157

Protein expression in plastids Peter B Heifetz* and Ann Marie Tuttle†

The genome of the plastid has generated much interest as a including roots, contain leucoplasts. The different plastids target for plant transformation. The characteristics of plastid are further distinguished by their varying capacities for the transgenes both reflect the prokaryotic origin of plastid expression of organelle-encoded genes. Illuminated organelles and provide a unique set of features that are chloroplasts, in particular, possess extraordinarily high currently lacking in genes introduced into the plant nucleus. rates of transcription and translation. These facilitate the Recent progress has been made in understanding plastid accumulation of large amounts of soluble ribulose bisphos- expression of recombinant proteins. phate carboxylase (thus insuring that enzyme levels do not limit photosynthetic carbon fixation) and allow the rapid Addresses turnover of redox-sensitive electron transfer components, *Torrey Mesa Research Institute, 3115 Merryfield Row, such as the D1 protein of photosystem II [4]. This feature San Diego, California 92121, USA; e-mail: [email protected] makes leaf chloroplasts suitable for the production of large † Syngenta Agribusiness Biotechnology Research Inc, 3054 Cornwallis amounts of recombinant proteins. In this review, we sum- Road, Research Triangle Park, North Carolina 27709, USA marize recent progress in understanding plastid protein Current Opinion in Plant Biology 2001, 4:157–161 expression and in transformation technology. 1369-5266/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Plastid transformation Boynton and colleagues [5,6] demonstrated that all copies Abbreviations aadA adenosyl-3′-adenyltransferase of the plastid genome in the single large chloroplast of the EPSPS 5-enolpyruvylshikimate-3-phosphate synthase green alga Chlamydomonas reinhardtii could, following GFP green fluorescent protein delivery of cloned DNA by particle bombardment, inte- NEP nuclear-encoded RNA polymerase grate such DNA in a targeted manner via homologous PEG polyethylene glycol recombination. These pioneering studies made possible PEP plastid-encoded RNA polymerase subsequent efforts to transform the plastids of higher plants [7]. They also laid out the unique ‘ground rules’ for Introduction plastid transformation as compared to nuclear transfor- One of the unique and defining characteristics of plants is mation: foreign DNA must be delivered across the double the presence of plastid organelles. These endosymbiotic membrane of the plastid envelope and across the cell remnants of a once free-living cyanobacterial progenitor [1] membrane (and across the cell wall, if present); homolo- have, over evolutionary time, given up the vast majority of gous (legitimate) recombination is the normal mode of their genes and cellular functions to become the energy DNA integration into the plastid genome. When suitable transduction and metabolic centers of the plant cell. Among regions of homology between the introduced DNA and the primitive features of the cyanobacterial progenitor that the target are provided, appropriate selectable markers are retained in plastids is a circular and largely prokaryotic for plastid transformation must facilitate the segregation chromosome. This genome of circa 50–290 kilobases is of transformed genome copies at the expense of non- remarkably similar across the algal and plant lineages with transformed ones; complete segregation of all the genome regard to the complement of genes encoded within it, their copies within a plastid, and within all of the plastids of a relative order and their sequence [2]. The plastid genome cell or tissue, must occur during clonal propagation in is also distinguished by its copy number, which even in the order to confer a stably transformed phenotype; and simplest of plants far exceeds the ploidy of the nuclear finally, bona fide plastid transformation events must be chromosomes. In a mature leaf cell, in which there may be readily distinguishable at the molecular level from spon- as many as 100 plastids each containing upwards of 10–100 taneous mutations or ectopic nuclear integrations of the genomes, it is possible to find 10,000 or more identical transforming DNA. copies of each plastid gene [3]. In addition to particle bombardment, polyethylene glycol Plastids are distributed ubiquitously throughout the differ- (PEG) treatment of protoplasts [8,9] has been found to entiated cells of diverse plant organs and tissues. Although be suitable for the delivery of DNA to plastids for stable each plant cell type contains identical copies of the plastid transformation. This method appears more prone to cre- genome because of their organelles’ shared biogenesis ating unexpected nuclear mutations than is particle from undifferentiated proplastids, the organelles them- bombardment, however, as demonstrated by the rela- selves can vary tremendously in morphology and function. tively high level of selection escapes [10] and Thus, leaves and green tissues contain photosynthetic physiological artifacts [11] observed in PEG-transformed chloroplasts; mature fruits and flowers contain pigmented tobacco. Recently, an alternative means of directly deliv- chromoplasts; tubers and other storage organs contain amy- ering DNA to plastids was developed by Knoblauch et al. loplasts or elaioplasts; and other non-green tissues, [12]. These workers used a sub-micron-diameter syringe 158 Plant biotechnology

driven by the controlled thermal expansion of a liquid Controlling plastid gene expression metal alloy to microinject DNA into chloroplasts. The plastid rpoA, rpoB, rpoC1 and rpoC2 genes encode the Although this method has not yet been shown to yield catalytic subunits of a eubacterial-type RNA polymerase regenerated and fertile transformed plants, transient that recognizes upstream sequences that have high homol- expression of the green fluorescent protein (GFP) trans- ogy to the consensus –10 and –35 regions typically found gene was achieved in tobacco without apparent harm to in eubacterial promoters. Promoter recognition by this the injected cells. plastid-encoded RNApolymerase (PEP) is mediated by sigma-like factors that are encoded in the plant nucleus. The ease with which plastid DNAcan undergo homo- These factors are expressed in a regulated manner in logous recombination between even short regions of response to environmental or developmental cues and are sequence identity [13] has significant implications for the imported post-translationally into plastids via transit pep- engineering of gene expression within plastids. First, tides [19]. Interestingly, plastids contain a second (and in targeted integration is the norm rather than the exception. spinach, likely a third) complete transcriptional apparatus This means that the location of transgenes can be predicted that is entirely nuclear encoded. This apparatus contains a with precision so as to minimize interference with endoge- single-subunit that is phylogenetically related to the RNA nous plastid genes and to prevent regulatory sequences polymerases of fungal and plant mitochondria, and of bac- near the integration site from influencing transgene teriophages such as T7 and T3 [20–22]. The promoter expression. An added benefit is that comparatively few elements recognized by the nuclear-encoded RNA poly- transgenic events need to be characterized as, in the merase (NEP) bear little similarity to eubacterial or PEP absence of illegitimate recombination, only single-locus promoters [23] and require one or more specificity factors insertion events occur. (An exception to this norm results for their correct interaction with the polymerase [24]. when the transforming DNA has been targeted to the Plastid genes can have only PEP promoters, only NEP inverted-repeat region of the plastid genome. Dual inte- promoters, or hybrid promoter regions that contain both gration then occurs due to homologous copy correction PEP and NEP elements. The significance for message between the two repeat regions.) Second, homologous accumulation of these multiple transcription initiation recombination occurring spontaneously between directly sites is unclear. A recent study suggests, however, that the repeated sequences flanking an integrated selectable NEP may recognize DNA promiscuously and, thus, could marker gene can result in the excision of the marker gene be capable of transcribing any plastid gene [25]. from the genome when selective conditions are relaxed Nonetheless, PEP and NEP elements are each capable [14]. This approach has recently been shown to be feasible of directing the expression of foreign genes in in plastids of higher plants [15••], raising the possibilities plastids [26,27]. of sequential transformation by marker recycling and of elimination of bacterial antibiotic resistance genes from Trans-activation expression systems using completely het- the final transgenic plants. erologous RNA polymerases, such as those from T7 phage, have also been developed for plastids [28,29•]. This A recent advance in selectable marker technology for plas- approach allows the efficient and controlled transcription tids was the discovery that GFP can be stably transformed of target plastid transgenes as long as appropriate promoters, and expressed in the leaf chloroplasts of tobacco [16,17••] which are recognized by the T7 polymerase but not by and potato [18••]. In potato, GFP was also expressed in NEP or PEP, are located upstream. The primary advantage tuber amyloplasts, albeit at a 100-fold lower level than in of trans-activation is that it allows the imposition of develop- chloroplasts. Transient expression of GFP has been mental, tissue-specific, or chemically-inducible regulation achieved in carrot, marigold and pepper [12], demonstrating upon the expression of plastid transgenes through control that DNA can be delivered to plastids in these plant of the polymerase by nuclear promoters of the desired species. An even more versatile use of GFP was developed specificity. Using the tobacco PR-1a promoter [30] to by Khan and Maliga [17••] who fused its coding sequence direct the expression of a plastid-targeted T7 polymerase, to that of the aadA gene, which encodes adenosyl-3′- β-glucuronidase (GUS) and a cellulose-degrading enzyme adenyltransferase. The resulting fusion molecule was were expressed at high levels in tobacco chloroplasts in functional both as an antibiotic-resistance gene and as a response to foliar application of an inexpensive field- fluorescent protein in multiple types of plastids in tobacco. registered compound [29•]. Moreover, following its introduction by bombardment and selection using streptomycin, activity of this fusion mole- The production of primary transcripts is not typically the cule was detectable at low levels in the plastids of rice limiting step for the expression of plastid genes, although suspension cells. These rice cells proved to be highly het- this step does limit plant nuclear expression and, to some eroplasmic, however, with only a small fraction of the degree, eubacterial expression. Instead, the rate of protein plastid genomes transformed and no plants regenerated. synthesis in plastids is much more dependent on post- Nonetheless, this remains the only successful demonstra- transcriptional processes, which include transcript tion of the stable delivery of foreign DNA to the plastids processing and stability, the conversion (by post-transcription of a cereal species. RNA-editing mechanisms) of cytidines to uridines in certain Protein expression in plastids Heifetz and Tuttle 159

editing-dependent plastid messages, and translational ini- [41] demonstrated that expression of a wild-type tiation or elongation on polyribosomes. These events are 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mediated at the RNA level by the binding of nuclear- gene and its complete transit-peptide sequence from petunia encoded factors to the 5′ untranslated leader and 3′ in the plastids of tobacco resulted in a ten-fold-greater untranslated trailer regions of the plastid messages [31] level of tolerance to the herbicide glyphosate. The expres- and, in the case of RNA editing, by the site-specific inter- sion of additional herbicide-tolerance genes in plastids, action of an editing complex with RNA segments flanking including mutant EPSPS, protoporphyrinogen IX oxidase, the targeted nucleotides [32,33]. The efficient expression and several bacterial genes, has been described recently in of transgenes in plastids thus requires not only the use of the patent literature [29•]. appropriate promoters but also the presence of the correct sequences in the 5′ and 3′ untranslated regions. The One of the most exciting potential applications of plastid occurrence of RNA editing also necessitates care in the protein expression is in the production of recombinant choice of the cloned sequences flanking a plastid expres- proteins for industrial, pharmaceutical or other value- sion cassette that are designed to direct homologous added uses. The biological containment afforded by integration into the plastid genome. Editing sites, but not plastid localization is attractive from the standpoint of editing itself, may be conserved among related plant reducing the risks of the pollen transmission of genes species [34]. Hence, it is important to prevent the inad- that may encode metabolically active proteins. vertent introduction of editing-dependent sequences into Moreover, the high levels of expression that have been a genetic background that lacks the capacity for correct reported for plastid-encoded proteins could effect sub- processing when flanking sequences from one plant stantial cost savings in cases in which large amounts of species are used to direct integration into the plastids of a tissue must be processed in order to yield purified mole- second plant species [34]. cules. Plastids are also capable of disulfide-bond formation and hydrolysis via their endogenous thiore- Uniparental inheritance of plastid genes doxin and protein disulfide isomerase enzyme systems. Another unusual feature of plastid genes is their non- Mammalian proteins, such as the growth hormone soma- Mendelian mode of inheritance. In gymnosperm plants, totropin, normally require passage through the plastid DNA is maintained in sperm cells but not egg cells endoplasmic reticulum in order to attain their mature and is therefore transmitted uniparentally by the male. In disulfide-bonded conformation. Recently, Staub et al. the majority of angiosperms (including most of the impor- [42••] demonstrated that metabolically active human tant crops with the exception of alfalfa and to a limited somatotropin could be expressed and correctly processed extent potato), however, it is the pollen that loses the plastid in the plastids of tobacco where it accumulated to 7% of DNA after mitosis I. Consequently, pollen cannot transmit total soluble protein. In contrast to this successful the contents of the plastid genome to the zygote [35]. This expression of a mammalian protein, attempts by Guda et could, in theory, prevent the spread of plastid transgenes al. [43] to express a protein biopolymer derived from through pollen to neighboring crops or related wild mammalian elastin were disappointing. These authors species [36•]. found that although the synthetic biopolymer gene con- sisting of multiple repeating units of the amino-acid Recombinant protein expression in plastids sequence GVGVP was transcribed effectively in tobacco The plastid genome is an attractive location for the engi- plastids, the accumulated message was either not readily neering of pest-resistance and herbicide-tolerance traits, translatable by the plastid ribosomes or was rapidly which typically benefit from high and stable levels of gene turned over. Consequently, the levels of polymer product expression. Insecticidal Bt proteins derived from Bacillus detected in plastids were low and thus highly inefficient thuringienesis cry genes are particularly amenable to plastid compared to expression of the same recombinant pro- expression in tobacco. McBride et al. [37] and, more tein in the plant nucleus. recently, Kota et al. [38•] demonstrated that plastid trans- formation with native bacterial cry sequences, produces Conclusions and future prospects extraordinarily high levels of protein accumulation as com- Plastid biotechnology has now progressed to the point at pared to nuclear transformation with codon-optimized which a range of proteins from prokaryotic and eukaryotic transgenes. The resulting leaf tissue is extremely toxic to sources have been expressed effectively in leaf chloroplasts target insect larvae. In a recent follow-up study [39••], it and other plastids of tobacco. Progress is still needed in the was found that the introduction of the complete Bt cry2Aa2 improvement of transformation technology (to expand the operon into tobacco plastids caused even higher levels of range of plant species that can be efficiently transformed) expression than did the single genes. This was accom- and in the control of heterologous gene expression in panied by the formation of cuboidal Bt protein crystals. plastids at the post-transcriptional level. Overcoming these This work is the first to demonstrate the expression of a challenges will result in a means of producing high levels natural bacterial operon in plastids and follows on from the of recombinant proteins or pathway-derived metabolites in initial report by Staub and Maliga [40] of chimeric operon a stable and predictable manner with the potential for expression from the tobacco plastid genome. Daniell et al. robust physical and genetic containment. 160 Plant biotechnology

Acknowledgements resistant to high levels of this antibiotic because of naturally occurring muta- tions in their 16S ribosomal RNA. The authors thank Eric Boudreau for a critical reading of 18. Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub JM, the manuscript. •• Nehra NS: Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J 1999, 19:209-216. References and recommended reading The authors describe both the first published use of GFP as a plastid marker Papers of particular interest, published within the annual period of review, and stable plastid transformation in a food crop. Their results show a greater have been highlighted as: than 100-fold difference in the expression of protein in chloroplasts and • of special interest amyloplasts. •• of outstanding interest 19. Allison LA: The role of sigma factors in plastid transcription. Biochimie 2000, 82:537-548. 1. Moreira D, Le Guyader H, Philippe H: The origin of red algae and the evolution of chloroplasts. Nature 2000, 405:69-72. 20. Gray MW, Lang BF: Transcription in chloroplasts and mitochondria: a tale of two polymerases. Trends Microbiol 1998, 6:1-3. 2. Gray MW: Evolution of organellar genomes. Curr Opin Genet Dev 1999, 9:678-687. 21. Hess WR, Borner T: Organellar RNA polymerases of higher plants. 190 3. Bendich AJ: Why do chloroplasts and mitochondria contain so Int Rev Cytol 1999, :1-59. many copies of their genome? BioEssays 1987, 6:279-282. 22. Lerbs-Mache S: Regulation of rDNA transcription in plastids of higher plants. 82 4. Heifetz PB, Lers A, Turpin DH, Gillham NW, Boynton JE, Osmond CB: Biochimie 2000, :525-535. dr and spr/sr mutants of Chlamydomonas reinhardtii affecting D1 23. Hajdukiewicz PTJ, Allison LA, Maliga P: The two RNA polymerases protein function and synthesis define two independent steps encoded by the nuclear and the plastid compartments transcribe leading to chronic photoinhibition and confer differential fitness. distinct groups of genes in tobacco plastids. EMBO J 1997, Plant Cell Environ 1997, 20:1145-1157. 16:4041-4048. 5. Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, 24. Bligny M, Courtois F, Thaminy S, Chang C-C, Lagrange T, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Baruah-Wolff J, Stern D, Lerbs-Mache S: Regulation of plastid rDNA Shark KB et al.: Chloroplast transformation in Chlamydomonas transcription by interaction of CDF2 with two different RNA with high velocity microprojectiles. Science 1988, 240:1534-1538. polymerases. EMBO J 2000, 19:1851-1860. 6. Newman SM, Boynton JE, Gillham NW, Randolph-Anderson BL, 25. Krause K, Maier RM, Kofer W, Krupkinska K, Herrmann RG: Johnson AM, Harris EH: Transformation of chloroplast ribosomal Disruption of plastid-encoded RNA polymerase genes in tobacco: RNA genes in Chlamydomonas: molecular and genetic expression of only a distinct set of genes is not based on characterization of integration events. Genetics 1990, 126:875-888. selective transcription of the plastid chromosome. Mol Gen 7. Svab Z, Hajdukiewicz P, Maliga P: Stable transformation of plastids Genet 2000, 263:1022-1030. in higher plants. Proc Natl Acad Sci USA 1990, 87:8526-8530. 26. Sriraman P, Silhavy D, Maliga P: The phage-type PclpP-53 plastid 8. Golds T, Maliga P, Koop H-U: Stable plastid transformation in promoter comprises sequences downstream of the transcription PEG-treated protoplasts of Nicotiana tabacum. Biotechnology initiation site. Nucleic Acids Res 1998, 26:4874-4879. 1993, 11:95-97. 27. Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop HU: In vivo analysis 9. O’Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P: Chloroplast of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation in plants: polyethylene glycol (PEG) treatment transformation: tobacco plastid gene expression is controlled of protoplasts is an alternative to biolistic delivery systems. by modulation of transcript levels and translation efficiency. Plant J 1993, 3:729-738. Plant J 1999, 19:333-345. 10. Koop HU, Steinmuller K, Wagner H, Rossler C, Eibl C, Sacher L: 28. McBride KE, Schaaf DJ, Daley M, Stalker DM: Controlled expression Integration of foreign sequences into the tobacco plastome via of plastid transgenes in plants based on a nuclear DNA-encoded polyethylene glycol-mediated protoplast transformation. Planta and plastid-targeted T7 RNA polymerase. Proc Natl Acad Sci USA 1996, 199:193-201. 1994, 91:7301-7305. 11. Nixon PJ, Maliga P: Reply… chlororespiration: only half a story. 29. Heifetz PB: Genetic engineering of the chloroplast. Biochimie Trends Plant Sci 1999, 4:51. • 2000, 82:655-666. This paper includes a description of a chemically inducible transactivation 12. Knoblauch M, Hibberd JM, Gray JC, van Bel AJ: A galinstan system for the expression of genes from the plastid genome. Such an expansion femtosyringe for microinjection of eukaryotic approach is important when heterologous gene products are deleterious to organelles and prokaryotes. Nat Biotechnol 1999, 17:906-909. the plant because of either their high expression levels or their biological activity. The use of induction in mature plants allows the expression of other- 13. Kavanagh TA, Thanh ND, Lao NT, McGrath N, Peter SO, Horvath EM, wise toxic molecules as transformation is performed in the absence of Homeologous plastid DNA transformation in Dix PJ, Medgyesy P: expression, which is only enabled once the nuclear transactivator and tobacco is mediated by multiple recombination events. Genetics plastid target are brought together in a sexual cross. 1999, 152:1111-1122. 30. Uknes S, Dincher S, Friedrich L, Negrotto D, Williams S, 14. Fischer N, Stampacchia O, Redding K, Rochaix JD: Selectable Thompson-Taylor H, Potter S, Ward E, Ryals J: Regulation of marker recycling in the chloroplast. Mol Gen Genet 1996, pathogenesis-related protein-1a gene expression in tobacco. 251:373-380. Plant Cell 1993, 5:159-169. 15. Lamtham S, Day A: Removal of antibiotic resistance genes from 31. Barkan A, Goldschmidt-Clermont M: Participation of nuclear genes •• transgenic tobacco plastids. Nat Biotechnol 2000, 18:1172-1176. in chloroplast gene expression. Biochimie 2000, 82:559-572. The work described in this paper and [14] demonstrates the feasibility of excising selectable marker genes or other sequences from the plastid 32. Chaudhuri S, Carrer H, Maliga P: Site-specific factor involved in the genomes of stably transformed plants. editing of the psbL mRNA in tobacco plastids. EMBO J 1995, 14:2951-2957. 16. Gray JC, Hibberd JM, Linley PJ, Uijtewaal B: GFP movement between chloroplasts. Nat Biotechnol 1999, 17:1146. 33. Bock R, Koop U: Extraplastidic site-specific factors mediate RNA editing in chloroplasts. EMBO J 16 17. Khan MS, Maliga P: Fluorescent antibiotic resistance marker for 1997, :3282-3288. •• tracking plastid transformation in higher plants. Nat Biotechnol 34. Corneille S, Lutz K, Maliga P: Conservation of RNA editing between 1999, 17:910-915. rice and maize plastids: are most editing events dispensable? Mol A technical advance resulting in the first stable integration of foreign DNA Gen Genet 2000, 264:419-424. into the plastid genome of a monocot plant. The work uses an approach in which the initial selection of cultured cells on streptomycin is followed by 35. Nagata N, Saito C, Sakai A, Kuroiwa H, Kuroiwa T: The selective screening for green fluorescence. The aadA gene product catalyzes the increase or decrease of organellar DNA in generative cells just covalent modification of both streptomycin and spectinomycin. Although the after pollen mitosis one controls cytoplasmic inheritance. Planta latter is a more effective plastid-selectable marker, most cereals are naturally 1999, 209:53-65. Protein expression in plastids Heifetz and Tuttle 161

36. Scott SE, Wilkinson MJ: Low probability of chloroplast movement These protoxins form crystalline inclusion bodies in the natural host, which • from oilseed rape (Brassica napus) into wild Brassica rapa. Nat increase their stability. The authors achieved the expression of one such Biotechnol 1999, 17:390-392. complete operon, which was encoded by three genes, in tobacco plastids. A theoretical study comparing the effect of nuclear and plastid transfor- This expression resulted in the production of cuboidal crystals within leaf mation on transgene flow in a readily out-crossed plant species. Plastid chloroplasts. The resulting proteins were immensely toxic even to recalcitrant expression blocks pollen transmission but still allows the production of pests such as beet armyworm and cotton bollworm. weedy hybrids if the female parent is pollinated by a related species. 40. Staub JM, Maliga P: Expression of a chimeric uidA gene indicates 37. McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P: that polycistronic mRNAs are efficiently translated in tobacco Amplification of a chimeric Bacillus gene in chloroplasts leads to plastids. Plant J 1995, 7:845-848. an extraordinary level of an insecticidal protein in tobacco. Biotechnology 1995, 13:362-365. 41. Daniell H, Datta R, Varma S, Gray S, Lee SB: Containment of herbicide resistance through genetic engineering of the 38. Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ: chloroplast genome. Nat Biotechnol 1998, 16:345-348. • Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against 42. Staub JM, Garcia B, Graves J, Hajdukiewicz PTJ, Hunter P, Nehra N, susceptible and Bt-resistant insects. Proc Natl Acad Sci USA •• Paradkar V, Schlittler M, Carroll JA, Spatola L et al.: High-yield 1999, 96:1840-1845. production of a human therapeutic protein in tobacco In a similar study to the pioneering work described in [37], a single bacterial chloroplasts. Nat Biotechnol 2000, 18:333-338. gene encoding a Bt toxin was expressed in tobacco plastids and found to Proteins from eukaryotic sources, which require complex folding or disulfide express at high levels, causing considerable mortality to target insect bond formation, are generally not easily expressed in prokaryotic systems. species. The use of highly insect-resistant plants as part of a ‘high dose The authors demonstrate that the tobacco chloroplast is capable of correctly resistance management strategy’ is discussed. processing fusions of human somatotropin and ubiquitin expressed from the plastid genome. Moreover, these transformed chloroplasts accumulate 39. De Cosa B, Moar W, Lee S-B, Miller M, Daniell H: Overexpression of metabolically active protein to high levels. •• the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 2001, 19:71-74. 43. Guda C, Lee SB, Daniell H: Stable expression of a biodegradable Insecticidal proteins from Bacillus thuringiensis are normally expressed in protein-based polymer in tobacco chloroplasts. Plant Cell Rep the insect as protoxins from operons, which can include a putative chaperonin. 2000, 19:257-262.