System for expression of genes in plants
The present invention provides trans-complementation systems for expressing gene products in plants. In general, the invention provides systems including a carrier vector and a producer vector, both based on plant viruses. The producer vector is defective for at least one function needed for successful systemic infection of a plant, e.g., replication, cell-to-cell movement, or long distance movement. The carrier vector supplies the missing function in trans. Certain producer vectors lack a functional coat protein coding sequence, in which case the corresponding producer vector supplies coat protein in trans. The invention also provides novel plant viral vectors and methods of use, e.g., to produce polypeptides or active RNAs in plants.
1. A producer vector comprising:
(a) a polynucleotide of interest, wherein the polynucleotide encodes an active RNA species or encodes an enzyme that synthesizes or modifies a biologically active agent; and
(b) one or more components of a first plant RNA virus,
wherein the vector is defective for systemic movement; and
wherein the vector encodes a replicase protein of a plant RNA virus and comprises sufficient non-coding portions to allow self-replication.
2. The producer vector of claim 1 , wherein the polynucleotide encodes an active RNA species.
3. The producer vector of claim 2 , wherein the active RNA species is a ribozyme.
4. The producer vector of claim 2 , wherein the active RNA species is an interfering RNA.
5. The producer vector of claim 4 , wherein the interfering RNA is a long double-stranded RNA, a short interfering RNA (siRNA), or a short hairpin RNA (shRNA).
6. The producer vector of claim 2 , wherein the active RNA species is targeted to a gene of the plant.
7. The producer vector of claim 1 , wherein the polynucleotide encodes an enzyme that synthesizes or modifies a biologically active agent.
8. The producer vector of claim 1 , wherein the plant RNA virus is a tobamovirus.
9. The producer vector of claim 8 , wherein the plant RNA virus is tobacco mosaic virus.
10. The producer vector of claim 1 , wherein the plant RNA virus is a bromovirus.
11. The producer vector of claim 10 , wherein the plant RNA virus is alfalfa mosaic virus.
12. The producer vector of claim 1 , wherein the enzyme that synthesizes or modifies a biologically active agent is an enzyme that modifies a protein.
13. The producer vector of claim 1 , wherein the enzyme that synthesizes or modifies a biologically active agent is an enzyme that modifies small molecule substrates.
14. The producer vector of claim 1 , wherein the enzyme that synthesizes or modifies a biologically active agent is targeted to a gene of the plant that encodes an enzyme that synthesizes a small molecule.
15. A method of expressing a polynucleotide of interest comprising:
(a) introducing the producer vector of claim 1 into a plant or plant cell; and
(b) maintaining the plant cell or plant cell under conditions and for a time sufficient that the polynucleotide is expressed.
16. A system for expressing polynucleotides of interest in a plant cell or whole plant, comprising:
the producer vector of claim 1 , and
a carrier vector comprising a second polynucleotide of interest and one or more components of a second plant RNA virus.
17. The system of claim 16 , wherein the first and second plant RNA viruses are the same plant RNA virus.
18. The system of claim 16 , wherein the first and second plant RNA viruses are different plant RNA viruses.