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

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.

Nanobody-Dependent Delocalization of Endocytic Machinery in Arabidopsis Root Cells Dampens Their Internalization Capacity

“…Recent results suggest that plant cells very likely contain a feedback loop controlling TPC expression, as carbon starved plants contained roughly the same amount of full-length TPLATE-GFP, next to an extensive amount of TPLATE-GFP degradation products (Wang et al, 2019). In case plant cells make more TPC upon depleting the complex at the PM, DeGradFP could provide a viable solution to this problem (Baudisch et al, 2018;Ma et al, 2019;Sorge et al, 2021). By applying this method in GFP-complemented tml-1(−/−) mutants, newly synthesized TML-GFP would be broken down immediately, preventing to achieve functional levels of TPC at the PM.…”

“…DeGradFP links an anti-GFP nanobody to an F-box protein, thereby targeting it for ubiquitin-dependent degradation (Caussinus et al, 2012). This approach was shown to be functional in plants (Baudisch et al, 2018) and successfully used to deplete WUSCHEL-GFP in the Arabidopsis flowering meristem (Ma et al, 2019) and the centromeric Histon H3 of Arabidopsis in transgenic tobacco plants (Sorge et al, 2021). Nanobodies have also been used in Arabidopsis seedlings to lock down vacuolar sorting receptors (VSRs) in cellular compartments upstream of trans-Golgi network/early endosomes, allowing to determine their retrograde trafficking pathway (Früholz et al, 2018).…”

Conditional and tissue-specific approaches to dissect essential mechanisms in plant development

“…An inducibly expressed anti-GFP Nb fused to the F-box domain of the E3 ligase complex was used to selectively degrade GFP-tagged POIs via the 26S proteasome [38] (Figure 2C). This deGradFP method was also used in transgenic tobacco plants to deplete the C-terminal centromeric histone H3 variant CENH3 of Arabidopsis, demonstrating the capacity of nucleus-specific protein degradation [39]. In Arabidopsis, anti-GFP Nbs were expressed under the control of the ethanol-inducible AlcR/AlcA system to achieve switchable degradation of GFP-tagged WUSCHEL, showing that WUSCHEL controls the auxin response in the stem cells of the shoot apical meristem [40*].…”

E3 Ligase for CENP-A (Part 2)

“…Currently, an endogenous E3 ligase for plant CENP-A (CENH3) is not yet identified. Sorge et al developed a synthetic biology approach to degrade plant CENP-A using E3-ligase adapter protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4) [201] (Table 1). To determine the function of proteins, CRISPR/Cas9-based methods and antisense/RNAi strategies are commonly used to remove the selected protein from all organs in a cell-and tissue-specific manner.…”