Binary viral expression system in plants

Details Binary viral expression system in plants Binary viral expression system in plants

1999-11-17

2003-10-14

Mehta, Ashwin (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S320100, C435S468000, C800S280000, C800S285000, C800S287000, C800S298000, C800S301000

Reexamination Certificate

active

06632980

ABSTRACT:

FIELD OF INVENTION
The present invention relates to the field of molecular biology and the genetic transformation of plants with foreign gene fragments. More particularly, the invention relates to a binary expression system useful for conditionally expressing transgenes in plants.
BACKGROUND OF THE INVENTION
Two serious technical problems beset plant transgenics. First, plant transgene expression attains only low and inconsistent levels. These poor expression levels are attributable in part to random chromosomal integration (‘position effects’) and in part to a general lack of gene copy number-dependent expression. Episomal vectors are expected to overcome these problems. In constrast to plants, microbes can attain high-level expression through episomal (plasmid) vectors because these vectors can be maintained by selection. Although plant viruses have been used as episomal expression vectors, their use has been restricted to transient expression because of lack of selection and/or their cellular toxicity (U.S. Pat. No. 4,855,237, WO 9534668).
Second, non-specific expression of transgenes in non-desired cells and tissues hinders plant transgenic work. This is important where the goal is to produce high levels of phytotoxic materials in transgenic plants. Conditional transgene expression will enable economic production of desired chemicals, monomers, and polymers at levels likely to be phytotoxic to growing plants by restricting their production to production tissue of transgenic plants either just prior to or after harvest. Therefore, lack of a commercially usable conditional expression system and the difficulty in attaining a reliable, high-level expression both limit development of transgene expression in plants.
Plant Viruses
Viruses are infectious agents with relatively simple organization and unique modes of replication. A given plant virus may contain either RNA or DNA, and may be either single- or double-stranded.
RNA Plant Viruses
Double-stranded RNA plant viruses include rice dwarf virus (RDV) and wound tumor virus (WTV). Single-stranded RNA plant viruses include tobacco mosaic virus (TMV) and potato virus X (PVX), turnip yellow mosaic virus (TYMV), rice necrosis virus (RNV) and brome mosaic virus (BMV). The RNA in single-stranded RNA viruses may be either a plus (+) or a minus (−) strand.
Although many plant viruses have RNA genomes, organization of genetic information differs between groups (the major groupings designated as monopartite, bipartite and tripartite). The genome of most monopartite plant RNA viruses is a single-stranded molecule of (+)-sense. There are at least 11 major groups of viruses with this type of genome. Examples of this type of virus are TMV and PVX. At least six major groups of plant RNA viruses have a bipartite genome. In these, the genome usually consists of two distinct (+)-sense single-stranded RNA molecules encapsidated in separate particles. Both RNAs are required for infectivity. Cowpea mosaic virus (CPMW) is one example of a bipartite plant virus. A third major group, containing at least six major types of plant viruses, is tripartite, with three (+)-sense single-stranded RNA molecules. Each strand is separately encapsidated, and all three are required for infectivity. An example of a tripartite plant virus is alfalfa mosaic virus (AMV). Many plant viruses also have smaller subgenomic mRNAs that are synthesized to amplify a specific gene product.
DNA Plant Viruses
Plant viruses with a double-stranded DNA genome include Cauliflower Mosaic virus (CaMV).
Plant viruses with single-stranded DNA genomes include geminiviruses, and more specifically, include African Cassava Mosaic Virus (ACMV), Tomato Golden Mosaic Virus (TGMV), and Maize Streak Virus (MSV). Geminiviruses are subdivided on the basis of whether they infect monocots or dicots and whether their insect vector is a leafhopper or a whitefly. Subgroup I geminiviruses are leafhopper-transmitted and infect monocotyledonous plants (e.g., Wheat Dwarf Virus); Subgroup II geminiviruses are leafhopper-transmitted and infect dicotyledonous plants (e.g., Beet Curly Top Virus); and Subgroup III geminiviruses are whitefly-transmitted and infect dicotyledonous plants (e.g., Tomato Golden Mosaic Virus, TGMV, and African Cassava Mosaic Virus, ACMV).
Subgroup I and II geminiviruses have a single (monopartite) genome. Subgroup III geminiviruses have a bipartite genome. For example, Subgroup III geminiviruses TGMV and ACMV consist of two circular single-stranded DNA genomes, A and B, of ca. 2.8 kB each in size. DNA A and B of a given Subgroup III virus have little sequence similarity, except for an almost identical common region of about 200 bp. While both DNA A and DNA B are required for infection, only DNA A is necessary and sufficient for replication and DNA B encodes functions required for movement of the virus through the infected plant.
In both TGMV and ACMV, DNA A contains four open reading frames (ORFs) that are expressed in a bidirectional manner and arranged similarly. The ORFs are named according to their orientation relative to the common region, i.e., complementary (C) versus viral (V) in ACMV and leftward (L) or rightward (R) in TGMV. Thus, ORFs AL1, AL2, AL3, and AR1 of TGMV are homologous to AC1, AC2, AC3, and AV1, respectively, of ACMV. Three major transcripts have been identified in ACMV DNA A and these map to the AV1 and AC1 ORFs, separately and the AC2/AC3 ORFs together. There is experimental evidence for the function of these ORFs. Thus, in ACMV AC1 encodes a replication protein that is essential and sufficient for replication; AC2 is required for transactivation of the coat protein gene, AC3 encodes a protein that is not essential for replication but enhances viral DNA accumulation; and AV1 is the coat protein gene. Except for the essential viral replication protein (encoded by AC1 and AL1 in ACMV and TGMV, respectively), geminivirus replication relies on host replication and transcription machinery. Although geminiviruses are single-stranded plant DNA viruses, they replicate via double-stranded DNA intermediate by ‘rolling circle replication’.
Viruses as Expression Vectors
Constructing plant viruses to introduce and express non-viral foreign genes in plants has been demonstrated (U.S. Pat. No. 4,855,237, WO 9534668). When the virus is a DNA virus, the constructions can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease in constructing the desired viral vector with the foreign DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The DNA plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA. The cDNA of RNA viral genome can be cloned behind a heterologous plant promoter. Such a chimeric gene, called an ‘amplicon’, can be introduced into a plant cell and used to transcribe the viral RNA that can replicate autonomously [Sablowski et al. (1995)
Proc. Natl. Acad. Sci. USA
vol 92, pp 6901-6905].
Geminiviruses have many advantages as potential plant expression vectors. These include 1) replication to high copy numbers, 2) small, well-characterized genomes, 3) assembly into nucleosomes, and 4) nuclear replication and transcription. The DNA A component of these viruses is capable of autonomous replication in plant cells in the absence of DNA B. Vectors in which the coat protein ORF has been replaced by a heterologous coding sequence have been developed and the heterologous coding sequence expressed from the coat protein promoter [Hayes et al., Stability and expression of bacterial genes in replicating geminivirus vectors in plants.
Nucleic Acids Res.
17:2391-403 (1989); Hayes et al., Gene amplification and expression in plants by a replicating geminivirus vector.
Nature
(London) 334:179-82 (1988)].
Greater than full length copies of wild-type TGMV A and

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