Investigation of the interaction of a papain-like cysteine protease (RD19c) with selenium-binding protein 1 (SBP1) in Arabidopsis thaliana

AtRD19c is a member of the papain-like cysteine proteases known for its participation in anther development after its maturation by βVPE (vacuolar processing enzyme). This papain-like cysteine protease was identified as an interacting protein of AtSBP1 (selenium binding protein 1) in a yeast two-hybrid screening. To confirm this interaction, we studied AtRD19c with respect to its expression and ability to interact with AtSBP1. The highest gene expression levels of AtRD19c were observed in the roots of 10-day-old seedlings, whereas minimum levels appeared in the hypocotyls of 10-day-old seedlings and flowers. AtRD19c expression was upregulated by selenium, and analysis of its promoter activity showed colocalization of a reporter gene (GUS) with AtSBP1. Additionally, the AtRD19c expression pattern was upregulated in the presence of selenite, indicating its participation in the Se response network. Confocal fluorescence microscopy revealed that AtRD19c localizes in the root tip, lateral roots, and leaf trichomes. Finally, we confirmed the physical interaction between AtRD19c and AtSBP1 and showed the importance of the first 175 aa of the AtSBP1 polypeptide in this interaction. Importantly, the AtRD19c-AtSBP1 interaction was also demonstrated in planta by employing bimolecular fluorescent complementation (BiFC) in a protoplast system.