High-Frequency Gene Transfer from the Chloroplast Genome to the Nucleus

High-frequency gene transfer from the chloroplast genome to the nucleus Sandra Stegemann, Stefanie Hartmann, Stephanie Ruf, and Ralph Bock* Westfa¨lische Wilhelms-Universita¨t Mu¨ nster, Institut fu¨r Biochemie und Biotechnologie der Pflanzen, Hindenburgplatz 55, D-48143 Mu¨nster, Germany Edited by Lawrence Bogorad, Harvard University, Cambridge, MA, and approved May 15, 2003 (received for review February 16, 2003) Eukaryotic cells arose through endosymbiotic uptake of free-living MS medium (21) containing 30 g͞liter sucrose. Homoplasmic bacteria followed by massive gene transfer from the genome of transformed lines were rooted and propagated on the same the endosymbiont to the host nuclear genome. Because this gene medium. transfer took place over a time scale of hundreds of millions of years, direct observation and analysis of primary transfer events Construction of Plastid Transformation Vectors. Plastid transforma- has remained difficult. Hence, very little is known about the tion vector pRB98 (Fig. 1A) is a derivative of the previously evolutionary frequency of gene transfer events, the size of trans- described vector pRB95 (22). A nuclear cauliflower mosaic virus ferred genome fragments, the molecular mechanisms of the trans- 35S promoter-driven nptII expression cassette was excised from fer process, or the environmental conditions favoring its occur- a plant transformation vector (23) and inserted into the rence. We describe here a genetic system based on transgenic polylinker of pRB95 yielding plasmid pRB98. chloroplasts carrying a nuclear selectable marker gene that allows the efficient selection of plants with a nuclear genome that carries Plastid Transformation and Selection of Transplastomic Tobacco Lines. pieces transferred from the chloroplast genome. We can select such Young leaves were harvested form sterile tobacco plants and gene transfer events from a surprisingly small population of plant bombarded with plasmid DNA-coated gold particles by using the cells, indicating that the escape of genetic material from the DuPont PDS1000He biolistic gun (24). Spectinomycin-resistant chloroplast to the nuclear genome occurs much more frequently shoots were selected on RMOP regeneration medium contain- than generally believed and thus may contribute significantly to ing 500 mg͞liter spectinomycin dihydrochloride (24). Primary intraspecific and intraorganismic genetic variation. plastid transformants were identified by a PCR strategy. Three independently generated transplastomic lines (designated Nt- he evolutionary origin of eukaryotic cells is characterized by pRB98-1, Nt-pRB98-11, and Nt-pRB98-12) were subjected to Tthe endosymbiotic uptake of bacteria and their gradual four additional rounds of regeneration on RMOP͞spectinomy- conversion into the DNA-containing cell organelles, mitochon- cin to obtain homoplasmic tissue (24). Homoplasmy was verified dria, and plastids (chloroplasts) (1–3). Genetically, the evolu- by restriction fragment length polymorphism analysis and highly tionary optimization of endosymbiosis was accompanied by the sensitive inheritance assays (see below). loss of dispensable and redundant genetic information and the large-scale translocation of genetic information from the endo- Selection for Gene Transfer from the Chloroplast to the Nucleus. symbiont to the host genome (4–6). Consequently, contempo- Young leaves from Nt-pRB98 plastid transformants were har- rary organellar genomes are greatly reduced and contain only a vested from sterile plants, cut into small pieces of Ϸ20–25 mm2, small proportion of the genes that their free-living ancestors had and placed onto the surface of a selectable plant-regeneration possessed. By using molecular methods, the origins of organelles medium containing kanamycin. Several independent screens have been traced back to specific taxa of eubacteria: Whereas were conducted with kanamycin concentrations ranging from cyanobacteria were identified as presumptive ancestors of 100 to 400 ␮g͞ml. With all concentrations, gene transfer plants plastids, ␣-proteobacteria are related most closely to mito- were recovered. However, because the lower kanamycin con- chondria (1). centrations produced a relatively high background of false- Interspecific variation in the gene content of organellar ge- positive clones, the final screen was conducted with 400 ␮g͞ml nomes (7–11) suggests that gene transfer from organelles to the kanamycin. All putative gene transfer plants were rescreened by nucleus is an ongoing process. Moreover, pieces of chloroplast placing small tissue pieces (Ϸ2 ϫ 2 mm) on kanamycin- and mitochondrial DNA are often found in nuclear genomes containing regeneration medium, a stringent selection step (12–18) and commonly referred to as promiscuous DNA. These (because of the small explant size) that eliminated essentially all sequences lack any apparent function but may provide the raw false-positive clones. Regenerated shoots then were transferred material for converting organellar genes into functional nuclear to sterile culture boxes and rooted on phytohormone-free MS genes, the products of which are reimported into the organelle, medium in the presence of kanamycin. Finally, rooted plants then allowing for subsequent loss of the genes from the organel- were taken out of the sterile containers, planted into soil, and lar genome (10). In addition, promiscuous DNA of mitochon- transferred to the greenhouse. drial origin has been implicated recently in DNA repair in yeast by patching broken chromosomes (19, 20). Isolation of Nucleic Acids. Total plant nucleic acids were isolated In the present study we developed an experimental system according to a miniprep procedure described by Doyle and suitable for selecting and analyzing DNA transfer events from Doyle (25). the chloroplast genome to the nuclear genome. We find that DNA escape out of the chloroplast and integration into the PCR Assays. DNA templates were amplified according to standard nuclear genome occurs much more frequently than generally protocols (45 s at 94°C, 1.5 min at 55–65°C, and 1.5 min at 72°C believed and thus provides a mechanism not only causing for 30 cycles) in an Eppendorf gradient thermocycler. Primers intraspecific but also intraorganismic genetic variation. Materials and Methods This paper was submitted directly (Track II) to the PNAS office. Plant Material. Tobacco plants (Nicotiana tabacum cv. Petit See commentary on page 8612. Havana) were grown under sterile conditions on agar-solidified *To whom correspondence should be addressed. E-mail: [email protected]. 8828–8833 ͉ PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1430924100 Downloaded by guest on September 28, 2021 SEE COMMENTARY Fig. 1. A genetic screen for gene transfer from the chloroplast to the nucleus. (A) Physical map of the chloroplast transformation vector pRB98. (Upper) The region of the tobacco chloroplast genome chosen for insertion of the two foreign gene constructs: a chimeric aadA gene conferring spectinomycin resistance as plastid selectable marker gene (24) and a nuclear expression cassette containing the kanamycin-resistance gene nptII.(Lower) Homologous recombination targets the two linked transgenes from pRB98 to the intergenic region between two tRNA genes (trnfM and trnG) (22). Location and orientation of PCR primers are indicated by arrows. ptDNA, plastid DNA. (B) Selection of cell lines that have transferred the kanamycin-resistance gene from the chloroplast genome to the nuclear genome. Putative gene transfer plants (arrow) were selected on plant-regeneration medium containing 400 ␮g͞ml kanamycin and typically appeared after 3–6 weeks of selection. (Scale bar, 1 cm.) specific for the nptII coding region (PnptIIf, 5Ј-GAG- transformed plants were transferred to soil and grown to matu- GCAGCGCGGCTATC-3Ј; PnptIIr, 5Ј-GCGGTCCGCCA- rity under greenhouse conditions. Seed pods were collected from CACCCA-3Ј) were used to assay for the presence of the trans- selfed plants and reciprocal crosses of the transplastomic lines gene in individual seedlings resulting from crosses of wild-type with wild-type plants. Surface-sterilized seeds were germinated plants and gene transfer plants, with the latter ones serving as the on spectinomycin-containing (500 mg͞liter) MS medium and pollen donor. Presence of the aadA gene in the nuclear genome analyzed for uniparental inheritance of the resistance trait. of the gene transfer plants was assayed with primers derived Selfed transformants and crosses with a chloroplast transfor- Ј Ј GENETICS from the 3 part of the coding region (P29, 5 -CGCTATG- mant as the maternal parent give rise to green (i.e., spectino- GAACTCGCCGCC-3Ј) and the downstream 3Ј untranslated mycin-resistant) progeny, whereas seeds collected from wild- region (P28, 5Ј-TAGCACCCTCTTGATAGAAC-3Ј). Linkage of nptII and aadA was analyzed by PCR by combining one primer type plants pollinated with pollen from transplastomic plants derived from the aadA coding region (P136, 5Ј-TCGAT- yield white (i.e., drug-sensitive) seedlings. Seeds from all crosses GACGCCAACTACC-3Ј) with a primer binding to the nptII were also assayed for kanamycin resistance. Wild-type plants coding region (PnptII5Ј,5Ј-GCTGCATACGCTTGATCC-3Ј) pollinated with pollen from transplastomic plants gave rise to (Fig. 1). progeny that were as kanamycin-sensitive as the wild type (assayed on 100 ␮g͞ml). In contrast, transplastomic plants Crosses and Inheritance Tests of Transplastomic Lines. For confir- displayed an enhanced kanamycin

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