Development of clover yellow vein virus as an efficient, stable gene‐expression system for legume species

Abstract

SummaryA highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green¯uorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC-Pro on pClYVV to create three recombinant plasmids: pClYVV-GFP, pClYVV-GFP-GS, and pClYVV-GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green¯uorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was con®rmed by RT±PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and¯owered early. As the GS gene, one of the nodulin genes for nitrogen ®xation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.

High expression of foreign proteins from a biosafe viral vector derived from Turnip mosaic virus

“…Potyviral-based vectors expressing full-length individual proteins have been developed previously (Dolja et al, 1992;Guo et al, 1998;Choi et al, 2000;GermanRetana et al, 2000;Masuta et al, 2000;Arazi et al, 2001;Fernández-Fernández et al, 2001;Beauchemin et al, 2005). However, the utility of most constructs designed for the overproduction of proteins of interest could be limited by their genetic instability due to RNA recombination events that rapidly eliminate foreign sequences (Dolja et al, 1993;Guo et al, 1998;Choi et al, 2000;German-Retana et al, 2000).…”

“…On top of the geometrical considerations, other attractive features of potyvirus-derived vectors are the fact that at least some potyviruses are infectious as cloned cDNA, thus facilitating the inoculation process, and viral expressed proteins can reach high percentages of the final protein present in the infected cells. All these traits have prompted several research groups to derive vectors form members of the Potyviruses (Dolja et al, 1992;Guo et al, 1998;Choi et al, 2000;GermanRetana et al, 2000;Masuta et al, 2000;Arazi et al, 2001;Beauchemin et al, 2005).…”

Virus-Induced Gene Silencing of Endogenous Genes and Promotion of Flowering in Soybean by Apple latent spherical virus-Based Vectors

“…In CIYVV-, SMV-, and BPMV-based vectors, rub-inoculation of DNA clones has been successfully conducted in soybean (Masuta et al, 2000;Seo et al, 2009;Zhang et al, 2010). Unfortunately, direct inoculations of an infectious cDNA clones of ALSV by mechanical or particle bombardment were less efficient in soybean plants.…”

Desarrollo De Un Vector Para La Expresión Simultánea De Múltiples Proteínas en Plantas Basado en El Potyvirus Del Grabado Del Tabaco

“…Con dicho vector se ha expresado la proteína estructural VP60 del virus de la enfermedad hemorrágica del conejo (Rabbit hemorrhagic disease vius, RHDV) en plantas de Nicotiana clevelandii, con cuyos extractos se logró obtener respuesta inmune a dicho virus en conejos . Genes reporteros como GFP o GUS se han usado para estudiar el mejor sitio de inserción dentro del genoma viral, medir la eficiencia de expresión y ensayar la posibilidad de insertar y expresar simultáneamente más de un gen. Ejemplos de ello son vectores basados en el virus del mosaico del nabo (Turnip mosaic virus, TuMV) , el TEV , el virus del amarilleo del trébol (Clover yellow vein virus, CYIVV) (Masuta et al, 2000) y el virus A de la patata (Potato virus A, PVA) (Kelloniemi et al, 2008).…”

“…If the heterologous proteins are flanked by the specific processing sites of a viral protease, they are efficiently released from the polyprotein . The use of different insertion sites allows for coexpression of several heterologous proteins from a single vector (Masuta et al, 2000;Kelloniemi et al, 2008). The elongated nature of the virion allows it to accommodate large amounts of extraneous genetic material (Kelloniemi et al, 2008).…”