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TRANSPLASTOMICS

TRANSPLASTOMICSPLASTID TRANSFORMATION

Muhammed Sadiq

2012-09-119

CHLOROPLAST

Plastids are the defining feature in plants and algae

Plastids were free-living cyanobacteria over one billion years ago, before becoming endosymbionts

The chloroplasts are the most common form of plastids in plants

Able to convert light into chemical energy through a process called photosynthesis

play an essential role in plant metabolism

Plastids produce fatty acids, aromatic and non-aromatic amino acids, purine and pyrimidine bases, isoprenoids

(carotenoids and sterols) and tetrapyrroles

There are up to 300 plastids in one plant cell.

Introduction

Endosymbiosis

CHLOROPLAST GENOME

The plastids have their own genome, known as plastome.

Plastomes from land plants are circular molecules of double stranded DNA, between 120 and 180 kb in size

Plastid DNA having high copy numbers and have their own transcription translation machinery.

plastids are highly polyploid and their plastomes are organized as nucleoids which are attached to the inner membrane.

The number of copies of plastomes per leaf cell ranges from 1000 to 1700 in Arabidopsis thaliana and reaches up to

50,000 in wheat (Bendich, 1987; Zoschke et al., 2007)

Chloroplast DNA

The plastome of higher plants is conserved in size, organization and sequence

Plastome contains an inverted repeat region A and B (IRA and IRB respectively) and two single unique regions

known as small single copy (SSC) and large single copy (LSC) regions

Chloroplast DNA is replicated transcribed and translated independently within the cell.

Plastids are usually strictly maternally inherited in most (80%) angiosperm plant species.

chloroplasts strongly depend on imported proteins that are encoded in the nucleus and imported into the organelle

In case of A. thaliana nucleus encode about 2100 chloroplast proteins and the whole chloroplast genome encodes

for 117 protein.

Chloroplast Gene Expression

Chloroplast gene organization, transcription and translation is similar to that of eubacteria.

Chloroplast genes are organized mainly in operons and can be expressed as polycistronic units.

Transcription in plastids is mainly executed by two or three DNA-dependent RNA polymerases

Plastid genome encodes for one RNA polymerase (PEP), Several RNA polymerases (NEP) like RPOTp and

RPOTpm are imported from the nucleus into plastids

Level of protein expression in plastids not only depends on the transcription level and mRNA-stability, it also

depends on protein stability

chloroplast translation utilizes 70S ribosomes which do not require 5´caps or 3´poly (A) tails(same as

bacteria).

The regulatory mechanisms of gene expression in plastids at the translational and posttranslational level

remain unknown.

The level of protein expression in plastids not only depends on the transcription level and mRNA-stability,

it also depends on protein stability

Most chloroplast genes are co-transcribed as polycistronic RNAs (i.e. they encode multiple proteins that

are separately translated from the same mRNA molecule)

Chloroplast Transformation

Plastid transformation was first achieved in a unicellular alga, Chlamydomonas reindhartii

First successful chloroplast transformation of a higher plant is in Tobacco

Tobacco plastid has been engineered to express the E7 HPV type 16 protein, which is an attractive candidate for

anticancer vaccine development.

Plastid transformation allows high expression levels of proteins and as it provides biological transgene containment

because of maternal inheritance of cytoplasmic genes in most crops.

Different methods are introduced for transferring the genes into plastids :

particle bombardment, PEG (polyethylene Glycol) mediated transfer and Micro injection.

Gene expression in plastids requires pro-and eukaryotic elements.

Most of the promoters are prokaryotic–but not all of them.

Up to date, plastid transformation has been extended to many other higher plants.

Such as Arabidopsis thaliana , potato , tomato , Lesquerella fendleri, a kind of oilseed Brassicaceae , oilseed rape, ,

petunia, lettuce , soybean , cotton , carrot, rice, poplar, tobacco, mulberry and eggplant.

If transgenic chloroplasts might be present in pollen, plastid DNA is eliminated from the male germ line at different

points during sperm cell development

Steps in chloroplast genetic engineering

Plants was asceptically grown from seed on MS medium (agar)

Collect the leaves and place it on the medium

Biolistic particle treated with vector and other chemicals

Recombinant DNA Plasmid are injected to the chloroplast of leaf using Gene gun or other methods.

After 2 days, leaves cut into section and transferred to medium containing an antibiotic ,for recombinant selection.

Green calli formed on the bleached leaf are subcultured on the same medium

Calli formed shoots

These shoots were rooted on MS medium to obtain plants, express the desired protein.

Vector design for chloroplast transformation

Selectable marker genes

Initially plastid 16S rRNA (rrn16) gene was used as a selection marker in chloroplast transformation. The transgenic

lines were selected by spectinomycin resistance but with low efficiency

Dominant markers confer high transformation efficiency. e. g. aadA (aminoglycoside 3′ adenylyltransferase) gene

confers resistance to streptomycin and spectinomycin by inactivation of antibiotics.

Marker, bacterial spectinomycin resistance gene aadA encoding for a 3´aminoglycoside-adenyltransferase (AADA).This protein inactivates spectinomycin and streptomycin antibiotics

Chloroplast specific expression cassette

Insertion sites

Plastid expression vectors possess left and right flanking sequences each with 1–2 kb in size from the host plastid

genome, which facilitates foreign gene insertion into plastid DNA via homologous recombination.

The insertion site in the plastid genome is determined by the choice of plastid DNA segment flanking the marker

gene and the gene of interest.

The foreign DNA is inserted in intergenic regions of the plastid genome.

Most commonly used insertion sites are trnV-3'rps12 ,trnI-trnA and trnfM-trnG

Regulatory sequences

The level of gene expression in plastids is predominately determined by regulatory sequences such as promoter as well

as 5′ UTR elements

Strong promoter is required to ensure high mRNA level for high-level of protein accumulation e.g. rRNA operon (rrn)

promoter (Prrn).

Most commonly used promoter is CaMV 35S promoter of cauliflower mosaic virus which drives high level of

transgene expression in dicots.

In plastid expression vectors, a suitable 5′ untranslated region (5′-UTRs) containing a ribosomal binding site (RBS) is

an important element.

Stability of the transgenic mRNA is ensured by the 5′ UTR and 3′ UTR sequences flanking the transgene.

Reporter genes used in plastids

GUS(β-glucuronidase), chloramphenicol acetyl transferase, and GFP have been used as plastid reporters

The enzymatic activity of GUS can be visualized by histochemical staining

GFP(Green Fluorescent Protien) is a visual marker that allows direct imaging of the fluorescent gene product in

living cells.

GFP has been used to detect transient gene expression.

GFP has also been fused with AadA and used as a bifunctional visual and selectable marker

Transformation Methods

In general, there are two DNA delivery methods for a stable introduction of foreign DNA into plastids.

The first one consists in bombarding of tissue or cells with DNA coated particles .

The second method treats isolated protoplasts (plant cells without cell wall) with polyethylene Glycol.

PEG induces pores in cell membranes allowing the entrance of DNA into plastids.

Micro injection is also used (femto syringe)

Biolistic Method

Plastid vector DNA is coated onto high-density tungsten or gold microprojectiles (0.6–1 μM diameter), which are

then delivered at high velocity first through the cell wall and membrane, and then through the double-plastid

membrane

This method yields a high efficiency rate, and can be used to transform a variety of explants. The first successful

chloroplast transformation in higher plants was achieved in 1989, when spectionomycin resistance was transferred

into tobacco

The insertion of foreign genes into plasmid DNA occurs by homologous recombination via the sequences flanking

at the insertion site

First successful chloroplast transformation was performed in Chlamydomonas reinhardtii by particle bombardment

method.

Advantages

Simple operation and high efficiency makes it a favourable way for plastid or chloroplast transformation

No need to obtain protoplast as the intact cell wall can be penetrated.

Manipulation of genome of sub-cellular organelles can be done

This device offers to place DNA or RNA exactly where it is needed into any organism.

Simple and convenient method involving coating DNA or RNA on to gold microcarrier, loading sample cartridges,

pointing the nozzle and firing the device

This device offers to place DNA or RNA exactly where it is needed into any organism.

Disadvantages

The transformation efficiency may be lower than Agrobacterium- mediated transformation.

Specialized equipment is needed. Moreover the device and consumables are costly.

Associated cell damage can occur.

The target tissue should have regeneration capacity

PEG Method

PEG Method Protoplasts are plant cells with their wall removed by enzyme treatment.

Treatment of freshly isolated protoplasts with PEG allows permeabilization of the plasma membrane and facilitates

uptake of DNA.

Plasmid DNA passes the plastid membranes and reaches the stroma where it integrates into the plastome as during

biolistic transformation.

A relatively small number of species have been transformed using this approach , mainly because it requires efficient

isolation, culture and regeneration of protoplasts, a tedious and technically demanding in vitro technology.

On the positive side, no special equipment is needed.

Advantages

A large number of protoplasts can be simultaneously transformed.

This can be successfully used for a wide range of plant species with adequate modifications.

Disadvantages

The DNA is susceptible for degradation and rearrangement.

Random integration of foreign DNA into genome may result in undesirable traits.

Regeneration of plants from transformed protoplasts is a difficult task.

Micro injection

The DNA is microinjected directly into chloroplasts using a very small syringe.

Delivery of foreign DNA into a living cell (e.g. a cell, egg, oocyte, embryos of animals) through a fine glass

micropipette.

DNA microinjection was first proposed by Dr. Marshall A. Barber in the early of nineteenth century.

Delivery of foreign DNA is done under a powerful microscope using a glass micropipette tip of 0.5 mm diameter

Easy identification of transformed cells upon injection of dye along with the DNA

Cells to be microinjected are placed in a container. A holding pipette is placed in the field of view of the microscope

that sucks and holds a target cell at the tip. The tip of micropipette is injected through the membrane of the cell to

deliver the contents of the needle into the cytoplasm and then the empty needle is taken out

Integration of Transgene into Plastid Genome

Chloroplast transformation vectors are thus designed with homologous flanking sequences on either side of the

transgene cassette to facilitate recombination.

Targeting sequences have no special properties other than that they are homologous to the chosen target site and are

generally about 1 kb in size

Both flanking sequences are essential for homologous recombination.

Transformation is accomplished by integration of the transgene into a few genome copies, followed by 25 to 30 cell

divisions under selection pressure to eliminate untransformed plastids, thereby achieving a homogeneous population

of plastid genomes.

If the transgene is targeted into the IR region, integration in one IR is followed by the duplication of the introduced

transgene into the other IR as well.

Transgenes have been stably integrated at several sites ie., Transcriptionally inactive spacer region and

transcriptionally active spacer region.

Transcriptionally active spacer regions offer unique advantages, including insertion of transgenes without 5’or 3’

untranslated regions (UTRs) or promoters.

Most commonly used site of integration is the transcriptionally active intergenic region between the trnI-trnA genes,

within the rrn operon, located in the IR regions of the chloroplast genome.

Chloroplast vectors may also carry an origin of replication that facilitates replication of the plasmid inside the

chloroplast

Selection of Transplastomic

Common selection marker used for plastid transformation is the bacterial spectinomycin resistance gene aadA

Encoding for a 3´aminoglycoside-adenyltransferase (AADA).This protein inactivates spectinomycin and streptomycin

antibiotics.

Transplastomic clones are identified as green shoots on spectinomycin medium.

Spectinomycin inhibit greening and shoot regeneration of wild type.

After integration,

homoplastomic cells (uniform population of transformed plastids) are obtained by several rounds of cell division and

organelle segregation. homoplasmy can be achieved through several rounds of regeneration starting from leaves or

tissue explants on selection

Homoplasmic Cell

Confirmation

Confirmation of transgene integration into chloroplast genome

Integration of transgenes into the cotton cultures was confirmed by PCR using internal primers,

first primer anneals to the flanking sequence and second primer anneals to the transgene region.

An expected size of size PCR product was amplified and this confirmed integration of the transgenes in different cell

cultures of plant

Integration of the transgenes into plastid genome were investigated by Southern blot analysis.

Genomic DNA from transformed and untransformed cultures was digested with appropriate restriction enzymes,

transferred to nitrocellulose membrane and probed with P32-radiolabel .

Transformed chloroplast genomic DNA digested with restriction enzymes yielded an expected 3.3 kb size hybridizing

fragment.

Excision of selectable marker genes

Marker elimination system by CRE-loxP site specific recombination system

Cre-Lox recombination is a site-specific recombinase technology, used to carry

out deletions, insertions, translocations and inversions at specific sites in the DNA of cells

According to the CRE- loxP scheme, the marker gene (flanked by two directly oriented lox sites) and the gene of interest are introduced into the plastid genome in the absence of CRE activity.

When elimination of the marker gene is required, a gene encoding a plastid-targeted CRE site-specific recombinaseis introduced into the nucleus, excises sequences between the loxP sites

Cre could be introduced by a second, Agrobacterium mediated transformation

The nuclear Cre is subsequently removed by segregation in the seed progeny

In tobacco, introduction of the nuclear Cre gene into the nucleus of transplastomic plants by Agrobacterium

transformation extends the time needed to obtain marker-free plants by only one month.

In an ideal case, it takes about six months to obtain a marker-free transplastomic tobacco plant that expresses a

novel recombinant protein.

Cre/lox site-specific recombination system is derived from the P1 bacteriophage.

Case Study

Case Study

Advantages of Chloroplast Transformation

little risk of any transgene flow from transplastomic plants to the neighbouring weedy or wild relatives.

possibility of expressing multiple genes in operons, high expression levels

Ability to accumulate the toxic proteins or biosynthetic products .

possibility of expressing unmodified bacterial genes and human cDNA

lack of gene silencing and position effects

Integration via homologus recombination

potent applications in developing plants resistant to biotic and abiotic stresses, and for production of therapeutic

proteins and vaccines

Applications of Transplastomics

Agronomic traits expressed via the plastid genome

Resistant to herbicide, drought, pathogens etc.

Chloroplast Biotechnology for production of therapeutic proteins

Vaccines, hormones, other biomolecules etc.

Agronomic traits expressed via the plastid genome

Important Agronomic traits engineered such as, herbicide resistance, insect resistance, and tolerance to drought and salt.

Engineering the chloroplast genome for herbicide resistance

Glyphosate is a potent, broad-spectrum herbicide, not distinguish crops from weeds.

Transplastomics (which are maternally inherited in most crops) offers a solution to this problem.

The chloroplast genome can be engineered to confer herbicide resistance by expressing a petunia EPSPS nuclear gene

via the chloroplast genome

Resultant transgenic plants are resistant to tenfold higher levels of glyphosate than the lethal dosage

Amplification of EPSP synthase gene induces resistance to glyphosate.

250-fold higher levels of the glyphosate-resistant protein were achieved than nuclear transformation

Engineering the chloroplast genome for pathogen resistance

A synthetic antimicrobial peptide (MSI-99) was expressed via the chloroplast genome

MSI-99 is an amphipathic α helical molecule

Its affinity for the negatively charged phospholipids found in the outer membrane of bacteria and fungi

It causes individual peptides aggregate to form pores, resulting in microbial lysis

MSI-99 was expressed via the chloroplast genome to accomplish high-dose release at the point of infection.

In vitro and in plant assays confirmed that the peptide was expressed at high level and retained biological activity against Pseudomonas syringae.

Microbes outer membrane are highly conserved ,unlikely to develop resistance against these peptides

Engineering the chloroplast genome for drought tolerance

Trehalose is a non-reducing disaccharide of glucose whose synthesis is mediated by the trehalose-6-phosphate (T6P)

synthase.

It present in Saccharomyces cerevisiae

it accumulates under stress conditions such as freezing, heat, salt or drought

Trehalose protects against damage imposed by these stresses, engineering high levels of Trehalose in plants might confer drought tolerance.

The yeast trehalose phosphate synthase ( TPS1 ) gene was introduced into the tobacco chloroplast.

Chloroplast transgenic plants showed up to 25-fold higher accumulation of trehalose than nuclear transgenic plants.

Chloroplast transgenic plants also showed a high degree of drought tolerance by growing in 6% polyethylene glycol, whereas respective control plants were bleached.

Air-dried chloroplast transgenic seedlings and transgenic plants under extreme drought (not watered for 24 days) successfully rehydrated, whereas similarly treated control plants died.

Trehalose functions by protecting the integrity of biological membranes rather than by regulating water potential

Compartmentalization of trehalose within chloroplasts confers drought tolerance without undesirable phenotypes

Chloroplast Biotechnology for production of therapeutic proteins

Advantages

Low cost production

Ability to carry out post-translational modifications and minimize the risk of contamination from potential human pathogens.

Convenient storage

Use of renewable resources for their production.

No gene silencing both at transcriptional and translational levels

Engineering the chloroplast genome to overproduce biopharmaceuticals

highly polyploid genomes offer an ideal compartment for overproduction of foreign proteins

Able to process eukaryotic proteins, including folding and formation of disulfide bridges.

Such folding and assembly can minimize the need for expensive in vitro processing of pharmaceutical proteins after

their extraction.

60% of the total production cost of human insulin is associated with invitro processing

Chloroplast-derived therapeutic proteins Expression

Depends on :

the site of integration

Regulatory elements used to enhance transcription/translation

Stability of the foreign protein.

Genes coding for 20 amino acids or 83 kDa (PA) have been expressed in transgenic chloroplasts

Somatotropin (hST)

a human therapeutic protein, is used in the treatment of hypopituitary dwarfism, Turner syndrome, chronic renal

failure and HIV wasting syndrome

hST has been expressed in tobacco chloroplasts to levels of between 0.2 and 7.0% of total soluble proteins of

plant.

Chloroplast-expressed hST was shown to be correctly disulfide-bonded and biologically active

Human serum albumin (HSA): widely used intravenous protein, obtained by fractionation of blood serum, accounts for about 60% of the total

protein in the blood.

HSA was expressed in transgenic chloroplasts, the expression level was up to 11.1% of the total soluble protein. 500-fold greater than nuclear expression.

Engineering the chloroplast genome to develop edible vaccine

Major cost of biopharmaceutical production lies in purification; eg, in insulin production, chromatography accounts

for 30% of the production cost and 70% of the set-up cost.

Oral delivery of properly folded and fully functional biopharmaceuticals should significantly cut down the

production cost.

Bioencapsulation of pharmaceutical proteins within plant cells offers protection against digestion in the stomach but

allows successful delivery to the target tissues

Stability and functionality of many proteins expressed in plastids are determined by N- or O- glycosylation

Engineering the chloroplast genome to develop edible vaccine

The β subunits of enterotoxigenic E. coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine

antigens.

Integration of an unmodified CTB gene into the tobacco chloroplast genome results in accumulation of up to 4.1% of

total soluble leaf protein as functional CTB oligomers

Binding assays confirm that correct folding and disulfide bond formation of the plant-derived CTB pentamers.

Insulin like growth factor (IGF-1)

Therapeutic value not only in mediating the growth of muscle and other tissues, but its therapeutic value is being

currently evaluated in diabetes, IGF-I induced neuro protection, and in promoting bone healing.

IGF-1 gene has codons suitable for eukaryotic , codon optimization for chloroplast done to increase the levels of expression in transgenic tobacco plants (expression about 32%)

Human interferon alpha (IFN2b):

Used in the treatment of malignant carcinoid tumors and has been shown to be very effective in the reduction of the tumour size.

It also has other therapeutic values such as inhibition of viral replication, cell proliferation and enhancement of the immune response.

Interferon treatment is very expensive ($26,000–40,000 per treatment of hepatitis C).

Nuclear expression of this protein is (0.000017% fresh weight) in tobacco

Transgenic chloroplast expression is 18%.

Antimicrobial peptide

Magainin is a broad-spectrum topical agent, a systemic antibiotic, a wound-healing stimulant, and an anticancer agent.

A magainin analogue, MSI-99 was expressed in transgenic tobacco chloroplast and expression level was 21.5% of the total soluble protein.

Pseudomonas aeruginosa , a gram-negative bacterium was used for testing the efficacy of the lytic peptide and this resulted in 96% growth inhibition of this pathogen.

Unsolved problems in plastid transformation

These issues include the development of plastid transformation systems for species other than tobacco, the expression of transgenes in non-green plastids, the level of protein accumulation and pleiotropic effects.

Plastid Transformation in Different Plant Species

lack of information of intergenic sequences for integration of transgenes

In some species, an additional limitation to plastid transformation is the lack of efficient selection/regeneration protocols

The extent of sequence divergence can influence the frequency of homologous recombination

The integration efficiency, functionality of recombinant coding sequences, likely affecting the regeneration of viable plants.

Gene Expression in Non-Green Plastids

Low expression level of plastid gene in non green tissues such as fruits, tubers and seeds.

Genome wide analysis reveals that all plastid genes are strongly down regulated at both transcriptional and

translational level in this non green storage organs

Few genes maintained relatively high mRNA level but were poorly translated(psbA gene in tomato)

Other few genes have low transcript level but strong ribosome association can make active translation

( accl gene in tomato)

Construction of hybrid expression elements led significant gene expression in non green plastids

Protein accumulation

Pastid transformation usually allows high accumulation of recombinant proteins, up to 70%

Several factors affect the accumulation levels of foreign proteins in transgenic plastids such as protein type, the

transcriptional and translational regulatory elements, plant tissue, plant development stage, the insertion site in

the plastome, RNA and protein stability

N-terminal amino acid sequence harbour important determinants of plastid protein stability

Protecting N-terminus and/ or C-terminus with polypeptide sequences taken from the highly stable proteins GFP

(green fluorescent protein) and PlyGBS (phage endolysin protein) able to solve the problem of protein stability.

Pleiotropic Effects

Expression of foreign proteins in plastids caused phenotypic alterations that include male sterility, yellow

leaves and reduced growth of transformed plants

Expression of immunogenic proteins A27L of vaccinia virus in transplastomic tobacco plants, then the

accumulation of the Rubisco large and small subunits was reduced.

Transplastomic plants showed chlorotic phenotype, male sterility and growth retardation.

The aberrant phenotypes observed in these studies could be due to metabolite toxicities, interference with

photosynthesis or disturbance of the plastid endomembrane system

To minimize the detrimental effects caused by the accumulation of recombinant protein in transformed

plastids, it is possible to use several systems for inducible gene expression

Additional Challenges

A low-level leakage of plastids in pollen has been reported.

using the biolistic plastid transformation, it is possible to achieve occasional unintended co-transformation of the

nuclear and plastid genomes

Correct folding of the proteins by formation of disulphide bonds is often required for functional tertiary and

quaternary structures

Limitations of Chloroplast Transformation

Transformation frequencies are much lower than those for nuclear genes.

Prolonged selection procedures under high selection pressure are required for the recovery of transformants

The methods of transgene transfer into chloroplasts are limited, and they are either expensive or require

regeneration from protoplasts.

These transformation systems are far more successful with tobacco than with other plant species.

Products of transgenes ordinarily accumulate in green parts only.

Conclusion

Future of Plastid Biotechnology Transgene expression from the plant’s plastid genome has unique attractions to

biotechnologists, including the plastids potential to accumulate foreign proteins to extraordinarily high levels and the

increased biosafety provided by the maternal mode of plastid inheritance, which greatly reduces unwanted transgene

transmission via pollen.

To date, more than 50 different transgenes have been stably integrated and expressed via the plant plastid genome to

confer important agronomic traits, as well as to produce industrially valuable biomaterials and therapeutic proteins.

Thank you…