Recombinant plant viral vectors

Details Recombinant plant viral vectors Recombinant plant viral vectors

2000-02-01

2002-04-09

Schwartzman, Robert A. (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S280000, C800S288000, C435S320100, C424S093210, C514S04400A

Reexamination Certificate

active

06369296

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to recombinant plant viral nucleic acids, and methods of use thereof.
PRIOR ART
Recombinant plant viral nucleic acids are of interest generally for their utility as expression vectors in plants.
Additionally, such nucleic acids can be used to initiate virus induced gene silencing (VIGS). This phenomenon is based on the observation that virus infection in plants can initiate sequence-specific nucleic-acid based defence mechanisms that resemble either transcriptional, or post-transcriptional gene silencing (PTGS) (Covey, Al-Kaff 1997; Ratcliff, Harrison et al. 1997; Al-Kaff, Covey et al. 1998). PTGS is also manifest as an inhibition of nuclear gene expression when a virus is modified to carry sequence from a nuclear expressed gene (Kumagai, Donson et al. 1995; Kjemtrup, Sampson et al. 1998; Ruiz, Voinnet et al. 1998). PTGS can also cause recovery from viral infection when a plant expressing a transgene derived from viral cDNA is infected by a homologous virus (Lindbo, Silva-Rosales et al. 1993; Guo and Garcia 1997). Both the inhibition of nuclear gene expression, and recovery from viral infection are caused by sequence-specific RNA degradation.
Because modified viruses inhibit the expression of homologous plant genes, VIGS can be used to induce an apparent null-phenotype or a loss of function and therefore identify the function of any gene. Viruses that have been modified in this manner include tobacco mosaic virus (TMV) (Kumagai, Donson et al. 1995) potato virus X (PVX) (Ruiz, Voinnet et al. 1998), and tomato golden mosaic virus (Kjemtrup, Sampson et al. 1998).
DISCLOSURE OF THE INVENTION
The present invention is concerned with novel recombinant plant viral nucleic acids.
In preferred forms the present invention is concerned with providing VIGS-based methods and materials which may be more suitable as a tool for functional genomics than those which have been used in the past. For instance TMV, PVX and TGMV infections cause significant symptoms, such as a chlorosis, leaf-distortion and necrosis. Phenotypes caused by VIGS of a plant gene can therefore be hard to differentiate from these viral symptoms. Secondly, like most viruses, TMV, PVX and TGMV form mosaic, vein-based infections, and therefore do not cause confluent VIGS across the whole leaf. Leaves may therefore contain a mixture of cells with and without VIGS, complicating interpretation of any phenotype. Thirdly, TMV, PVX and TGMV do not infect meristems (Matthews 1991) and can not therefore inhibit expression of genes that determine the identity and development of plant tissue. Finally, although the first plant genome to be fully sequenced will be that of
Arabidopsis thaliana
, TMV, PVX and TGMV vectors do not infect this plant. Therefore the potential of VIGS to identify gene function in Arabidopsis is limited with available technology. A VIGS vector which overcame one or more of these drawbacks would therefore represent a contribution to the art.
The present inventors have developed novel recombinant cDNA viral constructs based on tobacco rattle virus (TRV) which in preferred forms are particularly adapted for use with VIGS. Such vectors may induce few or no symptoms, cause confluent VIGS across the leaf, operate in Nicotiana species and in Arabidopsis, and inhibit gene expression in meristems.
A viral expression vector based on TRV has previously been described in which non-viral proteins were expressed from a sub-genomic promoter (Ratcliff, MacFarlane et al. 1999). The viral RNA was synthesised in vitro and then inoculated into the plant. The TRV vectors of the present invention include inter alia modifications to facilitate both the insertion of plant gene sequences and the it subsequent infection of plants. Other TRV based vectors are disclosed by Hamilton & Baulcombe (1989) J. Gen. Virol 70: 963-968 and Mueller et al (1997) J. Gen. Virol 78: 2085-2088.
Thus in a first aspect of the present invention there is disclosed a nucleic acid vector which comprises:
(a) a transfer nucleotide sequence comprising (i) a plant active promoter, operably linked to (ii) a recombinant tobacco rattle virus (TRV) cDNA which includes at least cis acting elements permitting replication of said cDNA; a subgenomic promoter operably linked to a sequence encoding a TRV coat protein; and a heterologous nucleotide sequence which is foreign to said virus;
(b) border sequences which permit the transfer of the transfer nucleotide sequence into a plant cell nucleus.
The transfer nucleotide sequence is situated between the border sequences and is capable of being inserted into a plant genome under appropriate conditions. Generally this may be achieved by use of so called “agro-infiltration” which uses Agrobacterium-mediated transient transformation. Briefly, this technique is based on the property of
Agrobacterium tumafaciens
to transfer a portion of its DNA (“T-DNA”) into a host cell where it may become integrated into nuclear DNA. The T-DNA is defined by left and right border sequences which are around 25 nucleotides in length. In the present invention the border sequences are included around the transfer nucleotide sequence (the T-DNA) with the whole vector being introduced into the plant by agro-infiltration, optionally in the form of a binary-transformation vector.
By “plant active promoter” is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3′ direction on the sense strand of double-stranded DNA). “Operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. Nucleic acid operably linked to a promoter is “under transcriptional initiation regulation” of the promoter.
The cDNA includes cis acting elements permitting replication of said cDNA. However the vector need not include all of the sequence required to replicate and move within the plant. The vectors of the present invention will generally require supplementary proteins and/or nucleic acids from TRV in order to achieve this. Thus the cDNA may correspond to part of TRV RNA 2, and will thus require proteins encoded by TRV RNA1 for replication.
The TRV coat protein (as with other defined or recited sequences herein) need not be ‘wild-type’, but may optionally be a variant (e.g. mutant, or other variant, or a substantially homologous derivative) provided that its function (to encapsulate and permit movement of the TRV genome) is not negated. By “Substantially homologous” is meant that the sequence in question shares at least about 70%, or 80% identity, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identity with the reference sequence. Identity may be at the nucleotide sequence and/or encoded amino acid sequence level. Homology may be over the full-length of the relevant sequence shown herein (e.g. in the sequence Annex) or may be over a part of it. Identity may be determined by the TBLASTN program, of Altschul et al. (1990)
J. Mol. Biol
. 215: 403-10, or BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA, Wisconsin 53711). Preferably sequence comparisons are made using FASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183: 63-98). Parameters are preferably set, using the default matrix, as follows: Gapopen (penalty for the first residue in a gap): −12 for proteins/−16 for DNA; Gapext (penalty for additional residues in a gap): −2 for proteins/−4 for DNA; KTUP word length: 2 for proteins/6 for DNA.
The heterologous nucleotide sequence is foreign (non-native) to TRV, which is to say that it does not occur naturally in the TRV viral genome at the position in which it is present in the VIGS vector. The sequence will generally be either a cloning site (to permit the insertion of a desired sequence) or a desired sequence itself.
Some preferred embodiments of the invention will now be discussed.
Vector
This is preferably

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