RUBY-BD: a highly efficient visual reporter system for assessing bidirectional promoter activity.

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conclusionWe developed a universal, one-step reporter system, RUBY-BD, enabling high-throughput examination of transcriptional activity of bidirectional promoters. Bidirectional promoters (BDPs), the intergenic regions between divergent gene pairs, enable simultaneous transcription of both genes and are highly valuable for multigene engineering. Conventional reporter systems like green fluorescent protein (GFP) and β-glucuronidase (GUS) are widely used to assess BDP activity; however, their reliance on cumbersome detection methods and high experimental costs hinders high-throughput screening. The RUBY reporter system offers an efficient alternative, requiring only the co-expression of three enzymes (AD1, DODA, GT) to convert tyrosine into betalain, a red-violet pigment visible to the naked eye. In this study, we separated the three genes and the intervening 2A sequence of the RUBY system into two bidirectional transcription units, constructing an RUBY-BD vector. Transient transformation assays in Nicotiana benthamiana leaves confirmed that inserting an active BDP leads to the expression of all three genes and results in betalain biosynthesis. Using the RUBY-BD system, we evaluated the activity of 20 putative plant BDPs in a single step. Betalain accumulation and expression analysis of the two flanking genes confirmed the bidirectional transcriptional activity of 11 BDPs. In summary, this study established a high-throughput visual screening tool for characterizing plant BDPs, which is expected to accelerate basic research into BDPs and their application in gene engineering.