el control de enfermedades virales. Los primeros estudios con plantas transgénicas expresando RNAs antisentido (algunos de ellos incluyendo una ribozima de cabeza de martillo) no tuvieron el éxito esperado pues la resistencia obtenida fue superada por el viroide poco tiempo después de su inoculación (Matusek et al, 1994;Atkins et al, 1995). Sin embargo, en una estrategia similar basada en la obtención de plantas de patata expresando una ribozima de cabeza de martillo contra los RNAs de polaridad negativa del PSTVd, se observó una notable resistencia en algunas de las líneas transgénicas.…”