System for expression of genes in plants
View Patent ↗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 system for expressing a polynucleotide of interest in a plant cell or whole plant, comprising:
(i) a carrier vector that includes a functional coat protein encoding component from a first plant virus; and a producer vector that includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional coat protein encoding component; or
(ii) a carrier vector that includes a functional replicase encoding component from a first plant virus; and a producer vector that includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional replicase encoding component; or
(iii) a carrier vector that includes a functional movement protein encoding component from a first plant virus; and a producer vector that includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional movement protein encoding component;
wherein the polynucleotide of interest encodes an active RNA species.
2. The system of claim 1 , wherein the carrier vector includes a functional coat protein encoding component from a first plant virus, and the producer vector includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional coat protein encoding component.
3. The system of claim 1 , wherein the carrier vector includes a functional replicase encoding component from a first plant virus, and the producer vector includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional replicase encoding component.
4. The system of claim 1 , wherein the carrier vector includes a functional movement protein encoding component from a first plant virus, and the producer vector includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional movement protein encoding component.
5. The system of claim 1 , wherein the first and second plant viruses are the same virus.
6. The system of claim 1 , wherein the first and second plant viruses are different viruses.
7. The system of claim 1 , wherein the carrier vector comprises a wild type viral genome.
8. The system of claim 1 , wherein the carrier vector comprises at least one polynucleotide encoding a replication protein and comprises sufficient non-coding portions to allow self-replication.
9. The system of claim 1 , wherein the carrier vector lacks one or more components required for self-replication.
10. The system of claim 9 , wherein the one or more components required for self-replication is a functional replication protein coding portion found in the first plant virus.
11. The system of claim 9 , wherein the one or more components required for self-replication of the carrier vector is supplied by the producer vector.
12. The system of claim 11 , wherein the carrier vector comprises a genomic promoter found in the second plant virus.
13. The system of claim 9 , wherein the one or more components required for self-replication is a noncoding portion found in the first plant virus.
14. The system of claim 1 , wherein the producer vector comprises at least one polynucleotide encoding a replication protein and comprises sufficient non-coding portions to allow self-replication.
15. The system of claim 1 , wherein the producer vector lacks one or more components required for self-replication.
16. The system of claim 15 , wherein the one or more components required for self-replication is a functional replication protein coding portion found in the second plant virus.
17. The system of claim 15 , wherein the one or more components required for self-replication of the producer vector is supplied by the carrier vector.
18. The system of claim 17 , wherein the producer vector comprises a genomic promoter from the first plant virus.
19. The system of claim 15 , wherein the one or more components required for self-replication is a noncoding portion found in the second plant virus.
20. The system of claim 1 , wherein the at least one component from a second plant virus comprises a polynucleotide that encodes a polypeptide selected from the group consisting of: a replication protein, a movement protein, and a coat protein.
21. The system of claim 1 , wherein the at least one component from a second plant virus comprises a non-coding portion of the genome of the second plant virus.
22. The system of claim 1 , wherein the at least one component from a second plant virus comprises a 5′ or 3′ untranslated region from a viral RNA.
23. The system of claim 1 , wherein the carrier vector lacks a functional movement protein encoding component and the producer vector comprises a functional movement protein encoding component.
24. The system of claim 1 , wherein the producer vector comprises a component from the first plant virus.
25. The system of claim 24 , wherein the component is an untranslated region from a viral RNA.
26. The system of claim 1 , wherein the carrier vector, the producer vector, or both comprise a portion of the first virus other than a coat protein coding portion, which portion facilitates systemic spread.
27. The system of claim 26 , wherein the portion that facilitates systemic spread comprises a recognition site for complex formation with coat protein.
28. The system of claim 1 , wherein each of the first and second plant viruses is independently selected from the group consisting of: a bromovirus and a tobamovirus.
29. The system of claim 28 , wherein the bromovirus is an alfamovirus, alfalfa mosaic virus, or an ilavirus, and wherein the tobamovirus is a tobacco mosaic virus.
30. The system of claim 1 , wherein the active RNA species is a ribozyme.
31. The system of claim 1 , wherein the active RNA species is an interfering RNA.
32. The system of claim 31 , wherein the interfering RNA is a long double-stranded RNA, a short interfering RNA (siRNA), or a short hairpin RNA (shRNA).
33. The system of claim 1 , wherein the active RNA species is targeted to a gene of the plant.
34. A system for expressing a polynucleotide of interest in a plant cell or whole plant, comprising:
(i) a carrier vector that includes a functional coat protein encoding component from a first plant virus; and a producer vector that includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional coat protein encoding component; or
(ii) a carrier vector that includes a functional replicase encoding component from a first plant virus; and a producer vector that includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional replicase encoding component; or
(iii) a carrier vector that includes a functional movement protein encoding component from a first plant virus; and a producer vector that includes the polynucleotide of interest, and further includes at least one component from a second plant virus, but lacks a functional movement protein encoding component;
wherein the polynucleotide of interest encodes an enzyme that synthesizes or modifies a biologically active agent.
35. The system of claim 34 , wherein the enzyme that synthesizes or modifies a biologically active agent is an enzyme that modifies a protein.
36. The system of claim 34 , wherein the enzyme that synthesizes or modifies a biologically active agent is an enzyme that modifies small molecule substrates.
37. The system of claim 34 , wherein the enzyme that synthesizes or modifies a biologically active agent is targeted to a gene of the plant an enzyme that synthesizes a small molecule.
38. The system of claim 34 , wherein the enzyme that synthesizes or modifies a biologically active agent is targeted to the product of a second polynucleotide of interest that is encoded by the carrier vector.