Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-07-21
2001-10-16
Wang, Andrew (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S320100, C435S468000, C536S023100, C536S023200, C536S024100, C800S278000
Reexamination Certificate
active
06303848
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology and viral genetics. Specifically, the present invention relates to methods for conferring herbicide, pest, or disease resistance in plant hosts.
BACKGROUND OF THE INVENTION
Major areas targeted for genetic improvement in plants include herbicide, insect, and disease resistance. Herbicide resistance can be obtained by genetically engineering plants to respond to toxic chemicals. Many herbicides act by inhibiting a key plant enzyme or protein necessary for growth. For example, the herbicide glyphosate destroys plants by inhibiting the activity of an enzyme necessary for synthesizing aromatic amino acids. Some bacteria contain enzymes that confer resistance to herbicides. A gene encoding a herbicide resistant enzyme from bacteria may be cloned, modified for expression in plants, and inserted crop plant genomes. When sprayed with such herbicide, plants containing the bacterial gene grow as well as unsprayed control plants. For example, the herbicide glyphosate acts by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS). Glyphosate tolerant plants have been produced by inserting EPSPS into the plant genome (U.S. Pat. No. 5,312,910).
Genetic engineering has also been used to protect plants from bacterial, fungal, and virus infection (D'Maris et. al.,
Trends in Microbiol.
6:54-61 (1998)). It has been shown that transgenic plants that express the coat protein of a virus become resistant to infection by that virus. Although the mechanism of resistance is unknown, the presence of viral coat protein in plant cells can interfere with the uncoating of viral particles containing that coat protein, thereby interrupting the virus replication cycle. Viral replicase and movement protein genes have also been used to confer resistance to viral pathogens. For example, mutated forms of viral genes may be added to the plant genome and expressed in plants for protection against viruses. U.S. Pat. No. 5,773,701 issued to Braun et al., describes the insertion of a potato virus X (PVX) replicase gene into a plant to confer resistance against PVX infection when the PVX replicase gene is sufficiently expressed. PCT publication WO 98/59046 describes the incorporation of &bgr;-glucanase gene in transgenic plants for increased resistance to fungal infection.
Proteins conferring insect resistance have also been genetically introduced into plants. Bt-toxins from
Bacillus thuringiensis
are toxic to moth, fly larvae, beetle, and mosquitoes. A Bt-toxin gene may be cloned into aplasmid vector under control of a chloroplast rRNA promoter and transferred into plant chloroplasts by particle bombardment. Plants that sufficiently express this protein are toxic to insect larvae.
The approach of using transgenic plants in acquiring herbicide, insect and disease resistance has many drawbacks. Because foreign genes are permanently inserted into the genome, development of transgenic plants can be costly and their durability could become a concern. In many cases, the targets of herbicides or pathogens are unknown and indirect. Therefore, finding suitable herbicide-specific or pathogen-specific targets is difficult.
The present invention features a method of using transient plant viral expression vectors to express enzymes or proteins to confer resistance to herbicides, pests, and diseases in plant hosts. Viruses are a unique class of infectious agents whose distinctive features are their simple organization and their mechanism of replication. Their hosts include a wide variety of plants. A complete viral particle, or virion, may be regarded mainly as a block of genetic material (either DNA or RNA) capable of autonomous replication, surrounded by a protein coat and sometimes by an additional membranous envelope. The coat protects the virus from the environment and serves as a vehicle for transmission from one host cell to another.
Virus-based expression systems, in which the foreign mRNA is greatly amplified by virus replication, can produce very high levels of proteins in leaves and other tissues. Viral vector-produced protein can also be directed to specific subcellular locations, such as endomembrane, cytosol, or organelles, or it can be attached to macromolecules, such as virions. In addition, virus vectors are ideal for shuttling libraries of sequence variants throughout plant hosts for selection of resistance to herbicides, pests, and diseases.
SUMMARY OF THE INVENTION
In one aspect of this invention, recombinant viral nucleic acids are used to express foreign proteins or enzymes that confer resistance to herbicides, pests, and diseases. These foreign proteins or enzymes may be the direct or indirect targets of herbicides or pathogens. In particular, viral expression vectors may contain one or more non-native subgenomic promoters. Herbicides may include any compounds that affect the plant growth. Pathogens may include bacteria, fungi, viruses, insects, nematodes or arachnids, among others, which affect the plant growth.
In another aspect of the invention, a library containing nucleic acid sequence variants may be generated by in vivo or in vitro methods. A population of nucleic acid sequence variants may be represented by a genomic library, a cDNA library, or a pool of mRNA derived from bacteria, fungi, viruses, plants, or animals. In vitro mutagenesis or recombination methods may also be used to construct a library containing nucleic acid sequence variants. These methods may include chemical treatment, oligonucleotide mediated mutagenesis, error-prone PCR, combinatorial cassette mutagenesis, DNA shuffling, random-priming recombination, restriction enzyme fragment induced template switching, staggered extension process, among others. A library containing sequence variants may be expressed in plant hosts to screen sequences that confer resistance to herbicides, pests or diseases. The screening of sequence libraries is typically followed by rescue of the viruses from populations conferring desired resistance. If necessary, re-screening of sub-libraries may be performed. In preferred embodiments, sequences of the viral nucleic acids conferring desired resistance may be determined and compared with the control sequences. The expression of sequence libraries to select recombinant viral nucleic acids that confer resistance in plant hosts is particularly suited when the targets of herbicides or pathogens are unknown or less defined. This aspect of the invention is also applicable when the level of resistance in plant hosts is in need of modulation or optimization.
In another aspect of the invention, antisense technology may be used as a method to determine the targets of herbicides or pathogens in plant hosts. In essence, antisense RNA or DNA molecules bind endogenous mRNA, thereby turning off certain endogenous genes. A library of sequence variants may be applied to the plants to turn off endogenous proteins or enzymes in plant hosts like herbicides or pathogens. Targets of herbicides or pathogens may be determined by isolating and sequencing viral nucleic acids that affect the plant growth. Once the target enzymes or proteins are identified, they may be expressed in plant hosts using viral expression vectors to screen for desired resistance.
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patent: 5316931 (
della-Cioppa Guy R.
Kumagai Monto H.
Gallegos Thomas
Halluin Albert P.
Kung Viola T.
Large Scale Biology Corporation
Wang Andrew
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