Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants

Khan, Muhammad Sarwar; Maliga, Pál

doi:10.1038/12907

Cited by 218 publications

(168 citation statements)

References 42 publications

(44 reference statements)

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“…To identify the reasons for the large increase in plastid-to-nucleus DNA transfer induced by heat stress, we examined the integrity of chloroplasts. A transplastomic tobacco line pMSK56 (27) that encodes and expresses GFP in its chloroplasts was treated at 45°C for 5 h. In seedlings without heat treatment, GFP fluorescence colocalized with chlorophyll autofluorescence in chloroplasts of stomatal guard cells and underlying mesophyll tissues (Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

Wang

Lloyd

Timmis

2012

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondria and chloroplasts (photosynthetic members of the plastid family of cytoplasmic organelles) in eukaryotic cells originated more than a billion years ago when an ancestor of the nucleated cell engulfed two different prokaryotes in separate sequential events. Extant cytoplasmic organellar genomes contain very few genes compared with their candidate free-living ancestors, as most have functionally relocated to the nucleus. The first step in functional relocation involves the integration of inactive DNA fragments into nuclear chromosomes, and this process continues at high frequency with attendant genetic, genomic, and evolutionary consequences. Using two different transplastomic tobacco lines, we show that DNA migration from chloroplasts to the nucleus is markedly increased by mild heat stress. In addition, we show that insertion of mitochondrial DNA fragments during the repair of induced double-strand breaks is increased by heat stress. The experiments demonstrate that the nuclear influx of organellar DNA is a potentially a source of mutation for nuclear genomes that is highly susceptible to temperature fluctuations that are well within the range experienced naturally.D NA in the highly energetic cytoplasmic organellar genetic compartments of eukaryotes is subjected to high levels of stress damage from the oxygen free radicals produced during respiration and photosynthesis. Nonetheless, for various reasons, the rate of accumulation of mutations is slower in plant cytoplasmic organelles than in the nucleus (1, 2). The vast majority of mitochondrial and plastid (chloroplast) genes have vacated their ancestral prokaryote genetic compartment in favor of the nucleus (2), with very few remaining within extant organelles. The first step in gene relocation for the organelle genomes is DNA escape, followed by insertion into nuclear chromosomes, a process shown to occur remarkably frequently under normal physiological conditions in tobacco (3, 4) and yeast (5), although an experimental screen in the unicellular alga Chlamydomonas reinhardtii did not detect any such transpositions (6). These experimental studies, together with bioinformatic and genomic analyses (7)(8)(9)(10)(11)(12)(13)(14), demonstrated that DNA transfer from cytoplasmic organelles is a frequent and continuous process in essentially all eukaryotes examined, although it may be very rare in organisms with very low organelle numbers (15). Although DNA transfer per se is very frequent in higher plants, genes that migrate are normally inactive in the nucleus because they lack the motifs required for nuclear expression. Nonetheless, on rare occasions nuclear integrants of organelle DNA (norgs) are activated by genomic rearrangements (16,17), that effectively duplicate the gene. Over evolutionary time, these frequent nonfunctional and rare functional DNA transfer processes have resulted in the net loss of redundant genes in the cytoplasmic organelles and have added to the genetic complexity of the nucleus.Environmental factors are known to modify mitoch...

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Section: Resultsmentioning

confidence: 99%

Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

Wang

Lloyd

Timmis

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Plastid transformation in rice has been shown using vectors containing a translational fusion of aadA with gfp [47]. Inspection of streptomycin-resistant rice shoots by confocal microscopy revealed a few proplastids with GFP fluorescence.…”

Section: Current Limitations Of Chloroplast Genetic Engineeringmentioning

confidence: 99%

“…Selectable markers and reporters aadA Aminoglycoside-3′-adenylyltransferase [14] nptII Neomycin phosphotransferase [52] codA Cytosine deaminase [53] BADH Betaine aldehyde dehydrogenase [4] uidA β-glucoronidase [12] cat Chloramphenicol acetyl transferase [9,11] gfp Green fluorescent protein [24,54] aadA:gfp Selectable or screenable fusion protein [47] Plant traits: herbicide resistance aroA Glyphosate resistance [2,19] bar Bialaphos resistance [18,20] Insect resistance…”

Section: Genes and Use Gene Products And Use Refsmentioning

confidence: 99%

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Daniell

Khan

Allison

2002

Trends in Plant Science

324

153

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Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.Chloroplast transformation is an environmentally friendly approach to plant genetic engineering that minimizes out-crossing of transgenes to related weeds or crops [1,2] and reduces the potential toxicity of transgenic pollen to non-target insects [3]. Because the plastid genome is highly polyploid, transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell, and generates extraordinarily high levels of foreign protein [3]. Chloroplast transformation vectors use two targeting sequences that flank the foreign genes and insert them, through homologous recombination, at a precise, predetermined location in the organelle genome (Fig. 1). This results in uniform transgene expression among transgenic lines and eliminates the 'position effect' often observed in nuclear transgenic plants. Gene silencing, frequently observed in nuclear transgenic plants, has not been observed in genetically engineered chloroplasts. The ability to express foreign proteins at high levels in chloroplasts and chromoplasts, and to engineer foreign genes without the use of antibiotic resistant genes [4,5],make this compartment ideal for the development of edible vaccines [6]. Moreover, the ability of chloroplasts to form disulfide bonds and to fold human proteins has opened the door to high-level production of biopharmaceuticals in plants [7]. Furthermore, foreign proteins observed to be toxic in the cytosol are non-toxic when accumulated within transgenic chloroplasts [6,8]. Chloroplast and nuclear genetic engineering are compared in Table 1.

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“…A similar approach was adopted by Wurbs et al (2007), who used the atpI promoter containing both PEP and NEP signals, and the rps16 3 0 -UTR, to express carotenoidrelated genes in tomato fruits. Nevertheless, the short rrn promoter, containing only the sequences for PEP recognition, was successfully used to express transgenes in fruit chromoplasts of transplastomic tomato, and in petal leucoplasts and root amyloplasts of tobacco and Nicotiana benthamiana (Khan and Maliga 1999;Ruf et al 2001;Apel and Bock 2009;Davarpanah et al 2009).…”

Section: Introductionmentioning

confidence: 99%

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

Valkov¹,

Gargano

Manna³

et al. 2010

Transgenic Res

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Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5 0 -UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5 0 -UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5 0 -UTR construct, described above, and another containing the same terminator, but with the promoter and 5 0 -UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

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Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants

Cited by 218 publications

References 42 publications

Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

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