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Collagen: Primer in Structure, Processing and Assembly (Topics in Current Chemistry)

Table of Contents Product Details J. Brinckmann: Collagens at a Glance. Engel, H. Ricard-Blum, F. Ruggiero, M. Koide, K. Nagata: Collagen Biosynthesis. Greenspan: Biosynthetic Processing of Collagen Molecules. Birk, P. Bruckner: Collagen Suprastructures. Eyre, J. Wu: Collagen Cross-Links. In Stock. Molecules The Elements and the Architecture of Everything. Organic Chemistry.

No More Heart Disease. An Introduction to Medicinal Chemistry. Type I collagen is the predominant collagen component of bone and tendon and is found in large amounts in skin, aorta, and lung. Type I collagen fibers provide great tensile strength and limited extensibility. The most abundant molecular form of type I collagen is a heterotrimer composed of two different alpha chains [alpha 1 I ] 2 and alpha 2 I Inkinen, All fibrillar collagen molecules contain three polypeptide chains constructed from a repeating Gly-X-Y triplet, where X and Y can be any amino acid but are frequently the imino acids proline and hydroxyproline.

Fibril forming collagens are synthesized as precursor procollagens containing globular N- and C-terminal extension propeptides. The biosynthesis of procollagen is a complex process involving a number of different post-translational modifications including proline and lysine hydroxylation, N-linked and O-linked glycosylation and both intra- and inter-chain disulphide-bond formation. The enzymes carrying out these modifications act in a coordinated fashion to ensure the folding and assembly of a correctly aligned and thermally stable triple-helical molecule.

Each procollagen molecule assembles within the rough endoplasmic reticulum from the three constituent polypeptide chains. As the polypeptide chain is co-translationally translocated across the membrane of the endoplasmic reticulum, hydroxylation of proline and lysine residues occurs within the Gly-X-Y repeat region.

Once the polypeptide chain is fully translocated into the lumen of the endoplasmic reticulum the C-propeptide folds. Three pro-alpha chains then associate via their C-propeptides to form a trimeric molecule allowing the Gly-X-Y repeat region to form a nucleation point at its C- terminal end, ensuring correct alignment of the chains. The temporal relationship between polypeptide chain modification and triple-helix formation is crucial as hydroxylation of proline residues is required to ensure stability of the triple helix at body temperature, once formed, the triple helix no longer serves as a substrate for the hydroxylation enzyme.

The C-propeptides and to a lesser extent the N-propeptides keep the procollagen soluble during its passage through the cell Bulleid et al. Following or during secretion of procollagen molecules into the extracellular matrix, propeptides are removed by procollagen N- and C-proteinases, thereby triggering spontaneous self-assembly of collagen molecules into fibrils Hulmes, Crucial to this assembly process are short non triple-helical peptides called telopeptides at the ends of the triple-helical domain, which ensure correct registration of the collagen molecules within-the fibril structure and lower the critical concentration for self-assembly Bulleid et al.

In nature, the stability of the triple-helical structure of collagen requires the hydroxylation of prolines by the enzyme prolylhydroxylase P4H to form residues of hydroxyproline within a collagen chain. Plants expressing collagen chains are known in the art, see for example, U. Although plants are capable of synthesizing hydroxyproline-containing proteins the prolyl hydroxylase that is responsible for synthesis of hydroxyproline in plant cells exhibits relatively loose substrate sequence specificity as compared with mammalian P4H and thus, production of collagen containing hydroxyproline only in the Y position of Gly -X-Y triplets requires plant co-expression of collagen and P4H genes Olsen et al, An attempt to produce human collagens that rely on the hydroxylation machinery naturally present in plants resulted in collagen that is poor in proline hydroxylation Merle et al.

Co-expression of collagen and prolylhydroxylase results with stable hydroxylated collagen that is biologically relevant for applications at body temperatures Merle et al. Lysyl hydroxylase LH,EC 1. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity Wang et al, A single human enzyme, Lysyl hydroxylase 3 LH3 can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation Wang et al, This suggests that plant endogenic Lysyl hydroxylase is unable to sufficiently hydroxylate lysines in collagen.

While reducing the present invention to practice, the present inventors uncovered that efficient hydroxylation of collagen chains relies upon sequestering of the collagen chain along with an enzyme capable of correctly modifying this polypeptide. According to one aspect of the present invention there is provided a method of producing collagen in a plant or an isolated plant cell comprising expressing in the plant or the isolated plant cell at least one type of a collagen alpha chain and exogenous P4H in a manner enabling accumulation of the at least one type of the collagen alpha chain and the exogenous P4H in a subcellular compartment devoid of endogenous P4H activity, thereby producing the collagen in the plant.

According to an additional aspect of the present invention there is provided According to further features in preferred embodiments of the invention described below, the method further comprises expressing exogenous LH3 in the subcellular compartment devoid of endogenous P4H activity.

Collagen CAS#:

According to still further features in the described preferred embodiments the at least one type of the collagen alpha chain includes a signal peptide for targeting to an apoplast or a vacuole. According to still further features in the described preferred embodiments the at least one type of the collagen alpha chain is devoid of an ER targeting or retention sequence.

According to still further features in the described preferred embodiments the at least one type of the collagen alpha chain is expressed in a DNA-containing organelle of the plant. According to still further features in the described preferred embodiments the exogenous P4H includes a signal peptide for targeting to an apoplast or a vacuole.

According to still further features in the described preferred embodiments the exogenous P4H is devoid of an ER targeting or retention sequence. According to still further features in the described preferred embodiments the exogenous P4H is expressed in a DNA-containing organelle of the plant. According to still further features in the described preferred embodiments the at least one type of the collagen alpha chain is alpha 1 chain.

According to still further features in the described preferred embodiments the at least one type of the collagen alpha chain is alpha 2 chain. According to still further features in the described preferred embodiments the plant is selected from the group consisting of Tobacco, Maize, Alfalfa, Rice, Potato, Soybean, Tomato, Wheat, Barley, Canola and Cotton. According to still further features in the described preferred embodiments the at least one type of the collagen alpha chain or the exogenous P4H are expressed in only a portion of the plant. According to still further features in the described preferred embodiments the portion of the plant is leaves, seeds, roots, tubers or stems.

According to still further features in the described preferred embodiments the exogenous P4H is capable of specifically hydroxylating the Y position of Gly-X-Y triplets of the at least one type of the collagen alpha chain. According to still further features in the described preferred embodiments the exogenous P4H is human P4H.

According to still further features in the described preferred embodiments the plant is subjected to a stress condition. According to still further features in the described preferred embodiments the stress condition is selected from the group consisting of drought, salinity, injury, cold and spraying with stress inducing compounds.

According to another aspect of the present invention there is provided a genetically modified plant or isolated plant cell capable of accumulating a collagen alpha chain having a hydroxylation pattern identical to that produced when the collagen alpha chain is expressed in human cells. According to yet another aspect of the present invention there is provided a genetically modified plant or isolated plant cell capable of accumulating a collagen alpha chain in a subcellular compartment devoid of endogenous P4H activity.

According to still further features in the described preferred embodiments the genetically modified plant further comprises an exogenous P4H. According to still further features in the described preferred embodiments the collagen alpha chain is alpha 1 chain. According to still further features in the described preferred embodiments the collagen alpha chain is alpha 2 chain.

According to still another aspect of the present invention there is provided a plant system comprising a first genetically modified plant capable of accumulating a collagen alpha 1 chain and a second genetically modified plant capable of accumulating a collagen alpha 2 chain. According to yet another aspect of the present invention there is provided a plant system comprising a first genetically modified plant capable of accumulating a collagen alpha 1 chain and a collagen alpha 2 chain and a second genetically modified plant capable of accumulating P4H.

According to still further features in the described preferred embodiments at least one of the first genetically modified plant and the second genetically modified plant further comprises exogenous P4H. According to yet another aspect of the present invention there is provided a method of producing fibrillar collagen comprising: a expressing in a first plant a collagen alpha 1 chain; b expressing in a second plant a collagen alpha 2 chain, wherein expression in the first plant and the second plant the is configured such that the collagen alpha 1 chain and the collagen alpha 2 chain are each capable of accumulating in a subcellular compartment devoid of endogenous P4H activity; and c crossing the first plant and the second plant and selecting progeny expressing the collagen alpha 1 chain and the collagen alpha 2 chain thereby producing fibrillar collagen.

According to still further features in the described preferred embodiments the method further comprises expressing an exogenous P4H in each of the first plant and the second plant. According to still further features in the described preferred embodiments each of the collagen alpha 1 chain and the collagen alpha 2 chain includes a signal peptide for targeting to an apoplast or a vacuole. According to still further features in the described preferred embodiments each of the collagen alpha 1 chain and the collagen alpha 2 chain is devoid of an ER targeting or retention sequence.

According to still further features in the described preferred embodiments steps a and b are effected via expression in a DNA-containing organelle of the plant. According to still further features in the described preferred embodiments the first plant and the second plant are subjected to a stress condition. According to still further features in the described preferred embodiments the stress condition is selected from the group consisting of drought, salinity, injury, heavy metal toxicity and cold stress.

According to yet another aspect of the present invention there is provided a method of producing fibrillar collagen comprising: a expressing in a first plant a collagen alpha 1 chain and a collagen alpha 2 chain, wherein expression in the first plant is configured such that the collagen alpha 1 chain and the collagen alpha 2 chain are each capable of accumulating in a subcellular compartment devoid of endogenous P4H activity; b expressing in a second plant an exogenous P4H capable of accumulating in the subcellular compartment devoid of endogenous P4H activity; and c crossing the first plant and the second plant and selecting progeny expressing the collagen alpha 1 chain, the collagen alpha 2 chain and the P4H thereby producing fibrillar collagen.

According to yet another aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide encoding a human P4H positioned under the transcriptional control of a promoter functional in plant cells. According to still further features in the described preferred embodiments the promoter is selected from the group consisting of the CaMV 35S promoter, the Ubiquitin promoter, the rbcS promoter and the SVBV promoter. According to still further features in the described preferred embodiments the collagen alpha 1 chain and the collagen alpha 2 chain are each capable of accumulating in a subcellular compartment devoid of endogenous plant P4H activity.

According to yet another aspect of the present invention there is provided a genetically modified plant or isolated plant cell being capable of accumulating collagen having a temperature stability characteristic identical to that of mammalian collagen. According to still further features in the described preferred embodiments the collagen is type I collagen.

According to still further features in the described preferred embodiments the mammalian collagen is human collagen. According to yet another aspect of the present invention there is provided a collagen-encoding sequence optimized for expression in a plant. According to still further features in the described preferred embodiments the collagen encoding sequence is as set forth by SEQ ID NO: 1. The present invention successfully addresses the shortcomings of the presently known configurations by providing a plant capable of expressing correctly hydroxylated collagen chains which are capable of assembling into collagen having properties similar to that of human collagen.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The invention is herein described, by way of example only, with reference to the accompanying drawings.

With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention.

In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

What is collagen (collagen protein structure and function)

The present invention is of plants expressing and accumulating collagen which can be used to produce collagen and collagen fibers which display characteristics of mammalian collagen. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Collagen producing plants are known in the art. Although such plants can be used to produce collagen chains as well as collagen, such chains are incorrectly hydroxylated and thus self-assembly thereof, whether in planta or not, leads to collagen which is inherently unstable. While reducing the present invention to practice, the present inventors have devised a plant expression approach which ensures correct hydroxylation of collagen chains and thus enables in-planta production of collagen which closely mimics the characteristics e.

Thus, according to one aspect of the present invention there is provided a genetically modified plant which is capable of expressing at least one type of a collagen alpha chain and accumulating it in a subcellular compartment which is devoid of endogenous P4H activity. As used herein, the phrase "genetically modified plant" refers to any lower e. As used herein, the phrase "collagen chain" refers to a collagen subunit such as the alpha 1 or 2 chains of collagen fibers, preferably type I fibers.

As used herein, the phrase "collagen" refers to an assembled collagen trimer, which in the case of type I collagen includes two alpha 1 chains and one alpha 2 chain. A collagen fiber is collagen which is devoid of terminal propeptides C and N. As is used herein, the phrase "subcellular compartment devoid of endogenous P4H activity" refers to any compartmentalized region of the cell which does not include plant P4H or an enzyme having plant-like P4H activity.

Any type of collagen chain can be expressed by the genetically modified plant of the present invention. For further description please see Hulmes, The expressed collagen alpha chain can be encoded by any polynucleotide sequences derived from any mammal. Typically, alpha collagen chains expressed in plants may or may not include their terminal propeptides i. Ruggiero et al. Cleavage of the C propeptide may take place on a procollagen peptide before the assembly of trimmer association of three C-Propeptides is essential for initiating the assembly of trimmers.

N-propeptide cleavage by plant proteolytic activity takes place in mature plants but not in plantlets. Such cleavage removes 2 amino acids from the N telopeptide 2 out of The C-propeptides and to a lesser extent the N-propeptides maintain the procollagen soluble during its passage through the animal cell Bulleid et al. Following or during secretion of procollagen molecules into the extracellular matrix, propeptides are removed by procollagen N- and C- proteinases, thereby triggering spontaneous self-assembly of collagen molecules into fibrils Hulmes, Crucial to this assembly process are short non triple-helical peptides called telopeptides at the ends of the triple-helical domain, which ensure correct registration of the collagen molecules within the fibril structure and lower the critical concentration for self-assembly Bulleid et al.

Prior art describe the use of pepsin to cleave the propeptides during production of collagen Bulleid et al However pepsin-damages the telopeptides and as a result, pepsin-extracted collagen is unable to form ordered fibrillar structures Bulleid et al Protein disulfide isomerase PDI that form the beta subunit of human P4H was shown to bind to the C-propeptide prior to trimmer assembly thereby also acting as a molecular chaperone during chain assembly Ruggiero et al, The use of human Procollagen I N-proteinase and Procollagen C-proteinase expressed in a different plants may generate collagen that is more similar to the native human collagen and can form ordered fibrillar structures.

In a case where N or C propeptides or both are included in the expressed collagen chain, the genetically modified plant of the present invention can also express the respective protease i. C or N or both. Such proteases can be expressed such that they are accumulated in the same subcellular compartment as the collagen chain. Accumulation of the expressed collagen chain in a subcellular compartment devoid of endogenous P4H activity can be effected via any one of several approaches.

For example, the expressed collagen chain can include a signal sequence for targeting the expressed protein to a subcellular compartment such as the apoplast or an organelle e. Examples of suitable signal sequences include the chloroplast transit peptide included in Swiss-Prot entry P, amino acids 1- 57 and the Mitochondrion transit peptide included in Swiss-Prot entry P, amino acids 1- The Examples section which follows provides additional examples of suitable signal sequences as well as guidelines for employing such signal sequences in expression of collagen chains in plant cells.

Alternatively, the sequence of the collagen chain can be modified in a way which alters the cellular localization of collagen when expressed in plants. As is mentioned hereinabove, the ER of plants includes a P4H which is incapable of correctly hydroxylating collagen chains. Collagen alpha chains natively include an ER targeting sequence which directs expressed collagen into the ER where it is post-translationally modified including incorrect hydroxylation. Thus, removal of the ER targeting sequence will lead to cytoplasmic accumulation of collagen chains which are devoid of post translational modification including any hydroxylations.

Example 1 of the Examples section which follows describes generation of collagen sequences which are devoid of ER sequences. Still alternatively, collagen chains can be expressed and accumulated in a DNA containing organelle such as the chloroplast or mitochondria. Further description of chloroplast expression is provided hereinbelow. As is mentioned hereinabove, hydroxylation of alpha chains is required for assembly of a stable type I collagen. Since alpha chains expressed by the genetically modified plant of the present invention accumulate in a compartment devoid of endogenous P4H activity, such chains must be isolated from the plant, plant tissue or cell and in-vitro hydroxylated.

Such hydroxylation can be achieved by the method described by Turpeenniemi-Hujanen and Myllyla Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro. Biochim Biophys Acta. Although such in-vitro hydroxylations can lead to correctly hydroxylated collagen chains, it can be difficult and costly to achieve. To overcome the limitations of in-vitro hydroxylation, the genetically modified plant of the present invention preferably also co-expresses P4H which is capable of correctly hydroxylating the collagen alpha chain s [i. P4H is an enzyme composed of two subunits, alpha and beta.

Both are needed to form an active enzyme while the Beta subunit also posses a chaperon function. In addition, P4H mutants which exhibit enhanced substrate specificity, or P4H homologues can also be used. Pairwise alignment of this protein sequence and a human P4H alpha subunit conducted by the present inventors revealed the highest homology between functional domains of any known P4H homologs of plants. Since P4H needs to co-accumulate with the expressed collagen chain, the coding sequence thereof is preferably modified accordingly addition of signal sequences, deletions which may prevent ER targeting etc.

In mammalian cells, collagen is also modified by Lysyl hydroxylase, galactosyltransferase and glucosyltransferase. These enzymes sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. A single human enzyme, Lysyl hydroxylase 3 LH3 can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. Thus, the genetically modified plant of the present invention preferably also expresses mammalian LH3.

P4H and LH3 positioned under the transcriptional control of plant functional promoters. Such a nucleic acid construct which is also termed herein as an expression construct can be configured for expression throughout the whole plant, defined plant tissues or defined plant cells, or at define developmental stages of the plant. Such a construct may also include selection markers e.

It will be appreciated that constructs including two expressible inserts e. In such a case, the chimeric transcript includes an IRES sequence between the two insert sequences such that the downstream insert can be translated therefrom. Numerous plant functional expression promoters and enhancers which can be either tissue specific, developmentally specific, constitutive or inducible can be utilized by the constructs of the present invention, some examples are provided hereinunder. As used herein in the specification and in the claims section that follows the phrase "plant promoter" or "promoter" includes a promoter which can direct gene expression in plant cells including DNA containing organelles.

Such a promoter can be derived from a plant, bacterial, viral, fungal or animal origin. Such a promoter can be constitutive, i. Thus, the plant promoter employed can be a constitutive promoter, a tissue specific promoter, an inducible promoter or a chimeric promoter. Examples of tissue specific promoters include, without being limited to, bean phaseolin storage protein promoter, DLEC promoter, PHS promoter, zein storage protein promoter, conglutin gamma promoter from soybean, AT2S1 gene promoter, ACT11 actin promoter from Arabidopsis, napA promoter from Brassica napus and potato patatin gene promoter.

The inducible promoter is a promoter induced by a specific stimuli such as stress conditions comprising, for example, light, temperature, chemicals, drought, high salinity, osmotic shock, oxidant conditions or in case of pathogenicity and include, without being limited to, the light-inducible promoter derived from the pea rbcS gene, the promoter from the alfalfa rbcS gene, the promoters DRE, MYC and MYB active in drought; the promoters INT, INPS, prxEa, Ha hsp Preferably the promoter utilized by the present invention is a strong constitutive promoter such that over expression of the construct inserts is effected following plant transformation.

It will be appreciated that any of the construct types used in the present invention can be co-transformed into the same plant using same or different selection markers in each construct type. Alternatively the first construct type can be introduced into a first plant while the second construct type can be introduced into a second isogenic plant, following which the transgenic plants resultant therefrom can be crossed and the progeny selected for double transformants. Further self-crosses of such progeny can be employed to generate lines homozygous for both constructs. There are various methods of introducing nucleic acid constructs into both monocotyledonous and dicotyledenous plants Potrykus, I.

Such methods rely on either stable integration of the nucleic acid construct or a portion thereof into the genome of the plant, or on transient expression of the nucleic acid construct in which case these sequences are not inherited by a progeny of the plant. In addition, several method exist in which a nucleic acid construct can be directly introduced into the DNA of a DNA containing organelle such as a chloroplast.

There are two principle methods of effecting stable genomic integration of exogenous sequences such as those included within the nucleic acid constructs of the present invention into plant genomes:. The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation.

Horsch et al. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledenous plants. There are various methods of direct DNA transfer into plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals, tungsten particles or gold particles, and the microprojectiles are physically accelerated into cells or plant tissues.

Following transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits.

Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants. Transient expression methods which can be utilized for transiently expressing the isolated nucleic acid included within the nucleic acid construct of the present invention include, but are not limited to, microinjection and bombardment as described above but under conditions which favor transient expression, and viral mediated expression wherein a packaged or unpackaged recombinant virus vector including the nucleic acid construct is utilized to infect plant tissues or cells such that a propagating recombinant virus established therein expresses the non-viral nucleic acid sequence.

Transformation of plants using plant viruses is described in U. Construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences in plants is demonstrated by the above references as well as by Dawson, W. EMBO J. Science ; and Takamatsu et al. FEBS Letters When the virus is a DNA virus, the constructions can be made to the virus itself.

On the control of collagen fibril organization and morphology

Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. The plasmid is then used to make all of the constructions.

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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. Construction of plant RNA viruses for the introduction and expression in plants of non-viral exogenous nucleic acid sequences such as those included in the construct of the present invention is demonstrated by the above references as well as in U. In one embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted.

Alternatively, the coat protein gene may be inactivated by insertion of the non-native nucleic acid sequence within it, such that a protein is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters.


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Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native foreign nucleic acid sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included. The non-native nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence. In a third embodiment, a recombinant plant viral nucleic acid is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid.

The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native nucleic acid sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that said sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.