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

Iman Tabatabaei,Stephanie Ruf,Ralph Bock

doi:10.1007/s11103-016-0560-x

Iman Tabatabaei, Stephanie Ruf + Show 1 more

Open Access

https://doi.org/10.1007/s11103-016-0560-x

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Abstract

Key messageA 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.

Highlights

The development of technologies to engineer plastid genomes (Boynton et al 1988; Svab et al 1990) has opened up new exciting opportunities to study virtually all aspects of plastid biology using in vivo approaches
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
Construction of plastid transformation vectors based on the bifunctional aac6-aph2 resistance marker and identification of suitable selection conditions for chloroplast transformation

Summary

Introduction

The development of technologies to engineer plastid (chloroplast) genomes (Boynton et al 1988; Svab et al 1990) has opened up new exciting opportunities to study virtually all aspects of plastid biology using in vivo approaches. A large number of proof-ofconcept studies have demonstrated the great potential of the transplastomic technology in molecular farming (Staub et al 2000; Tregoning et al 2003; Oey et al 2009b), metabolic engineering (Apel and Bock 2009; Bohmert-Tatarev et al 2011; Fuentes et al 2016) and resistance engineering (De Cosa et al 2001; Ye et al 2001; Zhang et al 2015). The lack of a suitable selectable marker gene is the main obstacle to the development of a mitochondrial transformation technology in plants (Li et al 2011)

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