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.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- buněčné linie MeSH
- geneticky modifikované rostliny genetika metabolismus MeSH
- histony genetika metabolismus MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteinové inženýrství * metody MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteolýza * MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- tabák genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ATP dependent 26S protease MeSH Prohlížeč
- histony MeSH
- proteasomový endopeptidasový komplex MeSH
- proteiny huseníčku MeSH
- rekombinantní fúzní proteiny MeSH
The SMC 5/6 complex together with cohesin and condensin is a member of the structural maintenance of chromosome (SMC) protein family. In non-plant organisms SMC5/6 is engaged in DNA repair, meiotic synapsis, genome organization and stability. In plants, the function of SMC5/6 is still enigmatic. Therefore, we analyzed the crucial δ-kleisin component NSE4 of the SMC5/6 complex in the model plant Arabidopsis thaliana. Two functional conserved Nse4 paralogs (Nse4A and Nse4B) are present in A. thaliana, which may have evolved via gene subfunctionalization. Due to its high expression level, Nse4A seems to be the more essential gene, whereas Nse4B appears to be involved mainly in seed development. The morphological characterization of A. thaliana T-DNA mutants suggests that the NSE4 proteins are essential for plant growth and fertility. Detailed investigations in wild-type and the mutants based on live cell imaging of transgenic GFP lines, fluorescence in situ hybridization (FISH), immunolabeling and super-resolution microscopy suggest that NSE4A acts in several processes during plant development, such as mitosis, meiosis and chromatin organization of differentiated nuclei, and that NSE4A operates in a cell cycle-dependent manner. Differential response of NSE4A and NSE4B mutants after induced DNA double strand breaks (DSBs) suggests their involvement in DNA repair processes.
- Klíčová slova
- Arabidopsis thaliana, NSE4 δ-kleisin, SMC5/6 complex, meiosis, mitosis, nucleus, phylogeny, super-resolution microscopy,
- Publikační typ
- časopisecké články MeSH
Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin ribosides from the root and their subsequent metabolic activation.
- Klíčová slova
- Antibody-mediated modulation, CKX, biosynthesis, cytokinin, homeostasis, organ specificity, tobacco,
- MeSH
- cytokininy fyziologie MeSH
- fenotyp * MeSH
- homeostáza * MeSH
- isopentenyladenosin analogy a deriváty metabolismus MeSH
- protilátky produkované rostlinami fyziologie MeSH
- regulátory růstu rostlin fyziologie MeSH
- semenáček fyziologie MeSH
- tabák fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cytokininy MeSH
- isopentenyladenosin MeSH
- protilátky produkované rostlinami MeSH
- regulátory růstu rostlin MeSH
- zeatin riboside MeSH Prohlížeč
With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.
- MeSH
- Arabidopsis genetika MeSH
- cytosol imunologie metabolismus MeSH
- fosfotransferasy biosyntéza genetika imunologie MeSH
- mapy interakcí proteinů genetika imunologie MeSH
- proteinkinasy biosyntéza genetika imunologie MeSH
- proteiny huseníčku biosyntéza genetika imunologie MeSH
- protilátky aplikace a dávkování imunologie MeSH
- regulace genové exprese u rostlin MeSH
- rekombinantní proteiny aplikace a dávkování imunologie MeSH
- signální transdukce MeSH
- umlčování genů imunologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- AHP3 protein, Arabidopsis MeSH Prohlížeč
- CKI1 protein, Arabidopsis MeSH Prohlížeč
- fosfotransferasy MeSH
- proteinkinasy MeSH
- proteiny huseníčku MeSH
- protilátky MeSH
- rekombinantní proteiny MeSH
