Auxin-inducible degron-mediated protein depletion in Diplonema papillatum

Abstract

Diplonemids are highly abundant unicellular flagellates that are evolutionarily divergent from traditional model eukaryotes. Diplonema papillatum is a model diplonemid for which several molecular tools have recently been established. However, no method for conditional protein depletion was available. Here, we have adapted an auxin-inducible degron (AID) system in D. papillatum. Depletion of the KIN-A motor protein (a glycomonad-specific component of the chromosomal passenger complex) causes defects in chromosome segregation and cell proliferation. The AID system also successfully depleted a divergent Mad1 protein that localizes at nuclear pores, and a flagellar component, CFAP20. Our study demonstrates the power of the AID system to perform loss-of-function studies in D. papillatum. Lay summary Diplonemids are single-celled microbes that live in the world’s oceans in enormous numbers, yet very little is known about their biology. They are only distantly related to humans and other well-studied model organisms used in cell biological research, but are close relatives of parasites that cause serious diseases such as African sleeping sickness, Chagas disease, and leishmaniasis. Studying diplonemids can therefore reveal both fundamental principles of cell biology and clues relevant to these related parasites. One diplonemid species, Diplonema papillatum, can be genetically manipulated in the lab, but researchers previously had no way to switch off individual proteins to study what they do. This work introduces such a tool, based on a plant-derived system that triggers controlled destruction of a target protein when a small chemical is added. We have used it to destroy three different proteins, showing that each is degraded efficiently and that one protein, called KIN-A, is required for cells to properly transmit their genetic material. This gives researchers a new way to study gene function in this understudied but important group of marine plankton.