Open AccessEngineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants
Author(s) -
Eberhard Sorge,
Dmitri Demidov,
Inna Lermontová,
Andreas Houben,
Udo Conrad
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0247015
Subject(s) - ubiquitin ligase , protein degradation , signal transducing adaptor protein , biology , microbiology and biotechnology , proteasome , transgene , drosophila melanogaster , ubiquitin , green fluorescent protein , ddb1 , computational biology , genetics , signal transduction , gene
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.