A bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient selectable marker for plastid transformation

Abstract

A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants. More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6')-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.

Keywords: Bifunctional enzyme; Nicotiana tabacum; Plastid transformation; Selectable marker; Tobramycin.

Fig. 1

The bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme has two active domains. The AAC(6′) domain catalyzes an acetylation reaction, using acetyl-CoA as the acetyl donor, to the 6′-amino group of ring I of aminoglycoside antibiotics. Exemplarily, the structure of tobramycin is shown here. The APH(2″) domain uses GTP (or ATP) as phosphate donor and phosphorylates the 2″-hydroxyl group of ring III of the aminoglycoside molecule (Frase et al. 2012)

Fig. 2

Generation of transplastomic tobacco plants by tobramycin selection. a Physical maps of the targeting region in the wild-type chloroplast genome (ptDNA) and the genomes of the transplastomic lines (Nt-IT) obtained with plastid transformation vectors pIT6, pIT19 and pIT20. Filled black boxes represent tobacco plastid genes, the aadA marker present in vector pIT6 is shown as an open box and the new aac6-aph2 selectable marker (present in all pIT vectors) as a blue-red box. Expression elements (promoters, 5′ UTRs and 3′ UTRs) are represented as grey boxes and denoted by the source organism (Nt: Nicotiana tabacum; Cr: Chlamydomonas reinhardtii) and the source gene (Prrn: rRNA operon promoter; G10L: Shine-Dalgarno sequence from the bacteriophage T7 gene 10; Trps16: 3′ UTR from the plastid rps16 gene; SD rbcL: Shine-Dalgarno sequence from the plastid rbcL gene; TpsbA: 3′ UTR from the plastid psbA gene; PpsbA: promoter from the psbA gene; TrbcL: 3′ UTR from the rbcL gene). Genes above the lines are transcribed from left to right, genes below the lines are transcribed in the opposite direction. A PCR product derived from the tobacco psaB gene was used as probe in restriction fragment length polymorphism (RFLP) analyses and is represented as a black bar. The expected sizes of plastid DNA fragments in RFLP analyses with the restriction enzyme BglII are indicated below each map. b RFLP analysis of transplastomic lines. DNA samples of the wild type (wt) and several independently generated antibiotic-resistant lines obtained from primary selection or the first regeneration round were digested with BglII, separated in 1% agarose gels, blotted and hybridized to the radiolabeled psaB probe shown in panel (a). The probe detects the expected 3.5 kb fragment in the wild type, a 6.8 kb fragment in the transplastomic lines obtained with vector pIT6, and a 5.8 and 5.7 kb fragment in the transplastomic lines produced with vectors pIT19 and pIT20, respectively

Fig. 3

Generation of transplastomic tobacco lines by selection for tobramycin resistance or gentamicin resistance. a Primary selection of transplastomic lines on medium with tobramycin or gentamicin. The lines were produced with vectors pIT6 and pIT19, respectively (see Fig. 2). As a control, a transplastomic Nt-IT6 clone obtained by selection for spectinomycin resistance is also shown. b Additional regeneration rounds conducted in the presence of the selection agent to obtain homoplasmic transplastomic shoots. Nt-IT6 plates were photographed after 4 weeks, the Nt-IT19 plate after 6 weeks. c Rooting and growth of transplastomic lines under aseptic conditions. Tobramycin or gentamicin-resistant shoots from selection plates were grown on phytohormone-free medium in the presence of the antibiotic (30 mg/L tobramycin or gentamicin), the spectinomycin-resistant plant was grown in the presence of 500 mg/L spectinomycin

Fig. 4

Growth of transplastomic plants to maturity and seed assays confirming maternal transgene inheritance. a Transplastomic plants with the inserted aac6-aph2 marker gene (right) grow like wild-type plants (left) under greenhouse conditions. Scale bars 10 cm. b Seed assays to confirm stable maternal inheritance of the chloroplast-encoded tobramycin resistance gene. A transplastomic Nt-pIT6 plant (tp) was selfed (tp × tp) and reciprocally crossed to a wild-type plant (tp × wt, wt × tp). As a control for antibiotic sensitivity, the selfed wild type (wt × wt) was also included. While the progeny from all crosses with the transplastomic line as maternal parent are homogeneously resistant to tobramycin (50 mg/L), the progeny from crosses with a wild-type plant as maternal parent are uniformly sensitive to tobramycin. Scale bar 1 cm

Fig. 5

Specificity test of the aadA and aac6-aph2 marker genes. The detoxification activity of the encoded enzymes towards the four aminoglycoside antibiotics tobramycin, gentamicin, spectinomycin and kanamycin was assayed by exposing leaf explants of transplastomic plants and a wild-type control to selective regeneration medium containing tobramycin, gentamicin, tobramycin + gentamicin, spectinomycin or kanamycin. Photographs were taken after 3 weeks. a An Nt-IT6 line harboring the aac6-aph2 gene under the control of strong expression elements and additionally the aadA marker. b An Nt-IT20 line containing the aac6-aph2 gene under the control of strong expression elements. c An Nt-IT19 line harboring the aac6-aph2 gene under the control of weak expression elements. d An Nt-DK305 line expressing only the aadA marker gene. e A wild-type plant. Note that transplastomic lines containing only the aadA gene (Nt-DK305) cannot detoxify tobramycin and gentamicin. See also Fig. S3