Patent Grant
8597950
The present invention relates to a viral vector system for replicating or for expressing a sequence of interest in a plant. The invention also provides a process for replicating and/or expressing a sequence of interest in a plant. This process can be used for expressing a protein of interest in plants, notably in crop plants. The system can be based on a large variety of different viral vectors.
Virus-based expression systems can be used for rapid protein production in plants (for review see: Porta & Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol, 240, 81-94) and are a powerful tool for functional genomics studies (Dalmay et al., 2000, Plant Cell, 12, 369-379; Ratcliff et al., 2001, Plant J., 25, 237-245; Escobar et al., 2003, Plant Cell, 15, 1507-1523). Numerous publications and patents in the field describe systems based on DNA and RNA viral vectors (Kumagai et al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Mallory et al., 2002, Nature Biotechnol. 20, 622-625; Mor et al., 2003, Biotechnol. Bioeng., 81; 430-437; U.S. Pat. Nos. 5,316,931; 5,589,367; 5,866,785; 5,491,076; 5,977,438; 5,981,236; WO02088369; WO02097080; WO9854342). The existing viral vector systems are usually restricted to a narrow host range in terms of their best performance and even the expression level of such vectors in their most favourable host is far below the upper biological limits of the system.
RNA viruses are the most suitable for use as expression vectors, as they offer a higher expression level compared to DNA viruses. There are several published patents which describe viral vectors suitable for systemic expression of transgenic material in plants (U.S. Pat. Nos. 5,316,931; 5,589,367; 5,866,785). In general, these vectors can express a foreign gene as a translational fusion with a viral protein (U.S. Pat. Nos. 5,491,076; 5,977,438), from an additional subgenomic promoter (U.S. Pat. Nos. 5,466,788; 5,670,353; 5,866,785), or from polycistronic viral RNA using IRES elements for independent protein translation (WO0229068). The first approach—translational fusion of a recombinant protein with a viral structural protein (Hamamoto et al., 1993, BioTechnology, 11, 930-932; Gopinath et al., 2000, Virology, 267, 159-173; JP6169789; U.S. Pat. No. 5,977,438) gives significant yield. However, the use of such an approach is limited, as the recombinant protein cannot be easily separated from the viral one. One of the versions of this approach employs the translational fusion via a peptide sequence recognized by a viral site-specific protease or via a catalytic peptide (Dolja et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10208-10212; Gopinath et al., 2000, Virology, 267, 159-173; U.S. Pat. Nos. 5,162,601; 5,766,885; 5,491,076).
Expression processes utilizing viral vectors built on heterologous subgenomic promoters provide a good level of protein production (U.S. Pat. No. 5,316,931). The most serious disadvantage of such vectors and many others is their limited capacity with regard to the size of DNA to be amplified. Usually, stable constructs accommodate inserts of not more than one kb. In some areas of plant functional genomics this may not be such a serious limitation as G. della-Cioppa et al. (WO993651) described the use of TMV-based viral vectors to express plant cDNA libraries with the purpose of silencing endogenous genes. Additionally, as such vectors are capable of systemic movement and produce coat protein, significant resources of the plant are diverted from the synthesis of recombinant protein. The low expression levels achieved so far with such plant viral expression systems are a major reason why these systems are hardly competitive with other expression systems like bacterial, fungal, or insect cell expression systems. Low expression levels give rise to very high downstream costs for protein isolation and purification in a huge background of plant material. Therefore, costs for downstream processing quickly decrease, as the yield of the protein or product of interest per unit plant biomass increases. Also, a biological safety of such vectors are an issue, as they are able to form infectious viral particles.
An alternative two-component system requiring a helper virus was developed by Turpen (U.S. Pat. No. 5,811,653; U.S. Pat. No. 5,889,191; U.S. Pat. No. 5,965,794); this approach relies on a system of a virus and a helper virus, whereby the helper virus provides a replicase function, whereas the main replicon is deficient in replicase activity. This system is not practical because viral RNA-dependent RNA polymerase (replicase) works inefficiently with substrate RNAs provided in trans. A possible explanation of such inefficiency is that TMV RNA-dependent RNA polymerase is a heterodimer consisting of a 126 kDa protein and a 183 kDa read-through protein (Watanabe et al., 1999, J. Virol. 73, 2633-2640). It was shown that at least one component of this heterodimer, the 126 kDa protein, appeared to work primarily in cis (Lewandowsky & Dawson, 2000, Virology, 271, 90-98). There are several publications concerning the complementation in trans of other viral functions, like cell-to cell and systemic movement. The MP and CP can be provided in trans either by a transgenic host or by another virus. For example, mutants of TMV with frameshifts within the MP or CP gene were unable to locally or systemically infect inoculated tobacco plants, but acquired the lost functions in transgenic tobacco plants expressing the wild-type MP or CP gene (Holt & Beachy, 1991, Virology, 181, 109-117; Osbourn, Sarkar & Wilson, 1990, Virology, 179, 921-925). These works did not address the issue of creating virus-based vectors for expressing a heterologous sequence of interest, but rather studied the biological functions of different viral proteins. Another work describes the complementation of long distance movement of a CP-deficient TMV expressing GFP by a chimeric TMV carrying ORF3 of groundnut rosette umbravirus (GRV) (Ryabov, Robinson & Taliansky, 1999, Proc. Natl. Acad. Sci. USA, 96, 1212-12170). However, as it follows from the results, the efficiency of GFP expression in systemic leaves of plants co-infected with CP-deficient TMV expressing GFP and TMV having CP replaced by ORF3 of GRV was significantly lower than in plants infected with systemic TMV expressing GFP. It appears that this low expression level may be due to the presence and competition of both viral vectors in systemic leaves. Moreover, all experiments mentioned above led to the formation of infectious viral particles in systemic leaves and are therefore not acceptable for use in the environment from the point of view of biological safety.
Another system proposed by C. Masuta et al. (U.S. Pat. No. 5,304,731) proposes to use a satellite CMV RNA virus to be used as a carrier of the heterologous sequence of interest, and a helper virus that provides functions necessary for CMV RNA replication. To the best of our knowledge, the system is highly inefficient.
A serious concern with prior art virus-based plant expression systems is biological safety. On the one hand, high infectivity of the recombinant virus is highly desired in order to facilitate spread of the virus throughout the plant and to neighbouring plants, thereby increasing the yield of the desired gene product. On the other hand, such a high infectivity compromises containment of the recombinant material since spread to undesired plants can easily occur. Consequently, safer virus-based plant expression systems are highly desired.
There is presently no biologically safe large-scale transgene expression system built on plant RNA viral vectors capable of moving systemically and providing for the yield and efficiency required for technical applications. The existing systemic vectors suffer from a low yield of recombinant product.
Therefore, it is an object of this invention to provide an environmentally safe plant viral expression system for high-yield production of a protein of interest. It is another object of the invention to provide a process of replicating and/or expressing a nucleotide sequence of interest in a plant or plant part, which is of improved ecological and biological safety. It is another object to provide a process of protein production in plants having efficiency enabling competitive large-scale protein production in plants.
The above objects are solved by a system for replicating or for replicating and expressing a sequence of interest in a plant, comprising:
The invention further provides a process of replicating or of replicating and expressing a sequence of interest in a plant, comprising providing cells of said plant with said RNA replicon (i) and said helper replicon (ii). The invention may be used for replicating a sequence of interest in a plant or for replicating a sequence of interest and expressing said sequence of interest, e.g. for producing a protein of interest like an industrial enzyme or a pharmaceutical protein in said plant. The invention also relates to proteins produced or producible by the process of the invention. Further, the invention provides a process of protein production in plants or in plant cells.
In important embodiments, said RNA replicon (i) and/or said helper replicon (ii) are provided to a plant via DNA precursors that are introduced into cell nuclei of said plant. These DNA precursors can generate the RNA replicon (i) and/or the helper replicon (ii) by transcription in cell nuclei. For improving formation and build-up of RNA replicon (i) and/or helper replicon (ii) in the cell cytoplasm, said precursor of said RNA replicon (i) and/or said precursor of said helper replicon (ii) may contain one or more introns. Generally, the DNA precursor of said RNA replicon (i) and/or the DNA precursor of said helper replicon (i) contain sequences for replicon function (like a replicase ORF or an MP ORF in the case of said RNA replicon (i)) being derived from sequences of an RNA virus, said sequences for replicon function preferably exhibiting at selected localities of said sequence of said RNA virus function-conservative differences from said sequence of said RNA virus, said differences causing an increased frequency of RNA replicon (i) and/or helper replicon (ii) formation compared to an RNA replicons not exhibiting said differences. This technology is described in detail in PCT/EP04/012743 for one RNA replicon and may be applied in the present invention to said RNA replicon (i) and to said helper replicon (ii).
The inventors have surprisingly identified a novel principle for replicating or for replicating and expressing a sequence of interest in a plant. The inventors have found that a helper replicon that is deficient in the ability to assemble virus particles, e.g. when provided to a plant by agrodelivery as a DNA copy, can express a sufficient amount of coat protein in locally transfected tissue to fully restore systemic movement of the said RNA replicon (i). It was further found that systemic movement of said RNA replicon (i) results in systemically infected leaves that express the sequence of interest but have a greatly reduced amount of coat protein compared with the case where a coat protein-expressing virus expresses a sequence of interest. The coat protein is usually the strongest expressed protein in virus-infected plant cells. With the system and process of the invention, however, resources of systemically infected cells are not used up by coat protein expression. Consequently, the expression levels of said sequence of interest in systemically infected plant cells are higher than in conventional viral expression systems. Further, systemically infected plant cells produce small amounts of assembled viral particles from said RNA replicon (i), whereby spread of the RNA replicon (i) to secondary host plants occurs with very low probability. If spreading of said RNA replicon (i) to an undesired plant occurs in a rare event, it cannot move systemically in such an undesired plant due to the absence of said helper replicon and poses therefore a negligible environmental risk. Thus, the system and process of the invention are of excellent biological/environmental safety. At the same time, the system and process of the invention maintain the important feature of viral expression systems that infection of a part of a plant is sufficient to achieve replication or replication and expression of a sequence of interest in other parts of the plant, preferably in the whole plant.
The advantageous features of the invention may be summarized as follows:
Further modifications of the RNA replicon (i) and of the helper replicon (ii) are described herein that minimize the risk of wild type virus reconstruction due to recombination between said RNA replicon (i) and said helper replicon (ii). Further, the invention has no detectable limit of the size of the sequence of interest to be expressed, it allows expressing multiple genes in the same cell and plant and it possesses high ecological and biological safety parameters.
The system and process of the invention can be used for replicating or for replicating and expressing a sequence of interest. Replicating refers to RNA production, namely amplification of said sequence of interest together with said RNA replicon (i). Expressing refers to the production of a protein of interest encoded in said sequence of interest. Preferably, the system and process of the invention is used for producing a protein of interest from a sequence of interest present in said RNA replicon (i).
The system of the invention may comprise said RNA replicon (i) and said helper replicon (ii). Preferably, said system comprises a DNA precursor of said RNA replicon (i) and a DNA precursor of said helper replicon (ii). More preferably, said DNA precursors are contained in T-DNA of Agrobacterial Ti plasmids. Most preferably, said system of the invention is a mixture of two Agrobacterium strains, one strain containing in T-DNA said DNA precursor of said RNA replicon (i), the other strain containing in T-DNA said DNA precursor of said helper replicon (ii). The system of the invention may be a kit for producing a protein of interest, said kit containing any of the replicon pairs, precursor pairs, or mixture of two Agrobacterium strains mentioned in this paragraph. Said kit may further contain a plant or seeds of a plant in which said sequence of interest is to be expressed. Further, the system of the invention may be a plant treated, infected or transformed with any of the replicon pairs, precursor pairs, or mixture of two Agrobacterium strains.
A first component of the system (or kit) of the invention comprises said RNA replicon (i). Said RNA replicon (i) is typically derived from a plus-sense single stranded RNA virus. Examples of such viruses are cowpea mosaic virus, potato virus X, and alfalfa mosaic virus. Preferred viruses are tobamoviruses, the most preferred ones being tobacco mosaic virus (TMV) and crucifer-infecting tobamovirus. Being derived from a plus-sense single stranded RNA virus means that said RNA replicon (i) is typically created using such a virus as a starting material. Alternatively, said RNA replicon (i) may be created using genetic functions (e.g. replicase, movement protein) from such a virus. Said RNA replicon (i) can also be created using components or genetic functions from different plus-sense single stranded RNA viruses. Said precursor of said RNA replicon (i) may be a DNA precursor encoding said RNA replicon (i), and said DNA precursor is capable of producing said RNA replicon (i) in cells of said plant. As further explained below, said DNA precursor of said RNA replicon (i) may contain one or more introns; or may have been modified relative to the virus it is derived from by changing the codon usage e.g. for removing splicing sites splicing at which would destroy the replicon capabilities of the RNA replicon.
For being a replicon, said RNA replicon (i) has to be capable of replicating autonomously in a plant cell. Autonomous replication means that the replicon codes for a replicase (RNA-dependent RNA polymerase) catalyzing replication of the replicon. A replicon may make use of functions of the host cell like the translation machinery needed for translating said replicase. Said replicase may be provided with one or several introns, notably if said RNA replicon (i) is provided as a DNA precursor to plant cell nuclei, for increasing the efficiency of RNA replicon build-up in the cytoplasm (cf. PCT/EP03/12530 and PCT/EP04/012743 that are incorporated herein by reference).
Further, said RNA replicon (i) contains said sequence of interest to be replicated or expressed. Preferably, said sequence of interest is expressed in the process of the invention to produce a protein of interest. Said sequence of interest is preferably heterologous to said plus-sense single-stranded RNA virus(es) from which said RNA replicon (i) is derived. Said RNA replicon (i) generally contains further genetic functions needed for expressing or replicating said sequence of interest like one or more subgenomic promoters, ribosome binding sites etc.
Said RNA replicon (i) is unable to move systemically in said plant in the absence of said one or more proteins expressed by said helper replicon (ii). This property can be achieved by modifying the nucleotide sequence encoding the protein necessary for systemic movement of said RNA virus from which said RNA replicon (i) is derived such that this protein cannot be expressed in a functional form from said RNA replicon (i). Expression in a functional form may be prevented by mutating or deleting parts of the sequence coding for said protein, or by mutating or deleting regulatory sequences (e.g. a subgenomic promoter) required for expressing said protein. In a preferred embodiment, said protein (or said proteins if said RNA virus contains more than one protein needed for systemic movement) is largely or totally deleted. Such a deletion adds several additional advantages to the system and process of the invention: Larger sequences of interest can be included in said RNA replicon (i) without compromising the efficiency of said RNA replicon (i), whereby said sequence of interest may be larger than 1 kb. Further, the homology between said RNA replicon (i) and said helper replicon (ii) is reduced. Thereby, recombination events between said replicons (i) and (ii) are unlikely.
In many plant viruses like tobamoviruses, said protein necessary for systemic movement is the coat protein. Thus, said RNA replicon (i) preferably does not contain a coat protein open reading frame (ORF) or lacks substantial parts of a coat protein ORF. Instead, said sequence of interest may take the position of the coat protein ORF. This position is 3′-proximal in many plant viruses, which is generally the position of the viral ORF that is expressed the strongest.
In order to be able to move systemically in said plant in the presence of said helper replicon, said RNA replicon (i) has to have a functional origin of viral particle assembly. In the case of tobamoviruses, the origin of viral particle assembly is located in the movement protein (MP) ORF. It is thus preferred that said RNA replicon (i) contains the sequence segment or ORF that harbors the origin of viral particle assembly in the RNA virus from which said RNA replicon (i) is derived.
The second component of the system and process of the invention is said helper replicon (ii). Said helper replicon (ii) helps said RNA replicon (i) in that it is capable of expressing in a plant one or more proteins necessary for systemic movement of said RNA replicon (i). The helper replicon (ii) may provide in said plant any protein or proteins that enable(s) said RNA replicon (i) to move systemically in said plant. Preferably, said protein necessary for systemic movement corresponds to that deleted or rendered unexpressible in said RNA replicon (i). Most preferably, it is a coat protein.
Said helper replicon (ii) may be a DNA replicon and may be derived from a DNA virus like gemini virus. Preferably, however, said helper replicon (ii) is an RNA replicon and may be derived from an RNA virus like a plus-sense single stranded RNA virus. Said helper replicon (ii) and said RNA replicon (i) may be derived from the same or from different plant viruses, e.g. from a tobamovirus like tobacco mosaic virus. Similarly as said RNA replicon (i), said helper replicon (ii) codes for a replicase capable of catalyzing replication of said helper replicon in plant cells.
Said helper replicon (ii) is incapable of systemic movement in said plant, independent of whether said RNA replicon (i) is present or absent in said plant. Being incapable of systemic movement may be achieved in various ways. In one embodiment, said helper replicon (ii) and said protein necessary for systemic movement are incompatible such that said protein necessary for systemic movement of said RNA replicon (i) cannot provide said helper replicon with the functionality of systemic movement. In this case, said RNA replicon (i) and said helper replicon (ii) are preferably derived from different plant viruses, and the helper replicon (ii) may be capable of expressing a coat protein that provides said RNA replicon (i) but not said helper replicon (ii) with the systemic movement functionality. Alternatively, said RNA replicon (i) and said helper replicon (ii) are derived from the same plant virus.
In a preferred embodiment said helper replicon (ii) is incapable of systemic movement due to lacking a functional origin of viral particle assembly. Thereby, the helper replicon (ii) cannot be packaged by said protein necessary for systemic movement, notably said coat protein. The origin of viral particle assembly may be rendered dysfunctional. In TMV, the origin of viral particle assembly is located in the movement protein (MP) ORF. The origin of viral particle assembly in the MP ORF may also be deleted. It is not required that the MP ORF of said helper replicon (ii) codes for a functional MP. If a functional MP for said helper replicon (ii) is desired, the MP may for example be provided by said RNA replicon (i); further, the MP may also be encoded by a plant host transgenic for MP. If the origin of viral particle assembly is located in the MP ORF, it is most preferred that said helper replicon lacks the MP ORF. This has the additional advantage that homology between a RNA replicon (i) having an MP ORF and the helper replicon (ii) is reduced, providing improved biologically safety to the system and process, since the probability of formation of wild-type like RNA virus by homologous recombination is negligible.
Whether said helper replicon (ii) is capable or incapable of systemic movement can be tested experimentally (cf. examples) by infecting a portion of a leaf of a plant with said replicon (or a precursor thereof) and observing the occurrence of the same replicon in other, non-infected, leaves (“systemic leaves”) of this plant. Being incapable of systemic movement is a relative property. Said helper replicon (ii) is considered to be incapable of systemic movement if the probability of systemic movement is substantially reduced compared to the virus it is derived from. In any case, the probability of systemic movement of said helper replicon (ii) is considerably lower than that of said RNA replicon (i), such that replication or replication and expression of said sequence of interest from said RNA replicon (i) in systemic leaves is not suppressed in systemic leaves in the typical time-frame of protein expression with plant viral expression systems (about 1 to 3 weeks). Most preferably, no systemic movement of said helper replicon (ii) in said plant is detectable (e.g. by Western or Northern blotting).
The plant used in the process or the system of the invention does preferably not contain a gene coding for a protein enabling systemic movement of said RNA replicon (i) or said helper replicon (ii), said gene being stably integrated into a nuclear chromosome of the plant. Such a situation would compromise biological safety of the process or system.
For improving the environmental safety and the efficiency of the system, said RNA replicon (i) and said helper replicon (ii) should not be prone to recombination with each other. This may be achieved by having a low homology between said replicons (i) and (ii). Preferably, said RNA replicon (i) and said helper replicon (ii) lack homology in functionally overlapping regions or do not overlap. Functionally overlapping regions are regions in said replicons having or coding for the same function, e.g. the replicase ORF, the MP ORF, or subgenomic promoters. Homology in such regions may be reduced e.g. by changing codons using the degeneracy of the genetic code and/or by using functional regions for said replicon (i) and (ii) that derive from different plant viruses.
Many potential recombination events do not change said replicons or lead to unfunctional replicons, depending on the specific replicons. Such recombinations may reduce the efficiency of the system but do not pose an environmental risk. For achieving the best environmental safety, said RNA replicon (i) and said helper replicon (ii) preferably lack a recombination-prone homology in a region, wherein recombination between said RNA replicon (i) and said helper replicon (ii) would create a replicon capable of, at the same time,
The sequence homology between said RNA replicon (i) and said helper replicon (ii) in any sequence segments having at least 100 nucleotides (preferably at least 150 nucleotides) should be at most 90%. Such sequence segments are preferably located downstream of the replicase ORFs of said RNA replicon (i) and said helper replicon (ii). Preferably, the sequence homology between said RNA replicon (i) and said helper replicon (ii) in sequence segments having at least 100 nucleotides (preferably at least 1550 nucleotides) is at most 80%. More preferably, the sequence homology between said RNA replicon (i) and said helper replicon (ii) in sequence segments having at least 100 nucleotides (preferably at least 150 nucleotides) is at most 70%. Most preferably, the sequence homology between said RNA replicon (i) and said helper replicon (ii) in sequence segments having at least 100 nucleotides (preferably at least 150 nucleotides) is at most 60%. In a highly preferred embodiment, these homology values apply to the sequence segment of the subgenomic promoter of the CP in the helper replicon (ii) and the subgenomic promoter of the sequence of interest in the RNA replicon (i).
The system of the invention thus contains at least said components (i) and (ii). The system of the invention may contain said RNA replicon (i) and/or said helper replicon (ii) in the form of a precursor from which said replicons (i) and (ii) are formed in cells of the plant. The precursor of said RNA replicon (i) will generally be DNA coding for said RNA replicon (i) and having a promoter functional in said plant for forming said RNA replicon (i) by transcription of said DNA in cells of said plant. Similarly, if said helper replicon (ii) is an RNA replicon (RNA replicon (ii)), the precursor of said helper-replicon (ii) may be DNA. Said DNA precursors may be flanked by T-DNA left and right border sequences and may be carried by agrobacteria. In a particularly preferred embodiment of the invention, said system of the invention comprises, in an Agrobacterium-carried T-DNA, a DNA precursor of said RNA replicon (i); and, in an Agrobacterium-carried T-DNA, a DNA precursor of said helper replicon (ii). Other precursors of said replicons (i) and (ii) for said system of the invention are DNA for biolistic transformation of said plant or for other transformation methods.
DNA precursors of said replicons typically have a sequence encoding said RNA replicon (i) and/or said helper replicon (ii) operably linked or linkable to a transcription promoter. If a DNA sequence encoding a replicon is operably linked to a transcription promoter, the transcription promoter may be a regulated promoter, like an inducible, tissue-specific or developmentally regulated promoter in order to make expression of said sequence of interest regulatable. More preferably, said promoter is a constitutive promoter. In this case, the process of the invention is switched on by applying said DNA precursors to said plant or parts thereof.
The system of the invention may further contain a plant or seeds thereof for carrying out the process of the invention. In principal, the invention may be carried out with any plant for which infectious viruses are known. Preferred are crop plants including monocot and dicot plants, whereby the latter are preferred. The invention is well-established with Nicotiana plants and may be applied to other plants of the family Solanaceae. Nicotiana tabacum and N. benthamiana are most preferred. These plants have the additional advantage that they typically do not enter the human food chain.
Said plant may be a wild-type plant or a transgenic plant. An MP gene stably and expressibly integrated in the genome of said plant may be used for complementing the MP function of said helper replicon (ii), as described above. A preferred MP for this purpose is the MP of tobacco mosaic virus.
Obviously, said plant used for the process of the invention, said RNA replicon (i), and said helper replicon (ii) have to be appropriately selected for giving a functional system. For example, said replicons (i) and (ii) have to be able to replicate in cells of said plants, the used MPs have to be functional for enabling cell-to-cell movement in said plant, the used coat protein has to able to provide systemic movement to said RNA replicon (i) in said plant, etc. These issues are familiar to people of skill in the art.
In the process of the invention, said RNA replicon (i) and said helper replicon (ii), or precursors thereof, are provided to cells of said plant. Said replicons (i) and (ii) may be provided directly to said plant, which may be as RNA molecules or as packaged viral particles. Alternatively, said RNA replicon (i) or said helper replicon (ii) or both said RNA replicon (i) and said helper replicon (ii) may be provided to said plant as DNA precursors of said RNA replicon (i) and/or said helper replicon (ii). The type of precursor depends on the type of transformation method to be used. Usable transformation methods are given below. A preferred transformation method is Agrobacterium-mediated transformation. In this case, said plant is provided with said RNA replicon (i) and/or said helper replicon (ii) by transfecting with agrobacteria containing in their T-DNA said precursor of said replicon (i) and/or with agrobacteria containing in their T-DNA said precursor of said helper replicon (ii).
When said plant is transformed with said RNA replicon (i) and said helper replicon (ii), or precursors thereof, a selected part of said plant should be treated with both replicons in order to allow complementation of said RNA replicon (i) by said helper replicon (ii). In the case of Agrobacterium-mediated transformation, this is most easily achieved by treating a selected part of said plant with a mixture of two Agrobacterium strains, one strain containing said RNA replicon (i) as a DNA precursor in T-DNA, and another strain containing said helper replicon (ii) as a DNA precursor in T-DNA. Since at least one of said replicons (i) and (ii) will generally be capable of cell-to-cell movement due to an MP, it is not absolutely required that a cell of said plants is transformed with both replicons (i) and (ii). For reasons of efficiency, it is, however, preferred that cells of said plant are transformed with both replicons (i) and (ii).
For making full use of the invention, selected parts of said plant like one or more leaves, preferably lower leaves, should be provided with said RNA replicon (i) and said helper replicon (ii) but not other parts of said plant. Other parts of said plants, notably systemic leaves, will then be reached by said RNA replicon (i) by way of systemic movement. Further, one or more plants may be sprayed with an Agrobacterium suspension containing two Agrobacterium strains (e.g. A. tumefaciens strains), one containing a DNA precursor of said RNA replicon (i) and the other containing a DNA precursor of said helper replicon (ii).
The plants that can be used for the process of the invention correspond to those that may be a component of the system of the invention.
Said replicated or expressed sequence of interest may be harvested from said plant by conventional means. These products may be isolated using the whole plant, i.e. plant material that was provided with said replicons (i) and (ii) and plant material that was not provided with said replicons (i) and (ii). Preferably, these products are harvested and isolated from plant parts that were not provided with said replicons (i) and (ii), like leaves systemically infected by said RNA replicon (i).
A system for replicating or for replicating and expressing a sequence of interest in a plant, comprising:
A process of expressing a sequence of interest in a Nicotiana plant, comprising co-transforming a leaf of said plant with a mixture of the Agrobacteria of the above system.
A process of expressing a interest in a plant, comprising providing cells of said plant with
Act2—promoter of the Arabidopsis ACTIN2 gene; RdRp—viral RNA-dependent RNA polymerase; MP—viral movement protein; NTR—viral 3′ non-translated region; CP—viral coat protein; Tnos—transcription termination region of nopalin synthase.
Lanes:
FIGS. 1 to 5 and 9 to 11 of PCT/EP04/012743 further illustrate the principle and examples of embodiments wherein sequences for replicon function of a DNA precursor of an RNA replicon exhibit at selected localities function-conservative differences from the sequence of the plant RNA virus, causing an increased frequency of replicon formation compared to an RNA replicon not exhibiting said differences.
The present invention provides a process of highly efficient and biologically safe systemic expression of a sequence or protein of interest using an RNA virus-derived replicon (said RNA replicon (i)). This process overcomes limitations of existing RNA viral vector-based expression systems, such as size limitation for heterologous sequences to be expressed systemically and high instability of said vectors. Further, said process offers better biosafety characteristics and prevents the formation of wild type viruses due to recombination of viral components. The replicons (i) and (ii) of the invention can be designed such that such recombinations are avoided. The approach described herein allows for a rapid and highly efficient expression of a sequence of interest in a whole plant including systemic leaves.
To our knowledge, there are no efficient two-component systems relying on the use of (a) vectors that are deficient in systemic movement (lacking functional coat protein) and (b) transgenic plants providing the missing coat protein in trans. Such systems are not practically useful, probably because the levels of coat protein expressed even under a strong constitutive promoter is insufficient to provide for efficient systemic movement of the vector. In addition, creation of transgenic plants is time consuming and should be avoided in those applications where rapid expression of small amounts of protein or RNA of interest is required. Also, in case where a constitutive promoter provides for the expression of a sufficient amount of coat protein to support systemic movement, the biological safety of the system would be low, as the assembly of infectious viral particles would take place in systemic leaves.
Many different RNA viruses belonging to different taxonomic groups are suitable for constructing said RNA replicon (i) and said helper replicon (ii) of this invention, subject to the identification of the viral elements responsible for systemic movement and to the possibility to reconstitute the systemic movement functionality of the RNA replicon (i) by expressing one of such elements in trans from the helper replicon (ii) of the invention. A list of RNA viruses which can be used for the creation of said RNA replicon (i) and said helper replicon (ii) of this invention is presented below. Taxa names in quotes (and not in italic script) indicate that this taxon does not have an ICTV international approved name. Species (vernacular) names are given in regular script. Viruses with no formal assignment to genus or family are indicated:
RNA Viruses:
ssRNA Viruses: Family: Bromoviridae, Genus: Alfamovirus, Type species: alfalfa mosaic virus, Genus: Ilarvirus, Type species: tobacco streak virus, Genus: Bromovirus, Type species: brome mosaic virus, Genus: Cucumovirus, Type species: cucumber mosaic virus;
Family: Closteroviridae, Genus: Closterovirus, Type species: beet yellows virus, Genus: Crinivirus, Type species: Lettuce infectious yellows virus, Family: Comoviridae, Genus: Comovirus, Type species: cowpea mosaic virus, Genus: Fabavirus, Type species: broad bean wilt virus 1, Genus: Nepovirus, Type species: tobacco ringspot virus;
Family: Potyviridae, Genus: Potyvirus, Type species: potato virus Y, Genus: Rymovirus, Type species: ryegrass mosaic virus, Genus: Bymovirus, Type species: barley yellow mosaic virus;
Family: Sequiviridae, Genus: Sequivirus, Type species: parsnip yellow fleck virus, Genus: Waikavirus, Type species: rice tungro spherical virus; Family: Tombusviridae, Genus: Carmovirus, Type species: carnation mottle virus, Genus: Dianthovirus, Type species: carnation ringspot virus, Genus: Machlomovirus, Type species: maize chlorotic mottle virus, Genus: Necrovirus, Type species: tobacco necrosis virus, Genus: Tombusvirus, Type species: tomato bushy stunt virus, Unassigned Genera of ssRNA viruses, Genus: Capillovirus, Type species: apple stem grooving virus;
Genus: Carlavirus, Type species: carnation latent virus; Genus: Enamovirus, Type species: pea enation mosaic virus,
Genus: Furovirus, Type species: soil-borne wheat mosaic virus, Genus: Hordeivirus, Type species: barley stripe mosaic virus, Genus: Idaeovirus, Type species: raspberry bushy dwarf virus;
Genus: Luteovirus, Type species: barley yellow dwarf virus; Genus: Marafivirus, Type species: maize rayado fino virus; Genus: Potexvirus, Type species: potato virus X; Genus: Sobemovirus, Type species: Southern bean mosaic virus, Genus: Tenuivirus, Type species: rice stripe virus,
Genus: Tobamovirus, Type species: tobacco mosaic virus,
Genus: Tobravirus, Type species: tobacco rattle virus,
Genus: Trichovirus, Type species: apple chlorotic leaf spot virus; Genus: Tymovirus, Type species: turnip yellow mosaic virus; Genus: Umbravirus, Type species: carrot mottle virus;
In addition to TMV-based expression systems, viral vectors for expressing heterologous genes of interest were developed on the basis of several other plus sense ssRNA viruses, such as potato virus X (Mallory et al., 2002, Nat. Biotechnol., 20, 622-625), alfalfa mosaic virus (Sanches-Navarro et al., 2001, Arbh. Virol., 146, 923-939), and cowpea mosaic virus (Gopinas et al., 2000, 267, 159-173). The strategy described in this invention for TMV-based vectors can also be employed to the viral expression systems mentioned above.
The construction of different types of TMV-based viral vectors used in this invention (
In order to address this problem, the helper viral vectors (helper replicon (ii)) pICH16601 and pICH16684 (
A minor amount of CP is also produced in systemic leaves by the two-component system of the invention (
The reduction or complete elimination of homologous functional regions being targets for homologous recombination can be achieved by several different approaches: deletions of said regions; changes of coding regions within the regions of homology by applying different codon usage; changing said regions by directed evolution (for review of approach see Tobin et al., 2000, Curr. Opin. Struct Biol., 10, 421-427). In case of TMV-based viral vector systems, one can use different RNA-dependent RNA polymerases (RdRp) for the RNA replicon (i) that expresses said sequence of interest and the helper replicon (ii). Well characterised RdRps for this purpose are e.g. the RdRps of TVCV, TMV-U1, TMV-U5, or crTMV.
A homology within RdRp coding regions does, however, not cause a serious recombination problem, since recombination between regions upstream of the origin of viral particle assembly (in replicon (i)) does not result in wild-type virus or virus having the system movement capability of a wild-type virus. More problematic is a short region of homology located at the 3′ end of the MP gene in front of GFP in case of the RNA replicon (i) (see
As was mentioned in the general description, in order to completely remove the homology region that can lead to the formation of autonomous viral vector capable of systemic movement, a subgenomic promoters can be used in order to drive CP expression from the helper replicon that is heterologous to the RNA virus from which said RNA replicon (i) is derived. More specifically, the subgenomic promoter used to drive CP expression from the helper replicon should be heterologous to the subgenomic promoter(s) used in the RNA replicon (i) to drive expression of MP and/or the sequence of interest. In the case of a TMV-based system, CP subgenomic promoters from different virus strains can be used in the helper replicon. Examples of such strains include TMV-U1, TMV-U5, crTMV, etc.
Interestingly, additional co-expression of MP in trans in infiltrated leaves increases the efficiency of systemic vector in our system. As it is shown in Example 7, transient expression of TVCV MP under control of a constitutive promoter significantly improves the efficiency of systemic movement of GFP-expressing replicon. This can be explained by production of larger quantities of CP by the helper replicon capable of cell-to-cell movement via said trans-complementation. Said additional CP might provide for packaging of larger number of RNA replicon (i) into viral particles capable of systemic movement.
Different methods may be used for providing cells of a plant with said RNA replicon (i) and/or said helper replicon (ii). Said vectors may be transformed into plant cells by a Ti-plasmid vector carried by Agrobacterium (U.S. Pat. Nos. 5,591,616; 4,940,838; 5,464,763) or particle or microprojectile bombardment (U.S. Pat. No. 05,100,792; EP 00444882B1; EP 00434616B1). Other plant transformation methods can also be used like microinjection (WO 09209696; WO 09400583A1; EP 175966B1), electroporation (EP00564595B1; EP00290395B1; WO 08706614A1) or PEG-mediated transformation of protoplasts etc. The choice of the method for vector delivery depends on the plant species to be transformed and the vector used. For example, microprojectile bombardment is generally preferred for vector delivery in monocot, while for dicots, Agrobacterium-mediated transformation gives better results in general.
In the examples described below, we used Agrobacterium-mediated delivery of vectors into Nicotiana cells. However, the vectors may be introduced into the plants in accordance with any of the standard techniques suitable for stable or transient transformation of the plant species of interest. Transformation techniques for dicotyledonous are well known in the art and include Agrobacterium-based techniques and techniques which do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. These techniques include PEG or electroporation mediated uptake, particle bombardment-mediated delivery and microinjection. Examples of these techniques are described in Paszkowski et al., EMBO J. 3, 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199, 169-177 (1985), Reich et al., Biotechnology 4:1001-1004 (1986), and Klein et al., Nature 327, 70-73 (1987). In each case, the transformed cells are regenerated to whole plants using standard techniques.
Agrobacterium-mediated transformation is a preferred technique for the transformation of dicotyledons because of its high transformation efficiency and its broad utility with many different plant species. The many crop species which may be routinely transformed by Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato), WO 87/07299 (Brassica), U.S. Pat. No. 4,795,855 (poplar)).
In the examples of this invention, we used agro-inoculation, a method of Agrobacterium-mediated delivery of T-DNA for transient expression of gene(s) of interest (Vaquero et al., 1999, Proc. Natl. Acad. Sci. USA, 96, 11128-11133). Agro-inoculation is an extremely useful tool not only for small-to-middle scale recombinant protein production systems, but as one of the elements of a vector optimisation system allowing to obtain fast results with different variants of constructs.
This invention is not limited to TMV-based vectors described in examples 1-5, but can be extended to replicons based on other plant RNA viruses.
Sequences or genes of interest, their fragments (functional or non-functional) and their artificial derivatives that can be expressed in plants or plants cells using the present invention include, but are not limited to: starch modifying enzymes (starch synthase, starch phosphorylation enzyme, debranching enzyme, starch branching enzyme, starch branching enzyme II, granule bound starch synthase), sucrose phosphate synthase, sucrose phosphorylase, polygalacturonase, polyfructan sucrase, ADP glucose pyrophosphorylase, cyclodextrin glycosyltransferase, fructosyl transferase, glycogen synthase, pectin esterase, aprotinin, avidcin, bacterial levansucrase, E. coli gIgA protein, MAPK4 and orthologues, nitrogen assimilation/methabolism enzyme, glutamine synthase, plant osmotin, 2S albumin, thaumatin, site-specific recombinase/integrase (FLP, Cre, R recombinase, Int, SSVI Integrase R, Integrase phiC31, or an active fragment or variant thereof), oil modifying enzymes (like fatty acids desaturases, elongases etc), isopentenyl transferase, Sca M5 (soybean calmodulin), coleopteran type toxin or an insecticidally active fragment, ubiquitin conjugating enzyme (E2) fusion proteins, enzymes that metabolise lipids, amino acids, sugars, nucleic acids and polysaccharides, superoxide dismutase, inactive proenzyme form of a protease, plant protein toxins, traits altering fiber in fiber producing plants, Coleopteran active toxin from Bacillus thuringiensis (Bt2 toxin, insecticidal crystal protein (ICP), CryIC toxin, delta endotoxin, polypeptide toxin, protoxin etc.), insect specific toxin AalT, cellulose degrading enzymes, E1 cellulase from Acidothermus celluloticus, lignin modifying enzymes, cinnamoyl alcohol dehydrogenase, trehalose-6-phosphate synthase, enzymes of cytokinin metabolic pathway, HMG-CoA reductase, E. coli inorganic pyrophosphatase, seed storage protein, Erwinia herbicola lycopen synthase, ACC oxidase, pTOM36 encoded protein, phytase, ketohydrolase, acetoacetyl CoA reductase, PHB (polyhydroxybutanoate) synthase, enzymes involved in the synthesis of polyhydroxyalkanoates (PHA), acyl carrier protein, napin, EA9, non-higher plant phytoene synthase, pTOM5 encoded protein, ETR (ethylene receptor), plastidic pyruvate phosphate dikinase, nematode-inducible transmembrane pore protein, trait enhancing photosynthetic or plastid function of the plant cell, stilbene synthase, an enzyme capable of hydroxylating phenols, catechol dioxygenase, catechol 2,3-dioxygenase, chloromuconate cycloisomerase, anthranilate synthase, Brassica AGL15 protein, fructose 1,6-biphosphatase (FBPase), AMV RNA3, PVY replicase, PLRV replicase, potyvirus coat protein, CMV coat protein, TMV coat protein, luteovirus replicase, MDMV messenger RNA, mutant geminiviral replicase, Umbellularia californica C12:0 preferring acyl-ACP thioesterase, plant C10 or C12:0 preferring acyl-ACP thioesterase, C14:0 preferring acyl-ACP thioesterase (luxD), plant synthase factor A, plant synthase factor B, D6-desaturase, proteins having an enzymatic activity in fatty acids biosynthesis and modifications, e.g. the peroxysomal β-oxidation of fatty acids in plant cells, acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, lipase, maize acetyl-CoA-carboxylase, etc.; 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), phosphinothricin acetyl transferase (BAR, PAT), CP4 protein, ACC deaminase, protein having posttranslational cleavage site, DHPS gene conferring sulfonamide resistance, bacterial nitrilase, 2,4-D monooxygenase, acetolactate synthase or acetohydroxyacid synthase (ALS, AHAS), polygalacturonase, Taq polymerase, bacterial nitrilase, many other enzymes of bacterial or phage origin including restriction endonucleases, methylases, DNA and RNA ligases, DNA and RNA polymerases, reverse trascryptases, nucleases (Dnases and RNAses), phosphatases, transferases etc.
The present invention can be used for the purpose of molecular farming and purification of commercially valuable and pharmaceutically important proteins including industrial enzymes (cellulases, lipases, proteases, phytases etc.) and fibrous proteins (collagen, spider silk protein, etc.). Human or animal health protein may be expressed and purified using described in our invention approach. Examples of such proteins of interest include inter alia immune response proteins (monoclonal antibodies, single chain antibodies, T cell receptors etc.), antigens including those derived from pathogenic microorganisms, colony stimulating factors, relaxins, polypeptide hormones including somatotropin (HGH) and proinsulin, cytokines and their receptors, interferons, growth factors and coagulation factors, enzymatically active lysosomal enzyme, fibrinolytic polypeptides, blood clotting factors, trypsinogen, a1-antitrypsin (AAT), human serum albumin, glucocerebrosidases, native cholera toxin B as well as function-conservative proteins like fusions, mutant versions and synthetic derivatives of the above proteins.
Sequences for Replicon Function that Exhibit Function-Conservative Differences from the Sequence of the RNA Virus, Causing an Increased Frequency of Replicon Formation
In this embodiment, said DNA precursor encoding said RNA replicon (i) contains sequences for replicon function of said RNA replicon (i), said sequences being derived from a sequence of said RNA virus, said sequences for replicon function exhibit at selected localities of said sequence of said RNA virus function-conservative differences from said sequence of said RNA virus, said differences causing an increased frequency of RNA (i) replicon formation compared to an RNA replicon not exhibiting said differences. Alternatively or additionally, said helper replicon may be derived from a plus-sense single stranded RNA virus and said DNA precursor encoding said helper replicon (ii) contains sequences for replicon function of said helper replicon (ii), said sequences being derived from a sequence of said RNA virus, said sequences for replicon function exhibit at selected localities of said sequence of said RNA virus function-conservative differences from said sequence of said RNA virus, said differences causing an increased frequency of helper replicon formation compared to a helper replicon not exhibiting said differences.
Said function-conservative differences are preferably present in the replicase ORFs of said DNA precursors. In cases where said RNA replicon (i) and said helper replicon (ii) are based on the same RNA virus, the replicase ORFs including said function-conservative differences in the RNA replicon (i) and the helper replicon (ii) may be identical. Further, function-conservative differences may be present in an MP ORF, notably of the DNA precursor of said RNA replicon (i).
Said function-conservative differences are causal for said increased frequency of RNA replicon (i) and/or helper replicon (ii) formation in plant cells. The causal connection between the increased frequency of replicon formation and said differences can be tested experimentally by comparing the frequency of replicon formation between sequences for replicon function having said differences and sequences for replicon function not having said differences. Such an experimental comparison can be made e.g. by counting protoplasts expressing said sequence of interest as described in the examples. Preferably, a sequence of interest coding for an easily detectable reporter protein like green fluorescent protein is used for this purpose. As further described below, it is also preferred to perform the experimental comparison with RNA replicons not capable of cell-to-cell spreading.
Said function-conservative differences are introduced into said sequences for replicon function at selected localities of said sequence of said RNA virus. Said selected localities are localities in sequences for replicon function of said RNA virus that are responsible for a low probability of an RNA replicon transcribed in the nucleus to appear in the cytosol as a functional replicon. Preferably, such selected localities have a high A/T(U)-content, i.e. a high A-content and/or a high T-content (a high U-content on RNA level), or have cryptic splicing sites, i.e. sequence portions that can be recognized by the nuclear splicing machinery as splicing sites. Said selected localities may be identified in an RNA virus on which an RNA replicon is based by analyzing the RNA profile of the RNA virus as exemplified below. Further, selected localities may be identified experimentally by analyzing the RNA formed in a plant cell after transformation with a heterologous DNA encoding an RNA replicon that does not exhibit said (function-conservative) differences according to the invention. This experimental analysis may be done by RT-PCR, preferably together with sequencing of the RT-PCR products. In the RT-PCR test, the replicase is preferably rendered dysfunctional e.g. by a frame-shift mutation in order to prevent RNA replicons reaching the cytoplasm from amplifying; such amplification may lead to contamination of RNA transcripts with wild type virus or to an overrepresentation of amplified RNA replicons in the cytoplasm. In this way, undesired splicing products that indicate splicing events destroying the RNA replicon may be identified. Further, the exact sites of undesired splicing may be identified and then remedied by introducing said function-conservative differences at said selected localities.
Thus, the invention also provides a process of expressing a sequence of interest in a plant, plant part, or plant cell culture, wherein (A) a plant, plant part, or plant cell culture is provided with a DNA precursor lacking said function-conservative differences, (B) testing RNA derived from said DNA precursor for undesired splicing products in said sequences for replicon function (e.g. by RT-PCR), (C) identifying (e.g. in the sequence of a product of said RT-PCR), a selected locality as a locality of an undesired splicing event, (D) introducing a function-conservative difference (e.g. an intron) according to the invention into or near said selected locality identified in step (C) into a DNA precursor of step (A) for producing said DNA precursor encoding said replicon (i) or (ii) of the invention, and expressing a sequence of interest in a plant, plant part, or plant cell culture according to the invention, e.g. from a plant transiently transformed with said heterologous DNA of the invention.
Said function-conservative differences cause an increased frequency of replicon formation by suppressing the deleterious effect of said selected localities on said frequency of RNA replicon formation. Said function-conservative differences may comprise a reduction of a high A/U-content in said RNA replicon by reducing a high A/T content in said sequences for replicon function of said sequence encoding said RNA replicon. Said high A/U content may be reduced by at least partial deletion or at least partial replacement by G/C bases (e.g. using the degeneracy of the genetic code), provided said differences are function-conservative. Further, cryptic splicing sites flanking A/U-rich regions of said sequences derived from a plant RNA virus may be removed. Such function-conserved differences may be introduced at one or at, preferably, several selected localities.
Preferred function-conservative differences comprise the insertion of one or more introns, most preferably nuclear introns, or one or more sequences capable of forming nuclear introns near or within A/U-rich localities of said sequences being derived from sequences of said plant RNA virus. It has surprisingly been found that the introduction of introns at or near A/U-rich localities results in an increased frequency of RNA replicon formation. Several introns may be introduced and examples are given herein for various numbers of introduced introns. The effects of more than one intron are cumulative. Further, intron insertion may be combined with other function-conservative differences at other selected localities.
The plant or the plant part (e.g. leaves) are preferably transiently transformed with said DNA precursors of the invention for transient expression of said sequence of interest. The term “transient transformation” means the introduction of said DNA precursors without selection of transformed cells for stable incorporation of said heterologous DNA into a plant chromosome. Transient transformation usually provides for transient expression of the sequence(s) encoded by the DNA precursor(s). Transient transformation can be achieved by any of the transformation methods given above. Agroinfiltration is preferred, e.g. by dipping the plant upside down in the Agrobacterium suspension, application of vacuum, and fast release of the vacuum.
The DNA precursors encoding an RNA replicon is preferably operably linked to a transcriptional promoter, preferably a constitutive transcriptional promoter. In another embodiment, said plant belongs to the genus Nicotiana and said sequences for replicon function are derived from a tobamovirus, preferably from tobacco mosaic virus. In a particularly preferred embodiment, tobacco plants including the stem and all leaves are transiently transformed by agroinfiltration. The latter embodiment can be used for large-scale applications of the process of the invention. In large-scale applications, said process is concomitantly applied to many plants (at least 5, preferably at least 10, more preferably at least 100 plants).
It is known that plant RNA viruses (an exception are viroids—small non-coding RNAs amplifying in plant cell nuclei—for a review see Diener, T. O., 1999, Arch. Virol. Suppl., 15, 203-220; Flores, R., 2001, CR Acad. Sci. III, 324, 943-952) never occur in the plant nucleus, but in the cytoplasm. Therefore, the sequences of RNA viruses are not adapted to withstand nuclear RNA processing events due to the presence of motifs that might be involved in complex series of processing steps including transport of processed RNA in cytoplasm, in which pre-mRNAs, rRNA and tRNA precursors are involved. The processing events, such as 5′ end capping, splicing, 3′ end generation, polyadenylation, degradation, base and sugar modification as well as editing (in plastids and mitochondria) are intensively studied. However, many elements of such events still remain unclear. The most dramatic changes to pre-mRNA in the nucleus happen during pre-mRNA splicing, the process by which intervening RNA sequences (introns) are removed from the initial transcript and exons are concomitantly ligated. Splicing is mediated by the splicesome, a complex structure comprising uridilate-rich small nuclear ribonucleoprotein particles. The splicesome carries out the splicing reaction in two consecutive steps: the first one—cleavage at the 5′ splice site of upstream exon/intron junction leading to lariat formation, and second step—cleavage at the 3′ splice site of intron/downstream exon junction followed by upstream and downstream exons ligation (for review see: Kramer, A., 1996, Annu. Rew. Biochem., 65, 367-409; Simpson, G G. & Filipowicz, W. 1996, Plant. Mol. Biol., 32, 1-41). The 5′ and 3′ splice site dinucleotides (5′/GU; AG/3′) flanking the intron sequences are highly conserved in higher plants and single G replacement might abandon the splicing activity at the site concerned. It is surprising that despite of a high conservation of splice sites between plants and animals, heterologous introns in plants are usually not spliced or spliced incorrectly (van Santen, V L. et al., 1987, Gene, 56, 253-265; Wiebauer, K., Herrero, J. J., Filipowicz, W. 1988, Mol. Cel. Biol., 8, 2042-2051). Considering that plant viral RNAs were not under evolutionary pressure to resist nuclear RNA processing machinery, these RNAs are very likely to become subject of such processing, including splicing, once they are placed into the nuclear environment. We address these problems by subjecting the expression vector to function-conservative modifications that significantly increase the frequency of functional RNA replicon formation, when the expression vector is introduced as a DNA precursor into plants or plant cells to provide for transient expression. We believe that such modifications of virus-derived sequences are the most profound solution for increasing the efficiency of RNA virus-based replicons. In this invention we predominantly focus on modifications (said function-conservative differences) within the plant RNA virus derived sequences, as they are crucial for increasing the efficiency of RNA replicon formation.
By introducing said function-conservative differences (e.g. introns), we have unexpectedly found an improvement of orders of magnitude. An analysis of the sequence derived from the RNA virus of expression vector pICH8543 (Reference Example 1,
Considering that all existing programs are not ideal and are subject to mistakes, the potential problematic regions can also be determined experimentally. This can be done by analyzing the transcripts derived from a DNA vector under test in a nuclear environment with the help of such a routine technique as RT-PCR (Frohman, M A., 1989, Methods Enzymol., 218, 340-356) or its more advanced version suitable for precise quantification of the concentration of different transcripts called real-time PCR (Gibson et al., 1996, Genome Res., 6, 995-1001), preferably followed by sequencing of the PCR-amplified products. The function-conservative differences of the invention change dramatically the RNA profile, for example by replacing intron-like sequences with exon-like ones, e.g. by introducing silent mutations with replacement of A/U-rich regions (intron-like) with G/C-rich regions (exon-like) (see FIG. 3 of PCT/EP04/012743, circled regions). Plant introns, unlike exons, are usually A/T(U) rich (Lorkovic, Z J. et al., 2000, Trends Plant Sci., 5, 160-167; Brown, J W. & Simpson, C G. 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol., 49, 77-95; Csank, C. et al., 1990, Nucl. Acid Res., 18, 5133-5141; Goodall & Filipowicz, 1989, Cell, 58, 473-483), but there are exceptions, for example when in monocotyledonous plants G/C rich introns were found (Goodall & Filipowicz, 1989, Cell, 58, 473-483; Goodall & Filipowicz, 1991, EMBO J., 10, 2635-2644). For practicing this invention, selected localities of high A/T(U) content include not only sequence stretches of at least 20 nucleotides in length with at least 55%, preferably at least 65%, most preferably 80% or a higher of A/T(U) content, but also shorter stretches (“islands”) of 6-19 nucleotides in a row of purely A/T(U)-containing sequences. Herein, localities of high A/U content include sequences which are more A- than U-rich, sequences which are A-rich, sequences which are more U- than A-rich, and sequences which are U-rich. Additionally, any transcribed sequence of interest can be tested for post-transcriptional modifications that cause a change in nucleic acids sequences (e.g. RNA splicing) by RT-PCR (Frohman, M A. 1989, Methods Enzymol., 218, 340-356). It is a trivial task for those familiar with the art to use RT-PCR for detecting the regions within RNA that are subject to post-transcriptional modifications like deletions of sequences from the original RNA transcript. In Reference Example 2 we demonstrate that the modification of A/U rich region increases the number of GFP expressing cells at least 10-fold. This is clearly demonstrated in
Such a high efficiency of replicon formation opens the door for expressing two or more different genes from two different RNA replicons (like sid RNA replicon (i) and said helper replicon (ii)) within the same plant cell, e.g. co-expressing different genes by using two or more plant RNA virus based vectors (Example 4 and 5). The achievement of synchronized release of two or more replicons concomitantly in the same cell is crucial for such co-expression, as the principle “first come, first served” is especially true for viral vectors. Systemic or cell-to-cell movement does not help, as different viral vectors do normally not overlap in their areas of spread or such overlap is insignificant. Simple calculations demonstrate the importance of the technology for achieving co-expression of two sequences of interest in the same plant cell from two replicons. In the case of a non-optimised viral vector with a frequency of functional replicon formation of only 0.2% of all cells, the proportion of cells co-expressing two genes from two different RNA replicons will be 0.2×0.2=0.04%, while for the construct with increased frequency of functional RNA replicon formation (50% or ½ of all cells), said proportion of cells will be 0.5×0.5=0.25 or 25%, e.g. about 625-fold higher. With some of the best performing vectors (e.g. pICH16191,
It is worth to note that function-conservative differences in heterologous sequences of interest to be expressed from said RNA replicon might also be used to increase the frequency of RNA replicon formation, notably in combination with differences in sequences for replicon function. For example, modifications within said sequences of interest can be introduced that are necessary for formation and/or processing of said replicon.
In an important embodiment of this invention, the frequency of replicon formation is improved by inserting nuclear introns in said sequences for replicon function (Reference Example 4). The incorporation of introns into the coding region of viral RNA-dependent RNA polymerase (RdRP) (Reference Examples 4 and 8) results in a significant (at least 50-fold) increase in the frequency of replicon formation from (FIG. 7A,B) vectors carrying function-conservative differences as defined herein (pICH15034, pICH15025, pICH15499 in
Many nuclear introns can be used to practice this invention. Examples of such introns include introns from rice tpi Act1, and salT genes (Rethmeier et al., 1997, Plant J., 12, 895-899; Xu et al., 1994, Plant Physiol., 100, 459-467; McElroy et al., 1990, Plant Cell, 2, 163-171); from the maize Adh1, GapA1, actin and Bz1 genes (Callis et al., 1987, Genes Dev., 1, 1183-11200; Donath et al., 1995, Plant Mol. Biol, 28, 667-676; Maas et al., 1991, Plant Mol. Biol., 16, 199-207; Sinibaldi & Mettler, 1992, in W E Cohn, K Moldave, eds, Progress in Nucleic Acids Research and Molecular Biology, vol 42, Academic Press, New York, pp 229-257), from petunia rubisco gene SSU301 (Dean et al., 1989, Plant Cell, 1, 201-208), Arabidopsis A1 EF1α, UBQ10, UBQ3, PAT1 genes (Curie et al., 1993, Mol. Gen. Genet. 228, 428-436; Norris et al., 1993, Plant Mol. Biol., 21, 895-906; Rose & Last, 1997, Plant J., 11, 455-464) and many others. Synthetic introns can also be used for this invention. The smallest usable introns or their parts may be limited to splice donor and acceptor sites which usually flank the internal intron sequences. Preferably, the introns should have a size of at least 50 nt., more preferably a size of 100 to 200 nt., but actually there are no limitations regarding the size of the introns. However, the size of the construct should be kept suitable for manipulations. The origin of the intron, its structure and size may be selected individually depending on the nature of the vector. Transient expression experiments may be used for testing the efficiency of a chosen intron or the corresponding intron parts.
The modifications described above have a cumulative effect, e.g. if intron insertion(s) are combined with a modification of the MP subgenomic promoter, the increase in frequency of replicon formation can be approx. 300-fold (Reference Example 5). The preferred regions for intron insertions in order to have an increase in the frequency of RNA replicon formation are called selected localities herein. Such localities may contain “intron-like” structures. This is confirmed by the insertion of introns in MP, actually in close proximity to such a problematic region as the MP subgenomic promoter (Reference Example 7). A 100-fold increase in frequency of replicon formation was observed. Insertion of introns into “exon-like” regions does not have such a pronounced effect as insertion in said intron-like regions (Reference Example 6).
The content of patent application European patent application No. 04001460.7, filed on Jan. 23, 2004, the priority of which is claimed by the present patent application and the content of International patent application PCT/EP03/012743 are incorporated herein by reference.
Information on the genetics of tobamoviruses like TMV and crucifer-infecting tobamovirus can be found in WO02/029068.
A cr-TMV-based viral vector containing GFP, pICH8543 (
Construction of Vector pICH8543
A replicon containing a green fluorescence protein (GFP) gene was made in several cloning steps. The resulting construct, pICH8543, contains in sequential order: a 787 bp fragment from the Arabidopsis actin 2 promoter (ACT2, ref An et al, 1996, GenBank accession AB026654, bp 57962 to 58748), the 5′ end of TVCV (GenBank accession BRU03387, bp 1 to 5455), a fragment of cr-TMV (GenBank accession Z29370, bp 5457 to 5677, with thymine 5606 changed to cytosine to remove the start codon of the coat protein, CP), sequences “taa tcg ata act cga g”, a synthetic GFP (sGFP) gene, cr-TMV 3′ nontranslated region (3′ NTR; GenBank accession Z29370, bp 6078 to 6312), and finally the nopaline synthase (Nos) terminator. The entire fragment was cloned between the T-DNA left (LB) and right (RB) borders of pICBV10, a CarbR pBIN19-derived binary vector.
Cloned cDNAs of the crucifer-infecting tobamovirus (cr-TMV; Dorokhov et al., 1994, FEBS Lett. 350, 5-8) and of the turnip vein-clearing virus (TVCV; Lartey et al., 1994, Arch. Virol. 138, 287-298) were obtained from Prof. Atabekov from Moscow University, Russia. A viral vector expressing TVCV CP was made by subcloning an EcoRI-ApaI fragment (containing part of MP, the complete CP coding sequence, and the 3′ non translated region of TVCV) from the TVCV cDNA into pICH8543. The resulting clone, pICH10595 (
To test whether CP produced by pICH10595 could package in trans the RNA replicon of pICH8543, both clones were co-infiltrated into a Nicotiana benthamiana leaf. Five days after infiltration, GFP could be detected in the inoculated leaf area. However, no GFP could be detected in upper leaves, even three weeks after infiltration. At this time, the inoculated plant had typical symptoms of a plant inoculated with wild-type virus: it was shorter than a control uninoculated plant, and upper leaves were wrinkled and yellowish. PAGE and coomassie staining of the gel showed that CP was expressed in the systemic leaf, but no GFP protein could be detected. Western blot analysis also failed to detect GFP in the systemic leaves, although GFP was detected in the inoculated leaf. In conclusion, a wildtype virus is unable to move a viral amplicon expressing a gene of interest (such as GFP) in trans.
In the previous example, only the wild type virus is detected in the upper uninoculated leaves when the lower leaves are first infiltrated with a mixture of CP and GFP-expressing clones. The CP clone is so efficient to replicate and move that the GFP-expressing clone cannot compete effectively and move systemically. To prevent the CP-expressing clone to move systemically, we removed the area that putatively contains the origin of assembly (OAS) of the virus (the CP-expressing clone), which corresponds to part of the MP located upstream of the CP subgenomic promoter. The resulting clone, pICH16601 (
To improve the efficiency of coexpression of the CP-expressing clone and of the clone of interest in the infiltrated area, introns were inserted in the RdRp and the MP of the construct containing the heterologous sequence of interest. pICH8543 was modified by addition of ten Arabidopsis introns in the RdRp (introns 1 to 10, sequence in the annex) at positions 1844, 2228, 2588, 2944, 3143, 3381, 3672, 3850, 4299, 4497 (coordinates relative to GenBank accession BRU03387) and of two introns in the MP (introns 11-12) at positions 5287 and 5444. The resulting clone, pICH17272 (
To improve expression of CP in a larger number of cells in the infiltrated area, introns 1 to 9 (described above) were added to pICH16601, at the same positions as described for the construction of pICH17272. In addition, the MP subgenomic promoter area was replaced by less T-rich and more GC-rich sequence. As a result, the sequence between bp 4585 to 5460 (coordinates relative to GenBank accession BRU03387) were replaced by Seq 1 given in the annex, resulting in construct pICH16684 (
An additional CP-expressing construct was made by the addition of 6 introns (introns 1 to 3 and 7 to 9, as described for construction of pICH17272) to pICH10595. This construct, pICH17501 (
To compare the approach described herein with systemic movement obtained using traditional vectors which express CP from the same molecule as the gene of interest, we made a control construct, pICH17344, that expresses both GFP and CP (
Some CP is made in systemic leaves of plants inoculated with a mixture of pICH16684 and pICH17272, albeit less than with pICH17344. This most likely occurs as a result of recombination between pICH16684 and pICH17272, producing a wild type CP-expressing virus. Reducing homology between both clones by making a CP-expressing clone based on a related but different virus (for example TMV strain U1) can reduce or eliminate such recombination events.
Clones with a mutated OAS, pICH16601 and pICH16684 (
The following Reference Examples correspond to examples 1 to 11 of PCT/EP04/012743.
Cloned cDNAs of the crucifer-infecting tobamovirus (cr-TMV; Dorokhov et al., 1994, FEBS Lett. 350, 5-8) and of the turnip vein-clearing virus (TVCV; Lartey et al., 1994, Arch. Virol. 138, 287-298) were obtained from Prof. Atabekov front Moscow University, Russia. A viral vector containing a green fluorescence protein (GFP) gene was made in several cloning steps. The resulting construct, pICH8543 (
To confirm this observation, we made a viral vector construct containing a mutation in the MP. This construct, called pICH14833, is similar to pICH8543 but differs by a deletion of 389 bp in the MP gene, upstream of the EcoRI site present in the MP. The sequence of the NcoI to EcoRI fragment that includes this deletion is given in the annex as SEQ ID No. 14. The entire viral construct (from the ACT2 promoter to the Nos terminator) was cloned between the T-DNA left and right borders of pICBV49, a pBIN19-derived KanR binary vector. Due to the deletion in the MP, replicons produced from this construct cannot move from cell to cell but are able to replicate autonomously within a cell. Cell to cell movement can be restored when MP is provided in trans, e.g. from a constitutive promoter such as the cauliflower mosaic virus 35S promoter.
To make an MP expression construct, the TVCV MP gene was amplified by PCR from cloned TVCV cDNA (GenBank accession Z29370, bp 4802 to 5628) and subcloned in a binary vector under control of the 35S promoter. The resulting construct, called pICH10745 (not shown), and pICH14833 were transformed into Agrobacterium strain GV3101 and various dilutions of an overnight culture were infiltrated in Nicotiana benthamiana leaves as described by English and colleagues (1997, Plant J., 12, 597-603), except that the infiltration media lacked acetosyringone. Infiltration of pICH14833 alone led to the appearance of a few GFP expressing cells within the infiltrated area. By counting protoplasts prepared from the infiltrated area, we found that only one to three protoplasts expressed GFP from a total of 500 protoplasts (0.2 to 0.6%). Coinfiltration of pICH14833 and pICH10745 led to the formation of GFP-expressing foci that grew from each initial GFP-expressing cell. Ultimately, due to cell-to-cell movement, a large proportion of cells in the infiltrated area expressed GFP (
RNA viruses such as tobamoviruses replicate in the cytoplasm and never enter the nucleus. Therefore, they have evolved in an environment where they are not exposed to the nuclear pre-mRNA processing machinery. As a result, it is not surprising that RNA replicon transcripts generated in the nucleus from artificial viral constructs may not be recognized and processed properly by the RNA processing machinery. Moreover, RNA replicons from viral vectors are very large: approximately 7,000 nt in the case of the replicon based on TMV. Very few plant genes have such a large size and the majority of such genes contains introns that facilitate processing of the pre-mRNAs, export from the nucleus, and that improve the stability of the processed transcripts. We therefore hypothesized that modifications of the pre-mRNAs that would increase the efficiency of accurate processing and of export of correctly processed transcripts from the nucleus to the cytosol would lead to an increase of the number of cells that would initiate viral replication. It turned out that there are two approaches can be used to make RNA virus-based vectors that can more efficiently initiate viral replication after DNA delivery to the nucleus: (1) one approach is the removal of sequence features that might induce unwanted processing events (such as alternative splicing events using cryptic splice sites, or premature termination events); (2) a second approach is the addition of introns to increase the amount of properly processed transcripts, to improve export of the RNA from the nucleus to the cytoplasm, and/or to improve stability of the transcripts.
We analyzed the sequence of the RNA replicon from pICH4351 using the Netgenell server program (Hebsqaard et al., 1991, J. Mol. Biol., 220,49-65) and noticed several intron-like sequence features that might induce alternative splicing events. One such feature is a 0.6 kb uridine-rich region (corresponding to nt 827 to 1462 in GenBank accession BRU03387) at the beginning of the RdRP (
A second potentially problematic region corresponds to the MP subgenomic promoter (
We tested whether the addition of introns into viral pro-replicon sequences would increase the frequency of initiation of replication. Two constructs were made, pICH15025 and pICH15034 (
Both constructs were separately agroinfiltrated into N. benthamiana leaves and compared to pICH14833. Both constructs significantly increased the number of cells initiating viral replication (
A third clone was made, pICH15499, which contained all 6 introns (6B, 7A, 7B). This construct was tested in N. benthamiana and N. tabacum. This construct was more efficient than each individual construct with 3 introns, but the improvement was however less than additive.
Removing intron-like features and adding additional introns in one construct showed that both types of modifications can contribute to improve initiation of viral replication. We subcloned the 6 introns of pICH15499 into pICH15900, which contains the mutagenized MP subgenomic promoter region. The resulting clone pICH15860 (
We inserted two different Arabidopsis introns at the beginning of the RdRP, resulting in clone pICH15477 (the sequence of this region is shown as SEQ ID No. 19 in the annex). The sequence in this region already looks very “exon-like” (e.g. GC-rich without cryptic splice sites) before the addition of introns. No improvement on replication of viral initiation was seen with this construct. Therefore, not any addition of an intron will result in an improvement of the viral vector. It appears that the position chosen for intron insertion or mutagenesis is an important parameter. For example, all intron insertions or nucleotide substitutions that were made in regions near problematic structures such as the MP subgenomic promoter resulted in large improvements, while insertions of introns into sequences that are already “exon-like” did not.
We first made a frameshift in the MP by digestion with the restriction enzyme AvrII, filling and religation. We then inserted two introns in the MP. The resulting clone pICH16422 (
A Kpn1 EcoRI fragment was subcloned from pICH15499 into pICH8543. The resulting clone, 16700 (
It is also possible to stably transform intron-containing viral vector constructs in transgenic plants. To avoid deleterious viral replication that would inhibit plant growth, an inactive clone (pro-replicon) can be made by having a part of the vector present in antisense orientation (
The flipped part can be located at the 3′ end of the vector (as shown in
The analysis of RNA profile of selected plant RNA viruses as well as one well characterised plant gene (AtDMC1) was performed by using the Netgenell server program (Hebsqaard et al., 1991, J. Mol. Biol., 220,49-65). The RNA profile shown in
A fully optimized construct containing the mutagenized region (described in pICH15466) and 16 introns (including the six introns of pICH15860, the two introns of pICH16422 and eight additional introns) was made. In summary this construct contains introns inserted at the following positions (given relative to TVCV sequence, GenBank accession BRU03387): nt 209, nt 828, nt 1169, nt 1378, nt 1622, nt 1844, nt 2228, nt 2589, nt 2944, nt 3143, nt 3381, nt 3672, nt 3850, nt 4299, nt 5287, nt 5444.
This construct was tested for expression in Beta vulgaris. Infiltration of the entire plant was performed as described next. Agrobacteria carrying pICH18711 were inoculated to 300 ml of LB containing 50 μg/ml Rifampicin and 50 μg/ml Kanamycin (selection for the binary vector) and grown until saturation. The bacteria were pelleted at 4800 g for 10 min and resuspended in 3 l of infiltration buffer (10 mM MES pH 5.5, 10 mM MgSO4) in order get a 10-fold dilution relative to the saturated Agrobacterium culture. A beaker containing the infiltration solution was placed in an exsiccator (30 mm diameter), with the aerial parts of a plant dipped in the solution. A vacuum was applied for two minutes using a Type PM 16763-860.3 pump from KNF Neuberger (Freiburg, Germany), reaching from 0.5 to 0.9 bar. The plants were returned to the greenhouse under standard conditions.
GFP expression was high in leaves of the plants infiltrated with pICH18711 (
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| Number | Date | Country | Kind |
|---|---|---|---|
| 04001460 | Jan 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2005/000492 | 1/19/2005 | WO | 00 | 7/21/2006 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2005/071090 | 8/4/2005 | WO | A |
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| 6632980 | Yadav et al. | Oct 2003 | B1 |
| 20070044170 | Marillonnet et al. | Feb 2007 | A1 |
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| 20070300330 A1 | Dec 2007 | US |
