Plant expression vectors

Plant expression vector system
By
Abhishek R. Indurkar
17PBT202

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.

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

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.

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

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.

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).

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.

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 D1 and vir D2 are involve in ssT-DNA production from Ti-plasmid and its export
The ssT-DNA (with associated vir D1 and vir D2 2) with vir E2 are exported through transfer apparatus vir B
In plant cell, T-DNA coated with vir E2
Various plant proteins influence the transfer of T-DNA + vir D1 + vir D2 + vir E2 complex and integration of T-
DNA to plant nuclear DNA.
(LB= left border; RB= Right border; pTi = Ti plasmid, NPC = nuclear pore complex)

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.

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

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.

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.

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.

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

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).

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.

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.

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

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.

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

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.

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

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.

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.

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.

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.

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.

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 (1992) 2(4), 549-557.
Kathleen H, “Plant Virus Expression Vectors: A Powerhouse for Global Health”,
Biomedicines 2017, 5, 44.

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