System For Expression Of Genes In Plants From A Virus-Based Expression Vector

Abstract

Modified expression vectors, including Tobacco Mosaic Virus (TMV) expression vectors, methods for modifying such vectors, and uses of the same are disclosed.

What is claimed is: 
     
         1 . A vector comprising pJL36 having the isolated nucleic acid sequence of SEQ ID NO:16. 
     
     
         2 . The vector of  claim 1 , wherein the isolate nucleic acid sequence is operatively linked to a promoter for expression in a plant cell, and includes a 35S driven Tobacco Mosaic Virus TMV expression vector in a binary vector backbone. 
     
     
         3 . The vector of  claim 1 , wherein a replicon generated from the transcription of pJL36 is lacking a coat protein open reading frame (CP orf) and is capable of expression of a desired product in plants. 
     
     
         4 . The vector of  claim 1 , wherein the pJL36 vector, when in the plant, increases expression rate of the desired product in comparison with corresponding wild type plants that do not contain the pJL36 vector. 
     
     
         5 . The vector of  claim 1 , wherein the pJL36 vector does not generate virion particles, yet demonstrates the ability to synchronously inoculate large numbers of cells in a leaf of the plant. 
     
     
         6 . The vector of  claim 1 , further including at least one promoter. 
     
     
         7 . The vector of  claim 6 , wherein the promoter is Cauliflower mosaic virus CaVM35S promoter. 
     
     
         8 . The vector of  claim 1 , wherein the vector includes a second sequence which encodes for a gene that has RNA silencing suppression functions. 
     
     
         9 . The vector of  claim 1 , wherein the vector includes a second sequence which encodes for protein P19 from Tomato bushy stunt virus. 
     
     
         10 . The vector of  claim 1 , wherein vector includes a second sequence which encodes for one or more: P0 protein gene from a polerovirus, HC-Pro protein gene from a potyvirus, CP from turnip crinkle or related viruses, or P21 from beet yellows closterovirus. 
     
     
         11 . An isolated polynucleotide for activating expression specifically in a plant cell, characterized in that the isolated polynucleotide comprises the vector of  claim 1 . 
     
     
         12 . A recombinant pJL36 vector having a plasmid map shown in  FIG. 5A . 
     
     
         13 . The recombinant JL36 vector of  claim 12 , wherein when stably integrated in the genome of a plant cell, has an increased expression rate of at least one product in comparison with corresponding wild type plants that do not contain the pJL36 vector; and, wherein the pJL36 vector does not generate virion particles, yet demonstrates the ability to synchronously inoculate large numbers of cells in a leaf of the plant. 
     
     
         14 . The vector of  claim 12 , having restriction sites: PAcI, AvrII and Nod. 
     
     
         15 . A plant cell transformed with the vector of  claim 1 . 
     
     
         16 . A method of agroinfection comprising infecting at least one plant cell with the vector of  claim 1 . 
     
     
         17 . The method of  claim 16 , wherein the infection occurs without any co-expression of an RNA silencing suppressor. 
     
     
         18 . The method of  claim 16 , comprising infecting the plant cell with pJL36 in  Agrobacterium  cultures diluted 10, 20, 50 or 100 or more fold, from an initial culture OD 600  of 1.0. 
     
     
         19 . The method of  claim 16 , wherein the vector includes a second sequence which encodes for a protein that has RNA silencing suppression functions. 
     
     
         20 . The method of  claim 16 , wherein the vector includes a second sequence which encodes for the protein P19 from Tomato bushy stunt virus. 
     
     
         21 . The method of  claim 16 , wherein the vector includes a second sequence which encodes for one or more of the following genes: P0 protein gene from a polerovirus, HC-Pro protein gene from a potyvirus, CP from turnip crinkle or related viruses, or P21 from beet yellows closterovirus. 
     
     
         22 . The method of  claim 16 , further including introducing a promoter driven version of a first gene of interest into the plant cells by agroinfiltration at the same time, or either before or after, pJL36 vector expressing a second gene of interest is introduced into the plant cells. 
     
     
         23 . The method of  claim 22 , wherein the second gene of interest comprises one or more of antibodies, or antibody fragments, enzymes, hormones, peptides. 
     
     
         24 . The method of  claim 22 , wherein the pJL36 vector enables high-level expression of secreted proteins in plants, or proteins targeted to various subcellular locations. 
     
     
         25 . The method of  claim 22 , wherein the pJL36 vector enables the expression of multiple proteins in the same cell. 
     
     
         26 . A method for producing a transgenic plant cell comprising: transforming the vector of  claim 1  into at least one plant cell to produce a transgenic plant cell or transgenic plant tissue; and, cultivating the transgenic plant cell or transgenic plant tissue to produce the transgenic plant or tissue or cell of the transgenic plant containing the vector of  claim 1 . 
     
     
         27 . The method as recited in  claim 26 , wherein transforming is achieved by an  Agrobacterium tumefaciens -mediating method. 
     
     
         28 . A method for improving agroinfiltration frequency comprising: deleting the CP gene sequence and the viral 3′ non translated sequences encoded in pJL36, and replacing the deleted sequences with TMV U1 nts 6177-6395.