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 method of expressing a polynucleotide of interest in a plant, the method comprising steps of:
(a) introducing into the plant:
(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, wherein the polynucleotide of interest encodes an active RNA species; 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, wherein the polynucleotide of interest encodes an active RNA species; 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;
(b) maintaining the plant under conditions and for a time sufficient to allow the carrier vector to complement the producer vector, so that the producer vector moves systemically in the plant; and
(c) maintaining the plant under conditions and for a time sufficient that the polynucleotide of interest is expressed in at least some plant cells.
2. The method of claim 1 , wherein the first and second plant viruses are the same virus.
3. The method of claim 1 , wherein the first and second plant viruses are different viruses.
4. The method 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.
5. The method of claim 4 , wherein the bromovirus is an alfamovirus, an alfalfa mosaic virus, a cucumber mosaic virus, or an ilarvirus, and the tobamovirus is a tobacco mosaic virus.
6. The method 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.
7. The method 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.
8. The method 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.
9. The method 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.
10. The method of claim 9 , wherein the non-coding portion comprises a 5′ or 3′ untranslated region from a viral RNA.
11. A method of expressing a polynucleotide of interest in a plant, the method comprising steps of:
(a) introducing into the plant:
(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, wherein the polynucleotide of interest encodes an enzyme that synthesizes or modifies a biologically active agent; 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, wherein the polynucleotide of interest encodes an enzyme that synthesizes or modifies a biologically active agent; 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;
(b) maintaining the plant under conditions and for a time sufficient to allow the carrier vector to complement the producer vector, so that the producer vector moves systemically in the plant; and
(c) maintaining the plant under conditions and for a time sufficient that the polynucleotide of interest is expressed in at least some plant cells.
12. The method of claim 11 , wherein the first and second plant viruses are the same virus.
13. The method of claim 11 , wherein the first and second plant viruses are different viruses.
14. The method of claim 11 , wherein each of the first and second plant viruses is independently selected from the group consisting of a bromovirus and a tobamovirus.
15. The method of claim 14 , wherein the bromovirus is an alfamovirus, an alfalfa mosaic virus, a cucumber mosaic virus, or an ilarvirus, and the tobamovirus is a tobacco mosaic virus.
16. The method of claim 11 , 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.
17. The method of claim 11 , 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.
18. The method of claim 11 , 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.
19. The method of claim 11 , wherein the at least one component from a second plant virus comprises a non-coding portion of the genome of the second plant virus.
20. The method of claim 19 , wherein the non-coding portion comprises a 5′ or 3′ untranslated region from a viral RNA.