Viral amplification of recombinant messenger RNA in transgenic plants

A transgenic plant containing a system comprising:

• a replicon comprising;
• a plus sense, single-stranded RNA plant virus derived replication origin for replicating the replicon,
• a nucleic acid cc non-native to the replicon encoding an RNA or protein product,
• the replicon not encoding for at least a plus sense, single stranded RNA plant virus replicase, and
• a plus sense, single-stranded RNA plant virus derived helper virus encoding;
• at least a plus sense, single stranded RNA plant virus replicase, and
• the helper virus not encoding at least a plus sense, single stranded RNA plant virus movement protein,
• wherein the replicon further encodes at least a plus sense, single stranded RNA plant virus movement protein, and
• wherein a DNA sequence of the replicon is integrated as a transgene into the chromosome of a host cell suitable for replicating the replicon.

A method for expressing a protein in plants comprising:

• (a) integrating a transgene into a chromosome of a plant cell for transcribing the transgene, the transgene encoding for a replicon which comprises;
• a plus sense, single-stranded RNA plant virus derived replication origin for replicating the replicon,
• a nucleic acid sequence non-native to the replicon encoding an RNA or protein product,
• the replicon not encoding for at least a plus sense, single stranded RNA plant virus replicase,
• wherein a DNA sequence of the replicon is integrated as a transgene into the chromosome of a host cell for replicating the replicon, and
• (b) infecting the plant cell with a plus sense, single-stranded RNA plant virus derived helper virus encoding;
• at least a plus sense, single stranded RNA plant virus replicase, and
• the helper virus not encoding at least a plus sense, single stranded RNA plant virus movement protein, wherein the replicon further encodes at least a plus sense, single stranded RNA plant virus movement protein, and
• (c) expressing said nucleic acid sequence.

A method for producing a primary or secondary metabolite accumulated in the tissues of a transfected plant comprising:

• (a) integrating a transgene into a chromosome of a host cell suitable for transcribing the transgene, the transgene encoding for a replicon which comprises:
• a plus sense single-stranded RNA plant virus derived replication origin for replicating the replicon,
• a nucleic acid sequence non-native to the replicon encoding an RNA or protein product,
• the replicon not encoding for at least a plus sense, single stranded RNA plant virus replicase, and
• (b) infecting the plant cell with a plus sense, single-stranded RNA plant virus derived helper virus encoding;
• at least a plus sense, single stranded RNA plant virus replicase, and
• the helper virus not encoding at least a plus sense, single stranded RNA plant virus movement protein wherein the replicon further encodes at least plus sense single stranded RNA plant virus movement protein,
• (c) expressing said nucleic acid sequence, and
• (d) producing a primary or secondary metabolite using the RNA or protein encoded by said nucleic acid sequence.

A system comprising:

• a replicon comprising:
• a TMV-derived replication origin,
• at least one gene non-native to TMV encoding a product non-native to TMV, and
• a gene encoding a TMV-derived viral movement protein,
• wherein said replicon lacks a gene encoding a TMV-derived replicase;
• and
• a TMV helper virus lacking a functional gene encoding a viral movement protein, said TMV helper virus comprising a gene encoding a TMV replicase;
• wherein a DNA sequence of the replicon is integrated as a transgene in the chromosome of a tobacco cell.

A transgenic plant containing a system comprising:

• a replicon comprising:
• a TMV-derived replication origin,
• at least one gene non-native to TMV encoding a product non-native to TMV, and
• a gene encoding a TMV-derived viral movement protein,
• wherein said replicon lacks a gene encoding a TMV-derived replicase;
• and
• a TMV helper virus lacking a functional gene encoding a viral movement protein, said TMV helper virus comprising a gene encoding a TMV replicase, and
• wherein the replicon is integrated as a transgene in the chromosome of a tobacco plant cell.

A method for expressing a protein in plants comprising:

• (a) integrating a transgene into a chromosome of a tobacco cell, the transgene comprising a replicon comprising:
• a TMV-derived replication origin,
• at least one gene non-native to TMV encoding a product non-native to TMV, and
• a gene encoding a TMV-derived viral movement protein,
• wherein said replicon lacks a gene encoding a TMV-derived replicase;
• and
• (b) infecting the tobacco cell with a TMV helper virus, the TMV helper virus comprising:
• a gene encoding a TMV replicase, and lacking a functional gene encoding a viral movement protein.

A system comprising:

• a replicon comprising:
• a tobamovirus derived replication origin,
• a nucleic acid encoding an RNA or protein product that is non-native to the replicon,
• a tobamovirus viral movement protein gene,
• wherein the replicon lacks a tobamovirus viral replicase gene, and a helper virus encoding;
• a tobamovirus viral replicase gene, and
• wherein the helper virus lacks a tobamovirus viral movement protein gene, and
• wherein a DNA sequence of the replicon is integrated as a transgene in the chromosome of a plant cell, suitable as a host for a tobamovirus.

A system comprising:

• a replicon comprising:
• a TMV derived replication origin,
• a nucleic acid encoding an RNA or protein that is non-native to the replicon,
• a TMV viral movement protein gene,
• wherein the replicon lacks a TMV viral replicase gene, and a helper virus encoding;
• a TMV viral replicase gene, and
• wherein the helper virus lacks a TMV viral movement protein gene, and
• wherein a DNA sequence of the replicon is integrated as a transgene in the chromosome of a plant cell, suitable as a host for TMV.