Fig 1.
Principle of ubiquitination by E2-E3 ligase complex and degradation of ubiquitinated proteins.
(A) The E2-E3 ligase complex mediates the transfer of ubiquitin to a target protein. The target specificity depends on the adaptor subunit NSlmb or SPOP. Natural binding domains of SPOP (MATH) or NSlmb (WD40) were replaced with an anti-GFP nanobody, which resulted in chimeric adaptor proteins with high binding affinity for GFP. (B) Schematic presentation of the chimeric proteins used in this study. EYFP-CENH3 located at the centromeres serves as a nucleus-specific substrate. The NSlmb construct originates from the Drosophila melanogaster protein Slmb. The sequence of the SPOP adaptor protein is derived from the human genome. Nanobody control to show the dependency of the directed degradation on the adaptor protein (NSlmb or SPOP). All sequences are under the control of the CaMV35S promoter for plant protein expression. Different selection markers (PPT for phosphinothricin and KMR for kanamycin resistance) enabled stable double transformant plant lines. C-terminal cmyc tag was used for immunodetection.
Fig 2.
Quantitative EYFP-CENH3 transcript analysis of transgenic N. tabacum lines using qRT-PCR.
Three independent transgenic lines of each genotype were tested for relative EYFP-CENH3 expression. The mRNA levels for EYFP-CENH3 varied between 0.2 and 1.2, except for the NSlmb-VHHGFP4 Line 25 with a value of 4. Fluorescence microscopy analysis confirmed that the high abundance of transcript in line 4 corresponds to the high amount of fusion protein (S3 Fig). Three biological and three technical replicates were analysed for each tested plant line.
Fig 3.
Target protein abundance in transgenic plants was analysed using Western blot analysis of nuclei extract proteins with anti-CENH3 and anti-EYFP antibodies.
(A) Detection using the anti-A. thaliana CENH3 antibody. Control plants expressing EYFP-CENH3 show signals for heterodimers of EYFP-AtCENH3 and N.tCENH3 at 68 kDa. The same results were found for transgenic plants that overexpress VHHGFP4 or NSlmb-VHHGFP4 together with EYFP-CENH3. These lines also exhibited a prominent signal at the range of 49 kDa, which represented EYFP-AtCENH3. The combination of VHHGFP4-SPOP and EYFP-CENH3 did not show either of these signals. All tested plants, including WT, resulted in an unspecific signal at 41 kDa. (B) Anti-EYFP detection. Control plants with EYFP-CENH3 expression show the same signals described in (a) except for an unspecific band at 41 kDa. This result confirms the signal reduction monitored by anti-CENH3 detection. (C) Anti-histone H3 detection as a loading control. An even distribution of signal intensity represents similar amounts of protein in the assay. Computational quantification of signal strength of single bands was done by “LI-COR Image Studio” software (LI-COR Biosciences–GmbH, www.licor.com) designed for the analysis of Western blot images.
Fig 4.
Confocal microscopy analysis of transgenic N. tabacum leaves.
(A and B) Transgenic plants accumulate EYFP-CENH3- within centromeric regions. (C and D) EYFP-CENH3- and VHHGFP4 co-expression. Nanobody without fusion partner binds EYFP but does not result in a signal reduction. Any signal modulation (reduction or translocation) must be induced by the fusion partner (SPOP/NSlmb). (E and F) Coexpression of YFP-CENH3- and NSlmb-VHHGFP4. We observed translocation of the fluorescence signal to the nucleoplasm. (G and H) Co-expression of EYFP-CENH3 and VHHGFP4-SPOP. Strong reduction of the target protein. Scale bar: 10 μm. Magnification 40x. For all genotypes leaf material of 10 independent transgenic lines was analysed.