Double-stranded RNAs (dsRNAs) targeted against essential genes can trigger a lethal RNA interference (RNAi) response in insect pests. The application of this concept in plant protection is hampered by the presence of an endogenous plant RNAi pathway that processes dsRNAs into short interfering RNAs. We found that long dsRNAs can be stably produced in chloroplasts, a cellular compartment that appears to lack an RNAi machinery. When expressed from the chloroplast genome, dsRNAs accumulated to as much as 0.4% of the total cellular RNA. Transplastomic potato plants producing dsRNAs targeted against the β-actin gene of the Colorado potato beetle, a notorious agricultural pest, were protected from herbivory and were lethal to its larvae. Thus, chloroplast expression of long dsRNAs can provide crop protection without chemical pesticides.

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.

SummaryThe chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially speci®es a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These ®ndings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the ®rst higher plant chloroplast genes identi®ed as being indispensable.

Eukaryotic cells arose through endosymbiotic uptake of free-living bacteria followed by massive gene transfer from the genome of the endosymbiont to the host nuclear genome. Because this gene transfer took place over a time scale of hundreds of millions of years, direct observation and analysis of primary transfer events has remained difficult. Hence, very little is known about the evolutionary frequency of gene transfer events, the size of transferred genome fragments, the molecular mechanisms of the transfer process, or the environmental conditions favoring its occurrence. We describe here a genetic system based on transgenic chloroplasts carrying a nuclear selectable marker gene that allows the efficient selection of plants with a nuclear genome that carries pieces transferred from the chloroplast genome. We can select such gene transfer events from a surprisingly small population of plant cells, indicating that the escape of genetic material from the chloroplast to the nuclear genome occurs much more frequently than generally believed and thus may contribute significantly to intraspecific and intraorganismic genetic variation.T he evolutionary origin of eukaryotic cells is characterized by the endosymbiotic uptake of bacteria and their gradual conversion into the DNA-containing cell organelles, mitochondria, and plastids (chloroplasts) (1-3). Genetically, the evolutionary optimization of endosymbiosis was accompanied by the loss of dispensable and redundant genetic information and the large-scale translocation of genetic information from the endosymbiont to the host genome (4-6). Consequently, contemporary organellar genomes are greatly reduced and contain only a small proportion of the genes that their free-living ancestors had possessed. By using molecular methods, the origins of organelles have been traced back to specific taxa of eubacteria: Whereas cyanobacteria were identified as presumptive ancestors of plastids, ␣-proteobacteria are related most closely to mitochondria (1).Interspecific variation in the gene content of organellar genomes (7-11) suggests that gene transfer from organelles to the nucleus is an ongoing process. Moreover, pieces of chloroplast and mitochondrial DNA are often found in nuclear genomes (12-18) and commonly referred to as promiscuous DNA. These sequences lack any apparent function but may provide the raw material for converting organellar genes into functional nuclear genes, the products of which are reimported into the organelle, then allowing for subsequent loss of the genes from the organellar genome (10). In addition, promiscuous DNA of mitochondrial origin has been implicated recently in DNA repair in yeast by patching broken chromosomes (19,20).In the present study we developed an experimental system suitable for selecting and analyzing DNA transfer events from the chloroplast genome to the nuclear genome. We find that DNA escape out of the chloroplast and integration into the nuclear genome occurs much more frequently than generally believed and thus provides a mechan...

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 Ϸ1.58 ؋ 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.biosafety ͉ Nicotiana tabacum ͉ plastid transformation ͉ pollen transmission ͉ paternal leakage