tRUBY: A convenient in planta tool for the detection of protein-DNA and protein-protein interactions.

Elucidating molecular interactions such as protein-DNA (PDIs) and protein-protein (PPIs) has traditionally relied on yeast-based 1-hybrid (1H) and 2-hybrid (2H) systems. To provide an alternative platform that better reflects the native cellular environment of plants, we optimized the tRUBY reporter system for 1H and 2H assays in Nicotiana benthamiana, enabling direct in planta analysis of PDIs and PPIs. Specifically, the 2A peptide sequence used for co-expressing the 3 betalain biosynthetic genes-responsible for the visible RUBY coloration-was replaced with T2A from the Thosea asigna virus in place of P2A or F2A from mammalian-pathogenic Picornaviridae viruses, improving biosafety for agricultural applications. The resulting tRUBY-1H and tRUBY-2H systems operate under near-physiological conditions with physiologically relevant expression levels, enabling quantitative, multiplexed, and directly compatible protein-level analyses, thereby offering high sensitivity and flexibility for advanced molecular studies. Ultimately, these systems demonstrate that the streamlined, cost-effective, and visually scorable in planta platform provided by RUBY is well-suited for intuitive, non-destructive monitoring of molecular interactions in plant tissues.