Leibniz Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.
Mendel Centre for Plant Genomics and Proteomics, CEITEC, Masaryk University, Brno CZ, Czech Republic.
PLoS One. 2021 Feb 12;16(2):e0247015. doi: 10.1371/journal.pone.0247015. eCollection 2021.
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.
确定蛋白质的功能仍然是现代生物学的一项关键任务。在植物中敲除蛋白质功能的经典遗传方法仍然面临着一些限制，例如产生纯合转基因系的耗时性质，或者非生存性功能缺失表型的风险。我们旨在通过在蛋白质水平的下游作用来克服这些限制。嵌合 E3 连接酶在哺乳动物细胞系、黑腹果蝇胚胎和转基因烟草中降解感兴趣的蛋白质。我们成功地招募了 26S 蛋白酶体途径，直接降解位于植物核内的感兴趣的蛋白质。这一成功是通过用特定的抗 GFP 纳米抗体 (VHHGFP4) 替换 E3 连接酶衔接蛋白 SPOP（Speckle-type POZ adapter protein）的相互作用域来实现的。为了验证概念，将拟南芥的靶蛋白 CENH3 与 EYFP 融合，并在体内进行纳米抗体引导的蛋白酶体降解。我们的结果表明，修饰后的 E3 连接酶衔接蛋白 VHHGFP4-SPOP 在这方面具有潜力。我们能够指出它在植物中具有核特异性蛋白质降解的能力。
