Chloroplast transformation overview 2012aghunt00/PLS620/Lecture notes/2016/17... · 2015-11-16 ·...

Chloroplast transformation Why?

Biocontainment (dispersal of transgenes via pollen should be minimized or eliminated)

Better expression properties of transgenes (not susceptible to gene silencing, possibility of greater protein accumulation)

Multigene expression (chloroplast transcripts may be polycistronic) Elimination of “baggage” (selectable markers, other components that

may be regulated or forbidden in field-grown crops) Understanding mechanisms of chloroplast gene expression and

chloroplast gene function (reverse genetics)

How? General principle – homologous recombination Vector design

Sites for integration Selectable markers Gene expression signals

Amenable plant species

Schematic representation of the chloroplast-specific expression cassette.

Verma D , Daniell H Plant Physiol. 2007;145:1129-1143

©2007 by American Society of Plant Biologists



Targeting the cassette to a “silent” region of the genome

Target insertions so as to avoiud disrupting essential functions

Annual Reviews

Transcription:

Prokaryotic promoters and enzymes

Two distinct DNA-dependent RNA polymerases

PEP – analogous to the canonical bacterial multi-subunit DNA-dependent RNA polymerase (rpo), requires sigma factors for function

Land plants possess several distinct sigma factors, probably with somewhat different functions or roles

Sigma factors are nucleus-encoded

NEP – single-subunit phage-type DNA-

dependent RNA polymerase (T7 RNAP-like), nucleus-encoded

RNA polymerases in the chloroplasts

Chloroplasts have two different DNA-dependent RNA polymerases:

E. coli-like (α , β , β ' subunits, uses σ factors), chloroplast-encoded [PEP]

phage (T7 RNA polymerase)-type, single subunit, nucleus-encoded [NEP]

mature chloroplasts

expression

PEP-‐dependent

NEP-‐dependent

Chloroplast gene transcrip8on during the proplas8d-‐>chloroplast transi8on

§  A switch from NEP-‐dependent to PEP-‐dependent transcrip8on occurs early during seedling growth

§  NEP-‐dependent transcrip8on remains rela8vely constant while PEP-‐dependent transcrip8on seems to be coupled to photosynthe8c capabili8es

Onset of germina8on

Use Class I or Class II promoters



Translational optimization



Extended dark adapta,on

Shi1 to light

measure

Mature plants grown in a normal light-‐dark cycle

Chloroplast gene expression in mature chloroplasts during a dark-‐>light transi8on

0.1

1

10

100

1000

0 25 50 75 100

125

hrs in light

PSII

psb transcr.

psb mRNA

psb prot. rate

psb prot.

0.01

0.1

1

10

100

1000

0 25 50 75 100

125

hrs in light

PSI

psa transcr.

psa mRNA

psa prot. rate

psa prot.

Changes in [protein] are not reflected in changes in [mRNA] -‐> transla8onal control Changes in [protein] may correlate with changes in transla8on rates No strong indica8ons of transcrip8onal control Ø  In mature chloroplasts, posLranscrip8onal and transla8onal controls are important

determinants of gene expression

Appropriate 3’ regions – processing and stabilization



RNA processing: Endo- and exo-nucleolytic processing of the primary transcripts The players – prokaryotic ribonucleases RNAse J RNAse E PNPase These nucleases have modest or no RNA sequence specificity Specificity is conferred by RNA structures or accessory gene-specific RNA binding proteins The results are a panoply of RNA isoforms

RNA processing: Maturation, editing, and removal of introns The players – prokaryotic ribonucleases RNAse J PNPase PPR/TPR proteins (RNA-binding proteins

that are usually specific for a given processing or maturation reaction)

RNA editing can change the coding capacity of mRNAs

The results: Populations of monocistronic and polycistronic mRNAs These mRNAs will have different 5’- and 3’- ends, as well as differing translatabilities and stabilities

Protein-coding regions



Desired characteristics Selectable markers

Desired characteristics

glyphosate

Bt toxins

(Dufourmantel et al., 2004; Kumar et al., 2004a). Yet anotherparameter is the optimal concentration of selection agent,which varies depending on the sensitivity of the species sub-jected to plastid transformation.

Selectable markers for plastid transformation

Over the years, a variety of selectable markers have been devel-oped (Table 1). They differ in various properties that conferadvantages and drawbacks, such as dominance, cell-autonomyor portability. Some markers are dominant, such as the aadAgene that confers resistance to spectinomycin and streptomycinby inactivating the antibiotics, while others are recessive, suchas the point mutation in the ribosomal RNA genes (rrnS andrrnL) that confer resistance to various antibiotics by relieving thesensitivity of individual ribosomes. Dominance is of particularrelevance for transformation of the highly polyploid plastome.Dominant markers increase the transformation frequencybecause they have an effect already at early stages during selec-tion even though they may only be present in a minority of theplastomes. Conversely, recessive markers only confer resistanceif random segregation has produced a plastid that has enough

transformed copies of the plastome for the selectable pheno-type to emerge. Because this is a rare event, recessive markersgive lower transformation efficiencies than dominant ones.

Another important property of selectable markers is whetherthey are plastid- and cell-autonomous, such as the antibiotic-resistant rrnS or rrnL genes, which confer their phenotype onlyto the organelle or cell in which they reside. In contrast, genesthat encode proteins that inactivate an antibiotic will also offerprotection to neighbouring plastids and cells by locally decreas-ing the effective concentration of the drug. In this case, linesthat emerge from a round of selection may still be heteroplas-mic and plant tissues may be chimeric, with both transformedand wild-type sectors (Figure 1).

Some markers must integrate in a specific locus of the plas-tome, such as the rrnS or rrnL genes, while others are portableand autonomous and can be inserted in virtually any locus ofthe plastid genome, such as the aadA gene driven by chloro-plast expression signals (promoter, 5¢UTR and 3¢UTR). Co-trans-formation with a marker and a gene of interest on separatevectors, followed by selection for the marker, can yield linesthat carry both (Kindle et al., 1991; Newman et al., 1991;Carrer and Maliga, 1995; Ye et al., 2003). However, if the two

Figure 1 Chloroplast transformation. (a) In the transformation vector, a selectable marker (red) is placed under the control of plastid expression signals

(promoter, 5¢UTR, 3¢UTR, shown in blue). Homologous recombination through the flanking targeting arms directs integration into the recipient plastid

genome (plastome). The resulting transformant carries an insertion of the marker, or a substitution of the target sequence between the two arms

(white bar). (b) The initial integration in only one copy of the polyploid plastome is heteroplasmic. In Chlamydomonas, the single chloroplast harbours

approximately one hundred copies of its genome. Several rounds of subcloning and selection allow the recovery of homoplasmic clones. (This is only

possible if no essential function of the plastome has been disrupted by the insertion, otherwise a heteroplasmic state retaining wild-type copies of the

target sequence is maintained by a balance of selection for the marker and for the essential function.) (c) In multicellular plants, a similar situation

prevails after transformation, but each cell contains multiple plastids. Repeated rounds of propagation and selection lead first to a homoplasmic plastid

in a cell that may also contain non-transformed plastids, then to a cell with only homoplasmic plastids within a chimeric tissue and eventually to a

non-chimeric homoplasmic plant. Regeneration from homoplasmic cells facilitates the recovery of homoplasmic plants.

ª 2011 The AuthorsPlant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 540–553

Anil Day and Michel Goldschmidt-Clermont542

sistance, drought tolerance, phytoremediation, salttolerance, and CMS through chloroplast genetic engi-neering (Table II). Genetically engineered tobacco plantsexpressing an insecticidal protein Cry2Aa2 have shownresistance against target insects and insects that de-veloped resistance against insecticidal protein (Kotaet al., 1999). Expression of the Cry2Aa2 resulted in theutmost expression levels on record (approximately46.1% of total leaf protein) and resulted in the detec-tion of cuboidal crystals using transmission electronmicroscopy (De Cosa et al., 2001). In addition, soybeanplastid transformants expressing Cry1Ab also conferredinsecticidal activity against velvetbean caterpillar(Dufourmantel et al., 2005). The antimicrobial peptideMSI-99, an analog of magainin 2, was expressed viathe chloroplast genome to obtain high levels of ex-pression in transgenic tobacco plants. In planta assayswith the bacterial pathogen Pseudomonas syringae pvtabaci and the fungal pathogen Colletotrichum destructi-vum showed necrotic lesions in untransformed controlleaves, whereas transformed leaves showed no lesions(DeGray et al., 2001).

Environmental stress factors such as drought, salin-ity, and freezing are perilous to plants generally be-cause of their sessile means of existence. Attempts toconfer resistance to drought by expressing trehalose

phosphate synthase 1 (tps1) gene via nuclear transfor-mation have proven futile because of undesirablepleiotropic effects even at very low levels of trehaloseaccumulation. However, hyperexpression of tps1 inthe chloroplasts has no phenotypic variation from theuntransformed control plants, and transgenic seedssprouted, grew, and remained green and healthy indrought tolerance bioassays with 3% to 6% PEG anddehydration/rehydration assays (Lee et al., 2003).High-level expression of BADH in cultured cells, roots,and leaves of carrot via plastid genetic engineering ex-hibited high levels of salt tolerance. Transgenic carrotplants expressing BADH grew in the presence of highconcentrations of NaCl (up to 400 mM), the uppermostlevel of salt tolerance reported so far among geneti-cally modified crop plants (Kumar et al., 2004a). Chlo-roplast genetic engineering has also been used for thefirst time to our knowledge to enhance the capacity ofplants for phytoremediation. This was accomplishedby incorporating a native operon containing the merAand merB genes, which code for mercuric ion reductase(merA) and organomercurial lyase (merB), respectively,into the chloroplast genome in a single transformationevent. Stable integration of the merAB operon into thechloroplast genome resulted in high levels of toleranceto the organomercurial compound phenylmercuric

Figure 2. Selection of transplastomic plants.Shown are representative photographs oftransplastomic tobacco and lettuce shoots un-dergoing first (A and D), second (B and E), andthird (C and F, rooting) rounds of selection,respectively.

Figure 3. Evaluation of transgene integration into the chloroplast genome. DNA isolated from putative transplastomic shoots areanalyzed by PCR and Southern-blot analysis. A, 3P/3M and 5P/2M primer pairs (Kumar and Daniell, 2004) are used for PCRanalysis; PCR products of 3P/3M primers. Lane 1, Untransformed plant; lanes 2 to 4, transformed lines (1.6 kb); lane 1kb1, DNAmarker; lanes 5 to 7, PCR product with 5P/2M primers (3.2 kb) in transformed lines. B, The chloroplast genome is probed with aradiolabeled flank fragment. Lane 1, Untransformed plant; lanes 2 and 4, homoplasmic transplastomic plant; lane 3,heteroplasmic transplastomic plant.

Verma and Daniell

1138 Plant Physiol. Vol. 145, 2007

are not closely genetically linked, they can segregate indepen-dently during selection, so that lines that are homoplasmic forthe gene of interest may be difficult to obtain. This is particu-larly true if the gene of interest is under negative selection pres-sure because it exerts an adverse effect on the chloroplast. Forchloroplast gene inactivation or site-directed mutagenesis, aportable selectable cassette inserted within or near the gene ofinterest favours co-segregation of the desired mutation with themarker and facilitates the recovery of homoplasmic mutantlines.

Some markers confer a phenotype that is strong enough toallow direct selection of transformants. Relevant to this issue iswhether selection is lethal, such that non-transformed cells ortissues are rapidly killed, or selection is non-lethal and growth isonly retarded. The latter is thought to allow the sorting andamplification of transformed plastomes until a sufficient propor-tion is attained to confer the selectable phenotype, so that vig-orous growth of the transformed cells will resume. In the caseof non-lethal selection with spectinomycin, untransformed cellssurvive but are bleached, while transformed sectors are greenand form shoots, so they are readily recognized. While specti-nomycin inhibits plastid protein synthesis and causes bleachingin dicots, its action on cell proliferation is variable within thedicots. Spectinomycin inhibits cell proliferation in Solanaceousspecies such as tobacco but not in Brassicas (Zubko and Day,

1998). It has been argued that non-lethal selection is importantfor successful transformation in tobacco (Svab and Maliga,1993), but this may not be essential (Maliga, 2004; Verma andDaniell, 2007). In some cases, a marker will confer a phenotypethat is not strong enough for primary selection after transfor-mation, but can still be used for secondary selection onceestablished in a sufficient proportion of plastomes. The markersthat confer tolerance to herbicides provide a typical example asdiscussed below (Iamtham and Day, 2000; Ye et al., 2001;Dufourmantel et al., 2007). Another possibility is to use suchmarkers to enhance selection with a primary marker, as wasexploited with photosynthesis markers (see next section).

When the marker is a foreign gene that is inserted in theplastid genome, it is necessary to provide native expression sig-nals such as a chloroplast promoter and a 5¢UTR with transla-tion initiation signals and a 3¢UTR. The choice of such elementsand of chloroplast vectors has been reviewed recently (Lutzet al., 2007; Verma and Daniell, 2007; Verma et al., 2008). Inplants, the Prrn promoter with the ribosome binding site fromgene 10 of bacteriophage T7 or the psbA promoter and 5¢UTRare commonly used for high levels of expression. In Chlamydo-monas, a number of different chloroplast expression signalshave been employed such as those from atpA, psaA, psbA, orpsbD (Ishikura et al., 1999; Barnes et al., 2005; Michelet et al.,2010).

Table 1 Selectable markers for plastid transformation

Marker Selection Organism References Notes

Photosynthesis

atpB Photoautotrophy Chlamydomonas Boynton et al. (1988)

tscA Photoautotrophy Chlamydomonas Goldschmidt-Clermont (1991);

Kindle et al. (1991)

psaA ⁄ B Photoautotrophy Chlamydomonas Redding et al. (1998)

petB Photoautotrophy Chlamydomonas Cheng et al. (2005) See Figure 2

petA, ycf3, rpoA Photoautotrophy Tobacco Klaus et al. (2003) Enhanced selection

rbcL Photoautotrophy Tobacco Kode et al. (2006) Enhanced selection

Antibiotic resistance

rrnS Spectinomycin streptomycin Chlamydomonas Kindle et al. (1991); Newman et al.

(1990); Roffey et al. (1991)

Tobacco Svab et al. (1990)

Tomato Nugent et al. (2005)

rrnL Erythromycin Chlamydomonas Newman et al. (1990)

aadA Spectinomycin streptomycin See Table 2 Goldschmidt-Clermont (1991) See Table 2

nptII Kanamycin Tobacco Carrer et al. (1993)

Cotton Kumar et al. (2004b) Double selection with aphA-6

aphA-6 Kanamycin, amikacin Chlamydomonas Bateman and Purton (2000)

Kanamycin Tobacco Huang et al. (2002)

Cotton Kumar et al. (2004b) Double selection with nptII

Herbicide resistance

psbA DCMU, metribuzin Chlamydomonas Newman et al. (1992); Przibilla et al. (1991)

bar Phosphinothricin Tobacco Iamtham and Day (2000) Secondary selection; aadA-free

AHAS Sulfometuron methyl Porphyridium sp. Lapidot et al. (2002)

EPSP Glyphosate Tobacco Ye et al. (2003) Secondary selection; aadA-free

HPPD Diketonitrile Tobacco Dufourmantel et al. (2007) Secondary selection; aadA-free

Metabolism

BADH Betaine aldehyde Tobacco Daniell et al. (2001b)

codA 5-fluorocytosine Tobacco Serino and Maliga (1997) Negative selection

ARG9 Arg autotrophy Chlamydomonas Remacle et al. (2009)

ASA2 Trp analogues Tobacco Barone et al. (2009)


Markers for chloroplast transformation 543

Photosynthesis

Chloroplast genes that encode subunits of the photosyntheticcomplexes are often strictly essential for photosynthesis. InChlamydomonas, after transformation of a mutant host withthe wild-type gene, selection for photoautotrophic growth isvery stringent. Furthermore, if a mutant with a chloroplast dele-tion is used, its genetic stability ensures that the reversion rateis extremely low. This scheme was used with atpB in the initialdemonstration of chloroplast transformation (Boynton et al.,1988). A deletion spanning the tscA gene, which encodes asmall RNA involved in psaA trans-splicing, was also used as ahost for transformation with the wild-type gene and selectionfor restoration of photoautotrophic growth (Goldschmidt-Clermont, 1991; Kindle et al., 1991).

Chlamydomonas mutants with defects in photosystem I (PSI)have a high sensitivity to light-induced oxidative damage. Thisproperty was used to facilitate site-directed mutagenesis ofpsaA and psaB, the chloroplast genes for the major subunits ofPSI (Redding et al., 1998). A host strain deleted for both geneswas first constructed by sequentially substituting the two chlo-roplast genes with an antibiotic resistance marker (aadA) thatwas subsequently excised. This double deletion line was thenco-transformed with mutant versions of both psaA and psaB,each flanked by an aadA marker. Selection for antibiotic resis-tance as well as tolerance to moderate levels of light thenallowed recovery of transformants with modifications in bothpsaA and psaB, as long as the desired mutations allowed resid-ual activity of PSI (Redding et al., 1998).

A similar approach was also developed in tobacco by gener-ating photosynthesis-deficient lines through the insertion of aselectable marker for antibiotic resistance (aadA) in one of sev-eral different chloroplast genes (Klaus et al., 2003) or by the

combined excision of a chloroplast gene (rbcL) and marker(Kode et al., 2006). The resulting lines were heterotrophic andhad visible phenotypes of pigment deficiency. The photosynthe-sis mutant plants containing aadA were transformed with anaph-A6 marker gene together with a wild-type copy of thechloroplast gene (Klaus et al., 2003). Marker-free rbcL deletionmutants were re-transformed using aadA and the wild-typerbcL gene (Kode et al., 2006). The photoautotrophic trans-formed cells formed recognizable green sectors that could bechosen for subcloning, and they also had a growth advantageso that the recovery of homoplasmic lines was facilitated. Earlytransfer of resistant plants to soil is possible, without prolongedantibiotic selection, because selection for photosynthesis main-tains transgenic plastid genomes.

Antibiotic resistance

The translation machinery of the plastid has retained prokary-otic features, so that mutations in ribosomal proteins or in rRNAcan afford resistance to several antibiotics, such as spectinomy-cin, streptomycin and erythromycin. These provide markers forchloroplast transformation, but can also cause problemsbecause the rate of spontaneous mutation to antibiotic resis-tance may be of the same order of magnitude or even higherthan the frequency of transformation. Mutations of the 16SrRNA gene (rrnS) to spectinomycin or streptomycin resistancecan be used singly or in combination to facilitate the discrimina-tion of true transformants from spontaneous mutants that willusually be resistant to only one of the antibiotics (Newmanet al., 1990; Svab et al., 1990). A mutation in rps12(3¢), encod-ing a protein component of the small subunit of the plastidribosome, can confer resistance to streptomycin, but whetherthis can be used as a marker for primary selection was notreported (Staub and Maliga, 1992). Another approach is to use

Table 2 List of species in which aadA-based plastid transformation has been demonstrated

Organism Marker Resistance References Notes

Chlamydomonas Chlamydomonas reinhardtii aadA Spectinomycin streptomycin Goldschmidt-Clermont (1991)

Tobacco Nicotiana tabacum aadA Spectinomycin streptomycin Svab and Maliga (1993)

Tobacco Nicotiana tabacum aadA_GFP Spectinomycin Khan and Maliga (1999)

Arabidopsis Arabidopsis thaliana aadA Spectinomycin Sikdar et al. (1998) Sterile

Rice Oryza sativa aadA_GFP Streptomycin Khan and Maliga (1999) Heteroplasmic

aadA Streptomycin Lee et al. (2006) Heteroplasmic

Potato Solanum tuberosum aadA Spectinomycin streptomycin Sidorov et al. (1999)

Euglena Euglena gracilis aadA Spectinomycin streptomycin Doetsch et al. (2001)

Tomato Solanum lycopersicum aadA Spectinomycin streptomycin Ruf et al. (2001)

Oilseed rape Brassica napus aadA Spectinomycin Hou et al. (2003) Heteroplasmic

aadA Streptomycin Cheng et al. (2005) Heteroplasmic

Lesquerella Lesquerella fendleri aadA_GFP Spectinomycin streptomycin Skarjinskaia et al. (2003) Grafted on B. napus

Carrot Daucus carota aadA Spectinomycin Kumar et al. (2004b)

Soybean Glycine max aadA Spectinomycin Dufourmantel et al. (2004)

Petunia Petunia hybrida aadA Spectinomycin streptomycin Zubko et al. (2004)

Physcomitrella Physcomitrella patens aadA Spectinomycin Sugiura and Sugita (2004)

Lettuce Lactuca sativa aadA Spectinomycin Lelivelt et al. (2005); Ruhlman

et al. (2010)

Polyethylene

glycol ⁄ protoplastCauliflower Brassica oleracea var. botrytis aadA Spectinomycin Nugent et al. (2006)

Poplar Populus alba aadA Spectinomycin Okumura et al. (2006)

Cabbage Brassica capitata aadA Spectinomycin streptomycin Liu et al. (2007)

Sugarbeet Beta vulgaris aadA Spectinomycin De Marchis et al. (2009)

Eggplant Solanum melongena L. aadA Spectinomycin streptomycin Singh et al. (2010)



plastomic rice plants were not homoplasmic, evenafter two generations of continuous selection. Plastidtransformation of carrot, cotton, rice, and soybeanopens the door for modification of the plastid genomeof several crops that require embryogenesis.

METHODS FOR CONSTRUCTION OF PLASTIDTRANSFORMATION VECTORS AND GENERATIONOF TRANSPLASTOMIC PLANTS

Plastid gene expression is regulated both at thetranscriptional and posttranscriptional levels. Proteinlevels in chloroplasts depend on mRNA abundance,

which is determined by promoter strength and mRNAstability. However, high mRNA levels do not result inhigh-level protein accumulation as posttranscriptionalprocesses ultimately determine obtainable proteinlevels. Therefore, we have designed expression cas-settes for transgene assembly to achieve optimal levelsof protein accumulation in leaves (Fig. 1). The basicplastid transformation vector is comprised of flankingsequences and chloroplast-specific expression cas-settes (Fig. 1). Species-specific chloroplast flankingsequence (e.g. trnI/trnA) is obtained by PCR usingthe primers designed from the available chloroplastgenomes. The chloroplast expression cassette is com-

Table II. Engineering of agronomic traits, biopharmaceuticals, vaccine antigens, and biomaterials via the plastid genome

Traits/Gene Products Gene Promoter/5#/3# UTRs Literature Cited

Agronomic traitInsect resistance cry1A(c) Prrn/rbcL/rps16 McBride et al. (1995)

cry2Aa2 Prrn/ggagg (native)/psbA Kota et al. (1999)cry2Aa2 operon Prrn/native 5# UTR/psbA De Cosa et al. (2001)cry1Aa10 Prrn/native 5# UTR/psbA Hou et al. (2003)cry1Ab Prrn/T7 gene10/rbcL Dufourmantel et al. (2005)cry9Aa2 Prrn/native 5# UTR/rbcL Chakrabarti et al. (2006)

Herbicide resistance aroA (petunia) Prrn/ggagg/psbA Daniell et al. (1998)bar Prrn/rbcL/psbA Iamtham and Day (2000)

Disease resistance MSI-99 Prrn/ggagg/psbA DeGray et al. (2001)Drought tolerance TPS1 (yeast) Prrn/ggagg/psbA Lee et al. (2003)Phytoremediation merA/merB Prrn/ggagg/psbA Ruiz et al. (2003); Hussein et al. (2007)Salt tolerance badh Prrn/ggagg/rps16 Kumar et al. (2004a)CMS phaA Prrn/psbA/psbA Ruiz and Daniell (2005)

Biopharmaceutical proteinshST hST Prrn/T7 gene10/Trps16

PpsbA/Trps16Staub et al. (2000)

Insulin-like growth factor IGF-1n IGF-1s Prrn/PpsbA/TpsbA Daniell et al. (2005a)IFNa2b IFNa2b Prrn/PpsbA/TpsbA Arlen et al. (2007)HSA hsa Prrn/PpsbA/TpsbA Fernandez-San Millan et al. (2003)IFN-g Gus-IFN-g PpsbA/TpsbA Leelavathi and Reddy (2003)Monoclonal antibody Guy’s 13 Prrn/ggagg/TpsbA Daniell et al. (2004)Human Pins CTB-Pins PpsbA/TpsbA Prrn/T7

gene10/Trps16Ruhlman et al. (2007)

Vaccine antigensCholera toxin ctxB Prrn/ggagg/TpsbA Daniell et al. (2001a)Tetanus toxin tetC bacterial and synthetic Prrn/T7gene 10/TrbcL

atpB/TrbcLTregoning et al. (2003)

CPV CTB-2L21 GFP-2L21 Prrn/PpsbA/TpsbA Molina et al. (2004, 2005)Anthrax PA pag Prrn/PpsbA/TpsbA Watson et al. (2004); Koya

et al. (2005)Amebiasis lecA Prrn/PpsbA/TpsbA Chebolu and Daniell (2007)Plague CaF1-LcrV Prrn/PpsbA/TpsbA Y. Ding, P. Arlen, J. Adamovicz,

M. Singleton, and H. Daniell(unpublished data)

Rotavirus VP6 Prrn/PpsbA/TpsbA Birch-Machin et al. (2004)Hepatitis C NS3 Prrn/PpsbA/TpsbA Daniell et al. (2005a)Lyme disease OspA OspA-T PpsbA/TpsbA Glenz et al. (2006)BiomaterialsElastin-derived polymer EG121 Prrn/T7 gene 10/TpsbA Guda et al. (2000)pHBA ubiC Prrn/PpsbA/TpsbA Viitanen et al. (2004)

Polyhydroxybutyrate phb operon PpsbA/TpsbA Lossl et al. (2003)Xylanase xynA PpsbA/TpsbA Leelavathi et al. (2003)Tryptophan ASA2 Prrn/rbcL/rpL32

rbcL/accD-ORF184Zhang et al. (2001)

Monellin monellin Prrn/PpsbA/TpsbA Roh et al. (2006)

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1136 Plant Physiol. Vol. 145, 2007

visualized by using direct repeats of 649 bp to excise aadA,gusA and the native plastid rbcL gene resulting in pigment-defi-cient sectors (Figure 5). The number and sizes of sectorsobtained illustrates the frequency and timing of excision, whichtakes place throughout plant development (Kode et al., 2006).Sector formation also requires segregation of marker-free plas-tid genomes and marker-free plastids by cytoplasmic sortingduring plant growth and development. In the schemesdescribed above, excision of the aadA gene results in loss ofantibiotic resistance, which is promoted by removing antibioticselection. An alternative strategy is to promote direct-repeat-mediated excision of aadA by gain of function, such as herbi-cide resistance (Dufourmantel et al., 2007). This is illustrated inFigure 6 where the aadA gene interrupts the coding region ofthe 4-hydroxyphenylpyruvate dioxygenase (hppd) gene. A par-tial duplication of the hppd gene creates 403-bp direct repeatsthat flank aadA. Recombination between these 403-bprepeats excises aadA and concomitantly restores the hppdcoding sequence that is expressed and confers resistance to the

herbicide DKN. Direct-repeat-mediated excision of marker genesexploits native homologous recombination pathways in plastidsand provides a simple and effective method to excise markergenes from plastids. A similar scheme using a split bar geneflanking aadA and GFP genes has been used to isolate aadA-free transplastomic soybean plants resistant to PPT. The lengthof the duplicated bar repeat used was 367 bp (Lestrade et al.,2009). Raising the length of direct repeats to over 600 bpincreases the efficiency of the process, without comprisingtransformation frequency. The efficiency of marker removalusing short direct repeats of less than 400 bp could beimproved by including a negative selection marker (Serino andMaliga, 1997) in the excision cassette. Negative selection wouldbe expected to favour segregation of plastids that have lost theexcision cassette. Multiple cycles of transformation and markerexcision are achievable by using unrelated direct-repeatsequences to flank the marker gene at each transformationstep. Rigorous proof for aadA removal by direct-repeat excisionhas been obtained by re-transforming a transplastomic plantwith the same aadA marker gene (Kode et al., 2006). Predomi-nance of homologous DNA recombination in plastids rangingfrom those found in algae to flowering plants suggests thatdirect-repeat-mediated excision will be widely applicable. Of themarker removal methods that have been demonstrated intobacco, direct-repeat-mediated excision is the first to be alsoimplemented in a major crop, namely soybean (Lestrade et al.,2009).

Excision of marker genes using site-specific recombinases. TheCre site-specific recombinase promotes strand-exchangebetween 34-bp loxP sites and is derived from the P1 bacterio-phage of Escherichia coli (Sternberg and Hamilton, 1981).When the aadA gene is flanked by directly repeated 34-bp loxPsites in plastid DNA, it can be removed by introducing Crerecombinase into plastids (Corneille et al., 2001; Hajdukiewiczet al., 2001; Lutz et al., 2006b; Oey et al., 2009b). The processappears to be efficient giving rise to a high frequency of aadA-free seedlings when a highly expressed nucleus-localized cregene, whose product is targeted to plastids, is introduced intotransplastomic plants. This can be achieved by stable or

Figure 4 The frequency of excision of marker genes is raised by increas-

ing the number of direct repeats (Iamtham and Day, 2000). Expression

of gusA, aadA and bar genes is driven by duplicated rrn promoter

(green) and triplicated 3¢ psbA UTR regulatory elements (blue). Selection

was switched from spectinomycin to herbicide (phosphinothricin) early

in the transformation process. Recombination between the two 174-bp

direct repeats (green dotted arrow) gave rise to herbicide (phosphino-

thricin)-resistant plants in the T0 generation. Marker-free tobacco plants

containing the gusA transgene are derived from recombination between

the outermost 418-bp repeats (blue dotted arrow), which takes place at

a higher frequency and gives rise to marker-free T1 seedlings (24% of

total seedlings).

Figure 5 Direct-repeat-mediated excision takes place at all stages of

leaf development in tobacco. The excision cassette contains rbcL, aadA

and gusA genes. Loss of the photosynthetic rbcL gene arrests chloro-

plast development and gives rise to yellow cells, which are easily visual-

ized (Kode et al., 2006).

Figure 6 Excision of aadA mediated by gain of herbicide resistance in

tobacco (Dufourmantel et al., 2007). Initial selection for aadA on specti-

nomycin is replaced by selection on the herbicide DKN promoting the

recombination event that deletes aadA and restores the hppd gene.



Review article

The chloroplast transformation toolbox: selectable markersand marker removalAnil Day1 and Michel Goldschmidt-Clermont2,*

1Faculty of Life Sciences, The University of Manchester, Manchester, UK2Departments of Plant Biology and of Molecular Biology, University of Geneva, Geneve, Switzerland

Received 8 November 2010;

revised 29 January 2011;

accepted 31 January 2011.

*Correspondence (Tel +41 22 379 6188;

fax +41 22 379 6868; email

[email protected])

Keywords: plastid transformation,

marker-free, aadA, aphA-6, Chla-

mydomonas reinhardtii, Nicotiana

tabacum.

SummaryPlastid transformation is widely used in basic research and for biotechnological applications.Initially developed in Chlamydomonas and tobacco, it is now feasible in a broad range of spe-cies. Selection of transgenic lines where all copies of the polyploid plastid genome are trans-formed requires efficient markers. A number of traits have been used for selection such asphotoautotrophy, resistance to antibiotics and tolerance to herbicides or to other metabolicinhibitors. Restoration of photosynthesis is an effective primary selection method in Chla-mydomonas but can only serve as a screening tool in flowering plants. The most successfuland widely used markers are derived from bacterial genes that inactivate antibiotics, such asaadA that confers resistance to spectinomycin and streptomycin. For many applications, thepresence of a selectable marker that confers antibiotic resistance is not desirable. Efficientmarker removal methods are a major attraction of the plastid engineering tool kit. Theyexploit the homologous recombination and segregation pathways acting on chloroplastgenomes and are based on direct repeats, transient co-integration or co-transformation andsegregation of trait and marker genes. Foreign site-specific recombinases and their target sitesprovide an alternative and effective method for removing marker genes from plastids.

Introduction

Plastid transformation offers an important tool to investigatemany aspects of plant physiology and the regulation of geneexpression. It has also gained strong interest for applications inbiotechnology because of several advantages compared withtransformation of the nuclear genome (Meyers et al., 2010).The most prominent is that plastid transgene expression can beremarkably high and the desired recombinant protein may rep-resent up to 70% of leaf protein (Daniell et al., 2009; Oeyet al., 2009a; Ruhlman et al., 2010). It is also important that inthe majority of flowering plants including major crops, inheri-tance of the plastid genome is through the maternal parent(Corriveau and Coleman, 1988), and transmission of plastidsthrough pollen is very rare (Ruf et al., 2007; Svab and Maliga,2007). Thus, plastid transformation provides a strong level ofbiological containment. Exceptional pollen transmission follow-ing transfer of a chloroplast marker to the nucleus is also a veryrare event (Huang et al., 2003; Stegemann et al., 2003; Rufet al., 2007; Bock and Timmis, 2008). Another advantage isthat the integration of a transgene in the plastid genome pro-ceeds by homologous recombination and is therefore preciseand predictable. Hence, variable position effects on geneexpression or the inadvertent inactivation of a host gene byintegration of the transgene are avoided. Furthermore, plastidgenes are not subject to gene silencing or RNA interference. Itis also noteworthy that multiple transgenes organized as a poly-cistronic unit can be expressed from the plastid genome (Stauband Maliga, 1995; De Cosa et al., 2001; Quesada-Vargas et al.,2005). Recent reviews have focused on the numerous applica-

tions of plastid transformation for the production of pharma-ceuticals or biofuels, and on the development of transformationprotocols in a rapidly increasing number of plant and algal spe-cies (Griesbeck et al., 2006; Bock, 2007; Verma and Daniell,2007; Verma et al., 2008; Daniell et al., 2009; Wang et al.,2009; Cardi et al., 2010; Meyers et al., 2010; Specht et al.,2010). Here, we focus on selectable markers, which are essen-tial tools for chloroplast transformation.

Chloroplast transformation

Chloroplasts are specialized plant organelles best known to hostphotosynthesis, but that also harbour many other importantbiosynthetic pathways. During plant development, they arise bydifferentiation of proplastids, precursors that are found in meri-stematic tissues and can also develop into many other formssuch as the amyloplasts in roots or the chromoplasts in fruits.Plastid transformation can involve delivery of DNA into chlorop-lasts or non-green plastids. Once stable transformation hasbeen achieved, all plastid types within the plant will contain thesame transgenic plastome. Thus, in flowering plants containinga variety of plastid developmental forms, the term plastid trans-formation is more accurate than chloroplast transformation.During evolution, plastids were most probably derived from anendosymbiotic cyanobacterium (Gould et al., 2008). From thisancestor, plastids have retained a small autonomous genomethat contains approximately a hundred genes in vascular plantsand Chlamydomonas. From the Gram-negative cyanobacterium,the plastids also inherited the two membranes that constitutethe envelope, which in secondary or tertiary endosymbiotes is

ª 2011 The Authors540 Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd

Plant Biotechnology Journal (2011) 9, pp. 540–553 doi: 10.1111/j.1467-7652.2011.00604.x

Update on Plastid Transformation Vectors

Chloroplast Vector Systems for Biotechnology Applications1

Dheeraj Verma and Henry Daniell*

Department of Molecular Biology and Microbiology, College of Medicine, University of Central Florida,Orlando, Florida 32816–2364

Chloroplasts are ideal hosts for expression of trans-genes. Transgene integration into the chloroplastgenome occurs via homologous recombination offlanking sequences used in chloroplast vectors. Iden-tification of spacer regions to integrate transgenes andendogenous regulatory sequences that support opti-mal expression is the first step in construction ofchloroplast vectors. Thirty-five sequenced crop chlo-roplast genomes provide this essential information.Various steps involved in the design and constructionof chloroplast vectors, DNA delivery, and multiplerounds of selection are described. Several crop specieshave stably integrated transgenes conferring agro-nomic traits, including herbicide, insect, and diseaseresistance, drought and salt tolerance, and phytore-mediation. Several crop chloroplast genomes havebeen transformed via organogenesis (cauliflower[Brassica oleracea], cabbage [Brassica capitata], lettuce[Lactuca sativa], oilseed rape [Brassica napus], petunia[Petunia hybrida], poplar [Populus spp.], potato [Sola-num tuberosum], tobacco [Nicotiana tabacum], and to-mato [Solanum lycopersicum]) or embryogenesis (carrot[Daucus carota], cotton [Gossypium hirsutum], rice [Oryzasativa], and soybean [Glycine max]), and maternal inher-itance of transgenes has been observed. Chloroplast-derived biopharmaceutical proteins, including insulin,interferons (IFNs), and somatotropin (ST), have beenevaluated by in vitro studies. Human INFa2b trans-plastomic plants have been evaluated in field studies.Chloroplast-derived vaccine antigens against bacterial(cholera, tetanus, anthrax, plague, and Lyme disease),viral (canine parvovirus [CPV] and rotavirus), andprotozoan (amoeba) pathogens have been evaluatedby immune responses, neutralizing antibodies, andpathogen or toxin challenge in animals. Chloroplastshave been used as bioreactors for production of bio-polymers, amino acids, and industrial enzymes. Oraldelivery of plant cells expressing proinsulin (Pins) inchloroplasts offered protection against development of

insulitis in diabetic mice; such delivery eliminatesexpensive fermentation, purification, low temperaturestorage, and transportation. Chloroplast vector sys-tems used in these biotechnology applications aredescribed.

ADVANTAGES OF PLASTID TRANSFORMATION

Chloroplasts are members of a class of organellesknown as plastids and are found in plant cells andeukaryotic algae. As the site of photosynthesis, chlo-roplasts are the primary source of the world’s foodproductivity and they sustain life on this planet. Otherimportant activities that occur in plastids includeevolution of oxygen, sequestration of carbon, produc-tion of starch, synthesis of amino acids, fatty acids, andpigments, and key aspects of sulfur and nitrogenmetabolism. Chloroplasts are generally considered asderivative of a cyanobacterial ancestor that was cap-tured early during the evolution of a eukaryotic cell.However, the chloroplast genome is considerably re-duced in size as compared to that of free-living cya-nobacteria, but the genomic sequences that are stillpresent show clear similarities (Martin et al., 2002).Land plant chloroplast genomes typically contain 110 to120 unique genes, whereas cyanobacteria contain morethan 1,500 genes. Many of the missing genes are presentin the nuclear genome of the host (Martin et al., 2002).

In most angiosperm plant species, plastid genes arematernally inherited (Hagemann, 2004), and therefore,transgenes in these plastids are not disseminated bypollen. This makes plastid transformation a valuabletool for the creation and cultivation of geneticallymodified plants that are biologically contained, thusposing lower environmental risks (Daniell, 2002, 2007).This biological containment strategy is therefore suit-able for establishing the coexistence of conventionaland genetically modified crops. Cytoplasmic male ste-rility (CMS) presents a further genetic engineering ap-proach for transgene containment (Ruiz and Daniell,2005). Furthermore, plant-derived therapeutic pro-teins are free of human pathogens and mammalianviral vectors. Therefore, plastids provide a viable al-ternative to conventional production systems such asmicrobial fermentation or mammalian cell culture.

Another advantage of plastid transformation is theability to accumulate large amounts of foreign protein(up to 46% of total leaf protein) when the transgene isstably integrated (De Cosa et al., 2001). This is due tothe polyploidy of the plastid genetic system with up to

1 This work was supported by the U.S. Department of Agriculture(grant no. 3611–21000–017–00D) and by the National Institutes ofHealth (grant no. 5R01 GM 63879–06).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Henry Daniell ([email protected]).

www.plantphysiol.org/cgi/doi/10.1104/pp.107.106690

Plant Physiology, December 2007, Vol. 145, pp. 1129–1143, www.plantphysiol.org � 2007 American Society of Plant Biologists 1129

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