Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants.

Eberhard Sorge,Dmitri Demidov,Udo Conrad,Inna Lermontova,Andreas Houben,Miguel A Blázquez

doi:10.1371/journal.pone.0247015

Abstract

Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes. We aimed to overcome these limitations by acting downstream of the protein level. Chimeric E3 ligases degrade proteins of interest in mammalian cell lines, Drosophila melanogaster embryos, and transgenic tobacco. We successfully recruited the 26S proteasome pathway to directly degrade a protein of interest located in plant nuclei. This success was achieved via replacement of the interaction domain of the E3 ligase adaptor protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4). For proof of concept, the target protein CENH3 of A. thaliana fused to EYFP was subjected to nanobody-guided proteasomal degradation in planta. Our results show the potential of the modified E3-ligase adapter protein VHHGFP4-SPOP in this respect. We were able to point out its capability for nucleus-specific protein degradation in plants.

Highlights

Understanding new aspects of molecular signalling pathways require the elimination of single key proteins within a complex framework
We performed transient transformation to test whether the system is capable of degrading stably expressed enhanced yellow fluorescent protein (EYFP)-Centromeric histone 3 (CENH3) by NSlmb-anti-GFP nanobody (VHHGFP4) or VHHGFP4-Speckle-type POZ protein (SPOP)
In transgenic N. tabacum lines with stable EYFP-CENH3 expression transiently transformed with a VHHGFP4-SPOP construct, a strong reduction of Green fluorescent protein (GFP) signals was recognised (S1 Fig)

Summary

Introduction

Understanding new aspects of molecular signalling pathways require the elimination of single key proteins within a complex framework. Rapid changes in protein levels in vivo allow us to decipher these pathways and address synthetic biology approaches. Trials to eliminate proteincoding genes via mutation or CRISPR/Cas9-based methods generally removed the selected protein from all organs, cells and cellular compartments of the organism [1, 2]. A recent study showed an elegant way to reduce protein function in a cell-type-specific manner in plants, based on CRISPR/Cas and oestrogen- induceable promotor [3]. CRISPR/ Cas is still error-prone and can show off-target effects [4]. Protein amounts have been lowered via the downregulation of the corresponding transcripts using antisense and RNAi strategies [5]

Objectives

Methods

Results

Conclusion