Plant expression vectors

What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.

1. Plant expression vector system By Abhishek R. Indurkar 17PBT202

2. Introduction An expression vector , is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively.

3. Plant gene structure A typical plant gene has the following region beginning with the 5’end: i). Promoter: For transcription initiation ii). Enhancer/silencer: Concerned with regulation of gene iii). Transcriptional start site or cap site: From here initiation of transcription take place iv). Leader sequence: It is untranslated region v). Initiation codon vi). Exons vii). Introns viii). The stop codon ix). A second untranslated region, and x). Poly A tail

4. Promoter is a region of DNA sequence which helps in the transcription of a particular gene. This contains specific DNA sequences as well as response elements which provide a secure initial binding site for RNA polymerase. These proteins called transcription factors that recruit RNA polymerase. The CAAT and TATA boxes represent consensus sequences within promoter for RNA polymerase II. ATG (AUG in mRNA) is initiation codon for mRNA translation, and mark the beginning of coding sequence of the gene. A sequence between the cap site and ATG is not translated and form the 5'-leader sequence of mRNA. Codon TAG/TAA/TGA are chain terminating codon and it is followed by a stretch of nontranslated region. At the end, poly-adenylation site is present which denotes the end of transcription.

5. Plant expression systems Plant expression vectors are mainly based on the Ti plasmid of Agrobacterium tumefaciens . Plant viruses are also used as expression vectors. DNA Vectors Cauliflower mosaic virus (CMV) Gemini viruses Mastreviruses Begomoviruses RNA Vectors Tobacco mosaic virus (TMV) Brome mosaic virus (BMV) Hordeiviruses Potexviruses Comoviruses

6. Agrobacterium Agrobacterium tumefaciens and Agrobacterium rhizogenes are common gram-negative soil borne bacteria causing induction of ‘crown gall' and ‘hairy root' diseases. These bacteria naturally insert their genes into the genome of higher plants. The studies on crown gall formation revealed that the virulent strains of bacteria introduce a part of their genetic material into the infected cells where it gets integrated randomly with the genetic material of the host cell. The bacterial genes are able to replicate along with the plant genome and uses the machinery of plants to express their genes in terms of the synthesis of a special class of compounds, called opines, which the bacterium uses as nutrients for its growth but are useless to the host cells. In the process, Agrobacterium causes plant tumors (gall formation) commonly seen near the junction of the root and the stem and is called ‘crown gall disease'. A. tumefaciens attracted to the wound site via chemotaxis, in response to chemicals (sugars and phenolic molecules) released from the damaged plant cells. The disease afflicts a great range of dicotyledonous plants, which constitute one of the major groups of flowering plants. Tumorous plant cells were found to contain DNA of bacterial origin integrated in their genome. Furthermore, the transferred DNA (named T-DNA ) was originally part of a small molecule of DNA located outside the chromosome of the bacterium. This DNA molecule called Ti ( tumor-inducing ) plasmid.

7. Organisation of T-DNA The transfer DNA (T-DNA) is the transferred DNA of the tumour inducing plasmid (pTi) of some Agrobacterium species of bacteria. T-DNA has both its side 24 kb direct repeat border sequence and contains the gene for tumor / hairy root induction and also for opines biosynthesis. pTi has three genes, two of these genes (iaaM and iaaH) encode enzymes which together convert tryptophane in to IAA (Indol-3-acetic acid) a type of auxin. If these two genes are deleted then shooty crown gall will produce. The third gene, ipt, encodes an enzyme which produces Zeatin-type cytokinin isopentenyl adenine. The deletion of ipt, causes rooty crown galls and the region was earlier designated as ‘rooty locus' and denoted by tmr (tumour having roots).

8. In addition to these, another locus called tml and the deletion of which results in large tumours. Besides, T-DNA also contains genes involved in opine biosynthesis which are located near the right border of T-DNA.

9. T-DNA transfer and integration Wounded plant cell releases phenolics substances and sugars Which are sensed by vir A, vir A activates vir G, vir G induces expression of vir gene of Ti-plasmid vir gene produce all the vir -protein vir D 1 and vir D 2 are involve in ssT-DNA production from Ti-plasmid and its export The ssT-DNA (with associated vir D 1 and vir D 2 2 ) with vir E 2 are exported through transfer apparatus vir B In plant cell, T-DNA coated with vir E 2 Various plant proteins influence the transfer of T-DNA + vir D 1 + vir D 2 + vir E 2 complex and integration of T-DNA to plant nuclear DNA. (LB= left border; RB= Right border; pTi = Ti plasmid, NPC = nuclear pore complex)

10. Agrobacterium virulence protein function

11. Ti plasmid The Ti plasmid contains all the genes which required for tumor formation. Virulence genes (vir-genes) are also located on the Ti plasmid. The vir genes encode a set of proteins responsible for the excision, transfer and integration of the T-DNA into the plant nuclear genome. The basic elements of the vectors designed for Agrobacterium-mediated transformation that were taken from the native Ti-plasmid The T-DNA border sequences , at least the right border, which initiates the integration of the T-DNA region into the plant genome The vir genes , which are required for transfer of the T-DNA region to the plant, and A modified T-DNA region of the Ti plasmid, in which the genes responsible for tumor formation are removed by genetic engineering and replaced by foreign genes of diverse origin, e.g., from plants, bacteria, virus. When these genes are removed, transformed plant tissues or cells regenerate into normal-appearing plants and, in most cases, fertile plants.

12. Ti plasmid is grouped into two general categories: i) Nopaline type pTi ii) Octopine type pTi

13. Ri plasmid Agrobacterium rhizogenes is a soil born gram negative bacterium. It causes hairy root disease of many dicotyledonous plants. The ability of A. rhizogenes to incite hairy root disease is confirmed by a virulence plasmid, which is similar to that found in Agrobacterium tumefaciens which causes Crown gall tumors of plants. The virulence plasmid of A. rhizogenes is commonly known as the Ri-plasmid (pRi). The pRi have extensive functional homology with the pTi. The pRi contains distinct segment(s) of DNA, which is transferred to plant genome during infection. The transfer T-DNA to the plant genome is mediated by another segment on the plasmid known as the virulence (vir) region. All strains of A. rhizogenes are known to produce agrocinopine.

14. I Cauliflower Mosaic Virus The Cauliflower Mosaic Virus (CaMV) is a double-stranded DNA virus which infects a wide range of crucifers, especially Brassicas, such as cabbage, cauliflower, oilseed rape or mustard. In order to get itself and its DNA replicated (multiplied) within a plant cell, the virus must trick the plant's own molecular ‘machinery' to do this task. For this purpose the virus has two promoters (35S and 19S) in front of its genes, which the plant cell believes to be its own. Furthermore, these promoters override the plant's own regulatory system, as they are constitutive, i.e. they are constantly switched on and can't be regulated or switched off by the plant. The CaMV 35S well known promoter is being used in almost all GM crops currently grown or tested, especially GM maize. It is the promoter of selection for plant genetic engineering, as it is a strong and constitutive promoter.

15. Initially, bacterial DNA sequence were used to probe the CaMV genome for potential sites for the insertion of heterologous genes. A 65bp fragment was inserted into naturally occurring XhoI site located within gene II, a region covered by a deletion in variant strain of CaMV and therefore not essential for viral infection. The first functional gene to be cloned into CaMV was dihydofolate reductase gene(dhfr ) from an E.coli plasmid, which encodes methotrexate intensive enzyme. Dhfr which is 234bp long was used to replace gene II (470bp) and resulted in recombinant CaMV, which could be propagated in turnip plants for three cycle of infection with viral DNA. Expression of dhfr gene was monitored by western blot analysis of leaf extract.

16. Genome organisation of CaMV. The circular dsDNA (8kbp) is represented by two concentric circles and contains there discontinuities adjacent to replication priming sequence. One open circle is (+) strand while the two are (-) strand. Outer arrow represents transcription from (+) strand by host DNA dependent RNA polymerase: the 35S which serves as template for both reverse transcriptase and translation of gene I to V and the 19S RNA from which gene VI is expressed. Inner arrow represents ORF which encode the movement protein (MP), the aphid transcription factor (ATF), a protein which has putative role in viral DNA folding during encapsidation (fold), the coat protein (CP), the reverse transcriptase (RT), and the translations transactivator protein (TrAP). Sequence which are dispensable for virus infection and which can be deleted and/or replaced with foreign gene are shown in black

17. II. Gemini viruses Gemini viruses are small circular DNA viruses that replicate in plant nuclei. The Gemini virus vectors lack a coat protein gene, they are not transmissible by insect vectors, which are required for plant-to-plant spread and, thus, use of the disarmed vectors does not require a permit. Viruses from the Gemini virus family normally infects a wide range of crop plants, including maize, cotton, wheat, bean and cassava and are, therefore, an ideal system of choice for VIGS-based gene function analyses in a broad range of crop plants. Now vectors have been developed for use in cotton, and work is also ongoing for suitable vectors for roses. Using these new VIGS vectors, recombinant virus bearing a partial sequence of a host gene is used to infect the plant. As the virus spreads, the endogenous gene transcripts, which are homologous to the insert in the viral vector, are degraded by post-transcriptional gene silencing. These VIGS virus vectors have been used in a range of studies to silence single or multiple genes, including the meristematic gene, Proliferating Cell Nuclear Antigen (PCNA).

18. A geminivirus vector based on the Beet Curly top virus (BCTV) has been used to generate a vaccine against hepatitis A. As another example, the dual-module in-plant activation (INPACT) expression platform was developed in tobacco yellow dwarf virus (TYDV) to control foreign gene expression. By interrupting an expression cassette by a plant intron, the protein of interest is expressed only after post-transcriptional processing, thus offering further control of expression of the gene of interest.

19. III. Mastreviruses Virus belongs to this genus are transmitted via leafhoppers, and most of them infects only monocotyledons. There genomes are monoparatite and comprises of both a large intrinsic region (LIR) and small intrinsic region (SIR). The complementary sense strand codes for protein C1 and C1:C2 has been shown only in viral protein necessary for replication. The vision sense strand encodes not only for CP, V2, which protects the viral genome and is required for insect transmission, but also the cell to cell movement protein (MP), V1. Both V1 and V2 are needed for systemic spread. Their replacement will therefore limit the use of resulting vectors to infect the cells in culture.

20. Types Wheat dwarf virus (WDV) Maize streak virus (MSV) Tobacco yellow dwarf virus (TYDV) Generic genome organisation of Mastrevirus

21. VI. Begomoviruses Viruses belonging to this group are whitefly transmitted infect dicotyledonous hosts and their majority of genome is made up of two molecules of ssDNA of similar size, termed as DNA A and B. The intergenic region, which is shared by the two molecules of DNA and therefore termed as common region (CR), contains site of initiation and termination of DNA replication, as well as the promoter virus for bidirectional transcription. While the two protein encoded by DNA B are required for virus movement, they are dispensable for viral replication. The coat protein (CP) of bipartite begomoviruses, encoded by DNA A, is dispensable both for viral DNA replication and systemic spread. This triggers widespread interest into replacement by foreign gene. Because DNA A encodes all the viral protein involved in replication, it can self replicate in protoplasts.

22. Types African cassava mosaic virus (ACMV) Tomato golden mosaic virus (TGMV) Generic genome organisation of bipartite begomovirus

23. V. Tobacco mosaic virus TMV have single-stranded RNA genome which also serves as mRNA. It encodes at least four proteins in three open reading frames. Its genome contains 4 genes, of these the coat protein (cp) gene seems to be nonessential and can be site of integration of transgene. Viral RNA promoters are successfully manipulated for the synthesis of recombinant messenger RNAs in whole plants. This vector consist of two steps, first, is the use of cDNA copy of viral genome for cloning in E. coli and, second, is in vitro transcription of the recombinant viral genome cDNA to produce infectious RNA copies to be used for plant infection.

24. Icon Genetics, a biotechnology company based in Germany, developed a technique for transfecting plants with these recombinant virus vector modules, known as Magnifection. Magnifection combines agro infiltration with the delivery of a deconstructed vector that lacks the ability to spread to other plants. A deconstructed TMV vector has been employed to generate Human papillomavirus HPV E7 protein and Norwalk virus-like particles (VLPs) in plants as well as the Influenza M2e epitope in plants. Foreign protein expression in TMV infected plants can also be enhanced significantly through the coexpression of the RNA silencing suppressor gene P19 of the Tomato bushy stunt virus. The recent discovery of adjuvant properties of TMV has sparked a renewed interest in the use of this virus as a delivery vehicle for immunotherapy. TMV particles have been demonstrated to be taken up by dendritic cells and to exhibit activation properties, resulting in robust CD8+ T cell responses

25. VI. Brome mosaic virus Brome mosaic virus (BMV) belongs to the family Bromoviridae of plant RNA viruses. BMV is a eukaryotic RNA virus, and its replication is entirely cytoplasmic. BMV genome is divided among three RNAs (1, 2 and 3) each packed into separate particle. Viral replication is dependent on well-organized interaction between nonstructural proteins 1a and 2a, encoded, respectively, by genomic RNA1 (gB1) and RNA2 (gB2). Genomic RNA3 (gB3) is dicistronic. Another nonstructural movement protein (MP) which promotes cell-to-cell spread encoded by 5′ half, while the capsid protein gene (CP) encoded in the 3′ half is translationally silent but is expressed from a subgenomic RNA (sgB4) that is synthesized from progeny minus-strand gB3 by internal initiation mechanisms. It was found in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1  +  RNA3 or RNA2  +  RNA3) as well as cellular RNAs. This indicates that placing a transgene downstream to the regulatory sequences of the cp gene of BMV will give high yields of the protein encoded by it.

26. VII. Hordeiviruses Barley stripe mosaic virus (BSMV) has a genome consisting of 3RNAs (alpha, beta and gamma) encapsidated in rod shaped virions, RNAs alpha and gamma encode protein essential for replication, while RNA beta contains four open reading frames. ORF beta-alpha encodes the viral CP, while other three encode triple block proteins involved in viral movement within an infected plant BSMV RNA beta can be modified to express heterologous sequence, a vector based on this RNA has been used to examine the function of MPs of several plant viruses.

27. VIII. Potexviruses Potato Virus X (PVX), a flexuous, rod-shaped virus containing a plus-sense RNA molecule, has also been engineered extensively as an expression vector for biopharmaceuticals. The genome of PVX consists of replicase and capsid protein genes, as well as a triple gene block, whose products are responsible for virus movement. PVX has been used to express full-length proteins, fusion proteins, epitopes that are displayed on the outer surface of the assembled virus particle, and more recently, PVX nanoparticles have been demonstrated to block tumour progression in animal models. PVX has been employed for the development of a universal influenza vaccine consisting of an epitope derived from the extracellular domain of H1N1 virus matrix protein 2 (M2e). The researchers fused M2e to bacterial flagellin, a strong mucosal adjuvant, in order to improve M2e immunogenicity PVX has produced other antigens as well. For example, Uhde-Holzem et al. (2010) used PVX to express the epitope HVR1 from the Hepatitis C virus (HCV) as a fusion protein. Recently, PVX has been designed to act as a nanoparticle for tumour immunotherapy.

28. IX. Comoviruses The Comovirus Cowpea mosaic virus (CPMV) has undergone extensive development as an expression vector. CPMV is an icosahedral virus of 30 nm in diameter, comprised of 60 large (L) and 60 small (S) capsid proteins. The genome of CPMV is bipartite, with RNA-2 being the principal component of expression vector development. CPMV has been utilized extensively in antigenic presentation and full-length protein expression as part of a fusion protein that can undergo proteolytic cleavage to release the therapeutic protein, as well as in material science research, such as the formation of magnetic clusters and biosensors. For example, Medicago, Inc. (Durham, NC, USA) has used the CPMV vector to generate virus-like particles (VLPs) carrying influenza virus HA antigens. These VLPs protect against lethal viral challenge in animal models, and are now undergoing a Phase 2 clinical trial with over 250 volunteers. Medicago’s CPMV production system enables a vaccine to be generated within 3 weeks of the release of the influenza strain sequence information, with an easily adaptable upscaling capacity.

29. A new vector, known as pCPMV-HT (Cowpea Mosaic Virus Hyper-Trans expression system) has been engineered that also provides high translational efficiency. These vectors have been used to generate vaccines against the bluetongue virus, HIV, Dengue, and the influenza virus. The Cowpea mosaic virus expression vector pEAG-HT has also been used to express the extracellular domain of the rat ErbB2 in tobacco plants. ErbB2 is an epidermal growth factor-related protein, and its aberrant expression can lead to a variety of cancers. Plant extracts expressing ErbB2 that were injected into mice elicited immune responses and potent antitumour activity.

30. Reference Walden R, Schell J (1990). "Techniques in plant molecular biology--progress and problems". European Journal of Biochemistry . 192 (3): 563–76. doi:10.1111/j 1432-1033.1990.tb19262.x Gemini virus vectors for gene expression in plants US 6392121 B1 http://nptel.ac.in/courses/102103016/module3/lec23/2.html Claudine P, George L, "Viruses as vectors for the expression of foreign sequence in plants”. Sean Chapman, Tony Kavanagh and David Baulcombe, “Potato virus X as a vector for gene expression in plants”, The Plant Journal (1 992) 2(4), 549-557. Kathleen H, “Plant Virus Expression Vectors: A Powerhouse for Global Health”, Biomedicines 2017 , 5, 44.

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