Genetic Suppressor Screen Using an Inducible FREE1-RNAi Line to Detect ESCRT Genetic Interactors in Arabidopsis thaliana

Abstract

FREE1 (FYVE domain protein required for endosomal sorting 1), a newly identified component of endosomal sorting complex required for transport I (ESCRT I), plays multiple roles in regulating protein trafficking and organelle biogenesis in Arabidopsis. Similar to other ESCRT components, FREE1 is essential for plant growth and development because free1 mutant is seedling lethal. To identify key components that genetically interact with FREE1, we performed forward genetic suppressor screening using a dexamethasone (DEX)-inducible FREE1-RNAi line. Here we describe the detailed protocol of identifying novel FREE1 regulators using an inducible FREE1-RNAi line for the genetic suppressor screen. This protocol represents a whole procedure for identifying ESCRT genetic interactors in Arabidopsis thaliana.