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. 2003 Jul 22;100(15):8828-33.
doi: 10.1073/pnas.1430924100. Epub 2003 Jun 19.

High-frequency gene transfer from the chloroplast genome to the nucleus

Sandra Stegemann  1 , Stefanie Hartmann, Stephanie Ruf, Ralph Bock
Affiliations

High-frequency gene transfer from the chloroplast genome to the nucleus

Sandra Stegemann et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Fig. 1.
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 μg/ml kanamycin and typically appeared after 3–6 weeks of selection. (Scale bar, 1 cm.)
Fig. 2.
Fig. 2.
Inheritance assays demonstrating gene transfer from the chloroplast to the nucleus. (A) Seed assay demonstrating Mendelian inheritance of the kanamycin resistance in a selected gene transfer line. Seeds from self-pollinated gene transfer plants were surface-sterilized and germinated on medium containing 1,000 μg/ml kanamycin. Phenotypic segregation of the progeny in the expected 3:1 ratio (96 resistant:34 sensitive seedlings) indicates nuclear localization of the kanamycin-resistance gene. Note that approximately one-third of the resistant seedlings display particularly rapid growth, suggesting that they are homozygous and thus show enhanced kanamycin resistance. (B and C) Mendelian inheritance and 1:1 segregation of the kanamycin resistance in reciprocal crosses of gene transfer plants with wild-type plants. (B) Pollination of a gene transfer plant with pollen from a wild-type plant yields 1:1 segregating progeny (65 resistant:72 sensitive seedlings) and, because of the continued presence of the transgenic chloroplasts, requires selection at elevated kanamycin concentrations (1,000 μg/ml; see text). (C) Pollination of a wild-type plant with pollen from a gene transfer plant separates the cell nucleus with the transferred nptII gene from the transgenic chloroplasts and thus allows phenotypic selection already at low kanamycin concentrations (100 μg/ml). As in the reciprocal cross, a 1:1 segregation (68 resistant:68 sensitive seedlings) typical of nuclear-encoded traits is observed, ultimately confirming that the formerly chloroplast-located nptII gene has moved to the nucleus. (D) Wild-type control. Self-pollination of wild-type plants does not yield kanamycin-resistant progeny. Kanamycin (Kan) concentrations used for selection are indicated in micrograms per milliliter.
Fig. 3.
Fig. 3.
Assay for cotransfer of the aadA gene with the selectable marker gene nptII. PCR with primers specific for the 3′ region of the chimeric aadA gene (Fig. 1 A) were performed to assay for transferred aadA sequences in individual seedlings from a cross of a wild-type plant (maternal parent) with a gene transfer plant (paternal parent). Although the five seedlings positive for nptII (samples 2, 3, 4, 7, and 9) also showed the aadA-specific 368-bp PCR product and displayed kanamycin resistance in growth assays on synthetic medium, the five PCR-negative seedlings (, , , , and 10) are kanamycin-sensitive, indicating cotransfer of the two foreign genes and Mendelian 1:1 segregation also for the aadA gene. Cotransfer of the two genes and their physical linkage in the nuclear genome was ultimately confirmed by combining an nptII-specific with an aadA-specific primer (Fig. 1), yielding a 1-kb product for all nptII-positive seedlings. +, positive control (Nt-pRB98-12 chloroplast transformant harboring both nptII and aadA in the plastid genome; Fig. 1 A); –, buffer control; M, molecular weight marker (fragment sizes in base pairs). Note much stronger PCR amplification in the chloroplast transformant (+) than in the gene transfer plants, which is due to the much higher copy number per cell of the chloroplast genome.
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