A Light-Dependent Selection Marker System in Plants

Burkholderia glumae produces toxoflavin which is essential for virulence factor causing panicle blight in rice. Toxoflavin is one the photosensitizers that are common in nature and play diverse roles as defense compounds and pathogenicity determinants, and as important molecules in many biological processes. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA is 24.56 kDa in size and similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn²⁺ and dithiothreitol were required for toxoflavin degradation by TflA. Our results demonstrated that the ability of toxoflavin degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high density screening of transgenic Arabidopsis seeds. Therefore, we propose the toxoflavin/tflA selection system provides a new robust non-antibiotic selection marker system for diverse plants.