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US 6,632,980 B1Granted
Binary viral expression system in plants
AssigneeE. I. Du Pont de Nemours and Company
InventorsNarendra S. Yadav, S. Carl Falco
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Abstract
The invention relates to two plant transgene expression systems. The first is comprised of two chromosomally-integrated components that are individually heritable. One component is an inactive replicon, which contains cis-acting viral sequences required for replication and is unable to replicate episomally. The other component is a chimeric transactivating gene comprising a regulated promoter operably-linked to the coding region for a protein that can transactivate replicon replication. Regulated expression of the transactivation protein in plant cells containing the inactive replicon triggers release of free replicon from the integrated inactive replicon and allows episomal replication.
Claims25
A binary viral expression system comprising:
- (i) a chromosomally-integrated inactive replicon, derived from viruses selected from the group consisting of geminiviruses and single-stranded RNA viruses, the inactive replicon comprising:
- a) cis-acting viral elements required for viral replication;
- b) a target gene comprising at least one regulatory sequence;
- and
- c) site-specific sequences responsive to a site-specific recombinase;
- and
- (ii) a chromosomally-integrated chimeric transactivating gene comprising a regulated plant promoter operably-linked to a site-specific recombinase coding sequence;
- wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in site-specific recombination which releases the inactive replicon from the chromosome, activates replicon replication, and increases expression of the target gene.
5 more elements
A method of altering the levels of a protein encoded by a target gene in a plant comprising:
- (i) transforming a first plant with a inactive replicon to form a first primary transformant, the inactive replicon derived from viruses selected from the group consisting of geminiviruses and single-stranded RNA viruses, comprising:
- a) cis-acting viral elements required for viral replication;
- b) a target gene comprising at least one regulatory sequence;
- and
- c) site-specific sequences responsive to a site-specific recombinase,
- (ii) transforming a second plant with a chimeric transactivating gene to form a second primary transformant comprising a regulated plant promoter operably-linked to a transactivating site-specific recombinase coding sequence;
- (iii) growing the first and second primary transformants wherein progeny from both seeds are obtained;
- and
- (iv) crossing the progeny of the first and second transformants wherein the target gene is expressed.
6 more elements
A binary transgenic viral expression system comprising:
- (i) a chromosomally-integrated inactive replicon derived from viruses selected from the group consisting of geminiviruses and single-stranded RNA viruses, the inactive replicon comprising:
- a) cis-acting viral elements required for viral replication;
- b) a target gene comprising at least one regulatory sequence;
- and
- c) site-specific sequences responsive to a site-specific recombinase;
- and
- (ii) a transiently-expressed chimeric transactivating gene comprising a plant or viral promoter operably-linked to a site-specific recombinase coding sequence;
- wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in the site-specific recombination, activation of replicon replication, and increased expression of the target gene.
5 more elements
A method of altering the levels of a protein encoded by a target gene in a plant comprising:
- (i) transforming a plant with a inactive replicon derived from viruses selected from the group consisting of geminiviruses and single-stranded RNA viruses, the inactive replicon comprising:
- a) cis-acting viral elements required for viral replication;
- b) a target gene comprising at least one regulatory sequence;
- and
- c) site-specific sequences responsive to a site-specific recombinase;
- (ii) infecting the transformant with a virus containing a chimeric transactivating gene comprising a regulated plant promoter operably-linked to a transactivating site-specific recombinase coding sequence;
- wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in the site-specific recombination, activation of replicon replication, and increased expression of the target gene.
4 more elements
A binary transgenic viral replication system comprising:
- (i) a chromosomally-integrated inactive replicon derived from viruses selected from the group consisting of geminiviruses and single-stranded RNA viruses, the inactive replicon comprising cis-acting viral elements required for viral replication and site-specific sequences responsive to a site-specific recombinase;
- and
- (ii) a chimeric transactivating gene, comprising a regulated plant promoter operably-linked to a site-specific recombinase coding sequence;
- wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in the site-specific recombination and activation of replicon replication.
1 more element
A method of increasing viral resistance in a plant comprising:
- (i) transforming a first plant with an inactive replicon, the inactive replicon derived from viruses selected from the group consisting of geminivirus and single-stranded RNA viruses, to form a first primary transformant, the inactive replicon comprising:
- a) cis-acting viral elements required for viral replication;
- a viral nucleotide sequence encoding a product that confers increased viral resistance, operably linked to a regulatory sequence;
- c) site-specific sequences responsive to a site-specific recombinase;
- (ii) transforming a second plant with a chimeric transactivating gene to form a second primary transformant comprising a regulated plant promoter operably-linked to a transactivating site-specific recombinase coding sequence;
- (iii) growing the first and second primary transformants wherein progeny from both seeds are obtained;
- and
- (iv) crossing the progeny of the first and second transformants wherein the viral nucleotide sequence is expressed, conveying increased viral resistance to the plant.
5 more elements
A binary viral expression system comprising:
- (i) a chromosomally-integrated inactive replicon, derived from viruses selected from the group consisting of geminiviruses and single-stranded RNA viruses, the inactive replicon comprising:
- a) cis-acting viral elements required for viral replication;
- b) a target gene comprising:
- 1) at least one regulatory sequence;
- and
- 2) a coding sequence operably linked to said regulatory sequence;
- and
- c) site-specific sequences responsive to a site-specific recombinase;
- and
- (ii) a chromosomally-integrated chimeric transactivating gene comprising a regulated plant promoter operably-linked to a site-specific recombinase coding sequence;
- wherein expression of the chimeric transactivating gene in cells containing the inactive replicon results in site-specific recombination which releases the inactive replicon from the chromosome, activation of replicon replication, and expression of the coding sequence, which causes post-transcriptional gene silencing of a transgene or endogenous gene.
8 more elements
Dependencies are inferred from the claim text.