Cloning was performed as previously described (Sambrook, 2001). DreamTaq polymerase, FastDigest restriction enzymes, and T4 DNA ligase from Thermo Fisher Scientific were used according to the manufacturer’s instructions.

A XbaI/XhoI fragment containing the CaMV-enhanced 35S promoter (d35S) was released from plasmid d35S-Nos-AB-M (Himmelbach et al., 2007) and inserted in the pGFP-Amp vector to form pGH102. The SfiI fragment of vector pGH102 harboring the full GFP expression cassette was introduced into the appropriate sites of plasmid p6d35S (Hensel et al., 2015) to generate the vector pGH219.

Template sequences (NSlmb-VHHGFP4 and NSnoFbox-VHHGFP4) were provided by Caussinus et al. (2011). NSlmb-VHHGFP4 comprises the sequence encoding the F-box domain containing in the N-terminal part of the F-box protein supernumerary limbs (Slmb) from Drosophila melanogaster fused to the GFP-binding nanobody VHHGFP4 sequence. NSnoFbox-VHHGFP4 lacks the F-box domain for SKP1 binding and represents the negative control used in later GFP-depletion assays. The primer pair Slmb-NcoI-for and Slmb-NotI-rev was used to add NcoI and NotI sites by PCR. The resulting fragments were subcloned using the TOPO Cloning Kit (Thermo Fisher Scientific). After the plasmids were sequenced and cut with NcoI and NotI, the fragment was inserted into the pRTRA vector. The resulting expression cassette was cut using HindIII and ligated into the binary vector pCB301-Kan. The resulting plasmids (containing the NSlmb-VHH-GFP4 or NSnoFbox-VHHGFP4 constructs) were used for transformation of Agrobacterium tumefaciens strain GV2260.

Transformation of Nicotiana tabacum was performed by agroinfection of leaf discs (Horsch, 1985) as described by Floss et al. (2010). The leaf discs were submerged for 1 h in an A. tumefaciens culture, plated on MS medium, and stored at 24°C in the dark for 2 days. Then, the explants were transferred to NBKC medium (MS medium containing 0.2 mg/L α-naphthaleneacetic acid, 1 mg/L 6-benzylaminopurine, 50 mg/L kanamycin, and 500 mg/L cefotaxime). Every 10–14 days, the plant material was removed and placed in fresh NBKC medium until plantlets appeared. Plantlets (2–3 cm in height) were placed onto MS medium containing 50 mg/L kanamycin for selection. Surviving putative transgenic plants were planted in soil and grown to maturity in a greenhouse.

Leaf discs were stored at -80°C, transferred to 2 mL safe-lock tubes along with two metal bullets, and disintegrated in a Retsch mill at a frequency of 28/s for 2 min under liquid nitrogen. Then, 150 μL of 2× SDS sample buffer (Conrad et al., 1998) was added and heated at 95°C for 10 min. Samples were then cooled on ice and centrifuged for 30 min at 21,000 × g. Extracted plant proteins (corresponding to 40 μg protein) were separated by reducing SDS–PAGE (12% polyacrylamide) and then electrotransferred to nitrocellulose membranes. The Western blotting procedure was carried out using monoclonal anti-cmyc antibodies following the protocol described by Gahrtz and Conrad (2009). The secondary antibody was sheep anti-mouse IgG horseradish-peroxidase-linked whole antibody (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, United Kingdom), and ECL was used for detection. GFP was detected with specific rabbit anti-GFP antibodies and goat anti-rabbit IgG horseradish-peroxidase-linked whole antibody. Specific signals were detected as described above.

Around 100 mg of tobacco leaves was transferred to 2 mL safe-lock tubes along with two metal bullets and disintegrated in a Retsch mill at a frequency of 28/s for 2 min under liquid nitrogen. Then, 300 μL of extraction buffer [50 mM Tris, pH 8.0, 5 mM EDTA, 1 mM DDT, 1× Complete proteinase inhibitor mix (Roche, Mannheim, Germany)] was added and the extracts were shaken end-over-end for 45 min at 4°C. To analyze the stability in solution, 50 μM MG-132 (Sigma, Steinheim, Germany) was applied.

Enhanced green fluorescent protein (kindly provided by Mario Jakob, Universität Halle/Saale, Germany) was diluted in Phage PBS (100 mM NaCl, 32 mM Na₂HPO₄, 17 mM NaH₂PO₄, pH 7.2) at a concentration of 0.05 μg/100 μL and put into Immunoplate MaxiSorp wells (Nalge Nunc International, Roskilde, Denmark). After overnight incubation at room temperature, the wells were saturated with 3% bovine serum albumin (BSA) in phosphate buffered saline, 0.05% Tween 20 (PBS-T) (Gahrtz and Conrad, 2009). Standards (0.01–100 nM eGFP) and appropriately diluted native extracts were mixed with rabbit anti-GFP antibodies (diluted 1:50,000 in 3% BSA–PBS-T) and incubated at 25°C for 30 min in a master plate. Samples were then transferred to the saturated eGFP plates and incubated for 1 h at 25°C. After extensive washing with PBS-T, goat anti-rabbit IgG alkaline-phosphatase-linked whole antibody (Sigma, Steinheim, Germany), diluted 1:2000 in 3% BSA and PBS-T, was applied for 1 h at 25°C. After further washing the enzymatic substrate, p-nitrophenyl phosphate (pNPP) in 0.1 M diethanolamine-HCl (pH 9.8) was added, and the absorbance signal was measured at 405 nm after 1 h incubation at 37°C. Measured values from control experiments performed in parallel (same handling procedure but without the antigen incubation step) were subtracted.

The OneTaq One-Step RT-PCR Kit (New England Biolabs) was used to identify GFP and actin mRNA. Analysis was performed according to the manufacturer’s instructions. Briefly, total RNA from plant tissue was extracted using the RNeasy Plant Kit (Qiagen). For cDNA synthesis the following solutions were mixed and denatured at 70°C for 5 min: 10 μL precipitated RNA (1 μg), 9 μL of H₂O, 2 μL of 10 μM GFP reverse primer or 2 μL of 10 μM actin reverse primer, and 25 μL of OneTaq One-Step Quick-Load Reaction Mix. Then, 2 μL of 10 μM GFP forward primer or 10 μM actin forward primer and 1.8 μL of OneTaq One-Step Enzyme Mix were added. PCR amplification was performed according to the following protocol for 40 cycles: 48°C 15 min, 94°C 1 min, 94°C 15 s, 63°C 30 s, 58°C 1 min, and 68°C 5 min. The resulting DNA fragments were separated on a 2% agarose gel in tris-acetate-EDTA, pH 8.0 (TAE) buffer (Sambrook, 2001).

ASK9/10-BamHI-for 5′-ATGGATCCTCGACGAAGAAGATCATA-3′

ASK9-XhoI-rev 5′-TTCTCGAGTTCAAAAGCCCATTTATTCTC-3′

ASK10-XhoI-rev 5′-TTCTCGAGTTCAAAACCCCATTGATTCT-3′

Slmb-NcoI-for3 5′-CCATGGCCATGATGAAAATGGAGACTGA-3′

Slmb-NotI-rev5 5′-TTGCGGCCGCGCTGGAGACGGTGACCTG-3′

Slmb-BamHI-for3 5′-GCGGATCCATGATGAAAATGGAGACTGAC-3′

Slmb-NotI-rev5 5′-TTGCGGCCGCGCTGGAGACGGTGACCTG-3′

Slmb-NdeI-for3 5′-CATATGATGATGAAAATGGAGACTGACAAAAT

AATGGACGAAACCAACTCCAATGCACAGGCC-3′

Slmb-cmyc-NotI-rev5 5′-GCGGCCGCATTCAGATCCTCTTCTGAGATGAG

TTTTTGTTCGTCGACGCTGGAGACGGTGACCTG-3′

VHHGFP4-NdeI-for 5′-GCATATGGATCAAGTCCAACTGGTGGAGT-3′

VHHGFP4-SalI-rev 5′-GTCGACGCTGGAGACGGTGACCTG-3′

BamHI-VHHGFP4-for 5′-GGATCCATGGATCAAGTCCAACTGGTG-3′

NotI-VHHGFP4-rev 5′-GCGGCCGCGCTGGAGACGGTGA-3′

Actin forward 5′-CTATTCTCCGCTTTGGACTTGGCA-3′

Actin reversed 5′-AGGACCTCAGGACAACGGAAACG-3′

GFP forward 5′-ATGGTGAGCAAGGGCGAGGAGCT-3′

GFP reversed 5′-TTACTTGTACAGCTCGTCCATGCCGA-3′

We constructed expression vectors allowing for the ubiquitous expression of either an NSlmb-VHHGFP4 (anti-GFP nanobody) or an NSnoFbox-VHHGFP4 fusion protein in the cytosol of plant cells. The CaMV35S promoter induces expression in nearly all plant cells, and the cmyc Tag enables detection of the fusion proteins by Western blot analysis (Figure 2). All expression cassettes were cloned into a binary plant expression vector allowing for the selection of transgenic plants via kanamycin resistance (Km^R). The second construct served as an internal control to show that the degradation of GFP was F-box domain dependent. We decided to use an animal-derived F-box variant and not a plant F-box variant to minimize the effects on general regulatory processes in plants. After leaf-disc transformation experiments were completed, 51 kanamycin-resistant plants transformed with NSlmb-VHHGFP4 were selected, grown, and analyzed by Western blot. Twenty lines with transgenic protein accumulation were detected. In addition, 51 kanamycin-resistant plants transformed with NSlmb-VHHGFP4 were selected, grown, and analyzed by Western blot. Seven lines with transgenic protein accumulation were detected. A binary plant expression vector (pGH219) allowing for the ubiquitous cytosolic expression of GFP, also driven by the CaMV35S promoter, was used for super-transformation of NSlmb-VHHGFP4 and NSnoFbox-VHHGFP4 lines. This vector allows for the selection of hygromycin-resistant transgenic plants (Figure 2). Wild-type N. tabacum (tobacco) plants were transformed as controls and GFP expression was detected by rabbit anti-GFP antibodies (Figure 3).

Four independent lines expressing the NSlmb-VHHGFP4 fusion protein and two independent lines expressing the NSnoFbox-VHHGFP4 fusion protein were super-transformed with a transgene encoding GFP as a reporter for protein degradation. Overall, 11 kanamycin- and hygromycin-resistant tobacco lines deriving from different super-transformation experiments were selected that showed NSlmb-VHHGFP4 accumulation, but little to no GFP accumulation was observed by Western blot analysis using highly specific rabbit anti-GFP antibodies (Figure 3 and Table 1). Conversely, strong GFP accumulation was detected in transgenic plants expressing NSnoFbox-VHHGFP4. Six kanamycin/hygromycin lines (derived from two NSnoFbox-VHHGFP4 fusion protein expressing lines) were analyzed that showed NSnoFbox-VHHGFP4 fusion protein expression and GFP expression. The results of the analysis of two NSnoFbox-VHHGFP4-GFP lines are demonstrated in Figure 3. To strengthen these results quantitatively, GFP accumulation was measured by a competitive ELISA, which could detect GFP concentrations between 4 and 0.1 nM (Figure 4). In all nine F-box transgenic plants investigated by the competitive ELISA, GFP concentrations were determined to be below 0.1 nM (detection limit, Figure 4 and Table 1). GFP concentration was significantly higher in NSnoFbox-VHHGFP4-GFP lines and in GFP expressing tobacco plants (Figure 4). In further experiments, we wanted to rule out that the lack of GFP protein expression was due to loss of the transgene or to downregulation at the transcriptional level. Using reverse transcription and PCR amplification with specific primers, we showed that NSlmb-VHHGFP4 plants contained GFP transcripts in similar amounts to NSnoFbox-VHHGFP4 plants (Figure 5). The detection of transcripts indicates the presence of the transgene coding for GFP. Consequently, the lack of GFP accumulation in NSlmb-VHHGFP4 plants detectable via Western blot analysis is not due to downregulation at the transcriptional level. To rule out, that the removal of GFP by directed proteolysis did occur after extraction, we added MG132, a potent inhibitor of the proteasome (Lee and Goldberg, 1998) to the extraction buffer. The samples, that have been either treated with MG132 or not, show identical bands for the NSlmb-VHHGFP4 plants, the NSnoFbox-VHHGFP4 plants, and the GFP plants, as well (Figure 3). These results clearly demonstrate that GFP is degraded at the protein level in the presence of the F-box domain in the plant cells, whereas the lack of the F-box domain leads to GFP accumulation (summarized in Table 1).

The interaction of the F-box domain with plant SKP1 (Vierstra, 2009) is presumed to have caused the effect described above. SKP1-like proteins, called ASKs, have been described in plants (Zhao et al., 2003). The specific interaction between the F-box domain of Drosophila and SKP1-like proteins in plants that act as adaptors to link the F-box protein to the scaffold of the SCF complex (Figure 1) has been shown by in vitro ELISA interaction studies (see Supplementary Material). This result shows that the F-box domain from Drosophila is capable of recognizing the SKP1-analogous ASK proteins of plants.