title: A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.
abstract: A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits.
authors.affiliation: Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India; Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
authors.forename: A Abdul Kader
authors.initials: AAK
authors.lastname: Jailani
authors.affiliation: Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Vikas
authors.initials: V
authors.lastname: Solanki
authors.affiliation: Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Anirban
authors.initials: A
authors.lastname: Roy
authors.affiliation: Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
authors.forename: T
authors.initials: T
authors.lastname: Sivasudha
authors.affiliation: Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India. Electronic address: leafcurl@rediffmail.com.
authors.forename: Bikash
authors.initials: B
authors.lastname: Mandal
mesh_terms: D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D018553:Cucumis sativus / Q000821:virology; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D016366:Open Reading Frames; D010935:Plant Diseases / Q000821:virology; D012093:Replicon; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D017898:Tobamovirus / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D022462:Vaccines, Edible / Q000096:biosynthesis / Q000235:genetics*; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D022462:Vaccines, Edible; D049452:Green Fluorescent Proteins
keywords: Cucumber green mottle mosaic virus; GFP expression; Nicotiana benthamiana; Plant viral vector; Replicon vector