Daniel Karcher

Determining the transgene containment level provided by chloroplast transformation.

Plastids (chloroplasts) are maternally inherited in most crops. Maternal inheritance excludes plastid genes and transgenes from pollen transmission. Therefore, plastid transformation is considered a superb tool for ensuring transgene containment and improving the biosafety of transgenic plants. Here, we have assessed the strictness of maternal inheritance and the extent to which plastid transformation technology confers an increase in transgene confinement. We describe an experimental system facilitating stringent selection for occasional paternal plastid transmission. In a large screen, we detected low-level paternal inheritance of transgenic plastids in tobacco. Whereas the frequency of transmission into the cotyledons of F1 seedlings was ≈1.58 × 10−5 (on 100% cross-fertilization), transmission into the shoot apical meristem was significantly lower (2.86 × 10−6). Our data demonstrate that plastid transformation provides an effective tool to increase the biosafety of transgenic plants. However, in cases where pollen transmission must be prevented altogether, stacking with other containment methods will be necessary to eliminate the residual outcrossing risk.

Generation of Chlamydomonas strains that efficiently express nuclear transgenes

The unicellular green alga Chlamydomonas reinhardtii is both an invaluable model organism for plant biology and an attractive biotechnological production system. Despite the availability of efficient methods for introduction of foreign genes into the nuclear genome of the alga, transgene expression levels are usually very poor. This is a serious limitation that has severely hampered both post-genomics research in Chlamydomonas and use of the alga in molecular farming. Here we report a solution to this problem. We have designed a genetic screen that facilitates isolation of algal strains that efficiently express introduced transgenes. The levels of accumulation of foreign protein in our expression strains are almost uniformly high in all transgenic clones and are little influenced by position effects. The possibility of expressing transgenes to high levels will greatly facilitate post-genomics research in Chlamydomonas, and will also boost exploitation of the alga as an inexpensive production host for biopharmaceuticals and other valuable compounds.

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons

The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as “vitamin E”) as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we (i) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, (ii) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and (iii) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

Horizontal genome transfer as an asexual path to the formation of new species

Allopolyploidization, the combination of the genomes from two different species, has been a major source of evolutionary innovation and a driver of speciation and environmental adaptation. In plants, it has also contributed greatly to crop domestication, as the superior properties of many modern crop plants were conferred by ancient allopolyploidization events. It is generally thought that allopolyploidization occurred through hybridization events between species, accompanied or followed by genome duplication. Although many allopolyploids arose from closely related species (congeners), there are also allopolyploid species that were formed from more distantly related progenitor species belonging to different genera or even different tribes. Here we have examined the possibility that allopolyploidization can also occur by asexual mechanisms. We show that upon grafting—a mechanism of plant–plant interaction that is widespread in nature—entire nuclear genomes can be transferred between plant cells. We provide direct evidence for this process resulting in speciation by creating a new allopolyploid plant species from a herbaceous species and a woody species in the nightshade family. The new species is fertile and produces fertile progeny. Our data highlight natural grafting as a potential asexual mechanism of speciation and also provide a method for the generation of novel allopolyploid crop species.

Inducible gene expression from the plastid genome by a synthetic riboswitch

Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology.

Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli

RNA thermometers are thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Naturally occurring RNA thermometers exhibit complex secondary structures which are believed to undergo a series of gradual structural changes in response to temperature shifts. Here, we report the de novo design of considerably simpler RNA thermometers that provide useful RNA-only tools to regulate bacterial gene expression by a shift in the growth temperature. We show that a single small stem-loop structure containing the ribosome binding site is sufficient to construct synthetic RNA thermometers that work efficiently at physiological temperatures. Our data suggest that the thermometers function by a simple melting mechanism and thus provide minimum size on/off switches to experimentally induce or repress gene expression by temperature.

Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

The amino acid sequence of a plastid protein is developmentally regulated by RNA editing.

RNA editing in plant organelles post-transcriptionally alters single nucleotides by C-to-U or U-to-C conversions at highly specific sites. Plant editing is generally viewed as a repair mechanism acting at the transcript level by restoring conserved amino acid residues. Here we report that an editing reaction within the ndhB transcript (encoding a plastid NAD(P)H dehydrogenase subunit) is strictly dependent on active photosynthesis. Employing non-photosynthetic mutants, we show that in the absence of photosynthesis, the site remains unedited, whereas it is fully edited when the photosynthetic apparatus is intact. Moreover, the site also remains unedited during the etiolated stage of seedling development, suggesting that two different NdhB proteins are synthesized under photosynthetic versus non-photosynthetic conditions. This is the first case where RNA editing in plants appears to regulate gene expression qualitatively, resulting in the production of two different proteins from one and the same gene in a developmental stage-dependent manner.

A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop

Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. DOI: http://dx.doi.org/10.7554/eLife.13664.001

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).