1. Targeting Plant Ethylene Responses by Controlling Essential Protein–Protein Interactions in the Ethylene Pathway 
Melanie M.A. Bisson, Georg Groth 
Molecular Plant 
Volume 8, Issue 8, Pages (August 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
Nuclear Localization Signal (NLS) in EIN2 Is Required for EIN2-ETR1 Complex Formation.
(A–D) Subcellular localization of C-terminal EIN2479−1294 and EIN2479−1294(ΔNLS) transiently expressed in Nicotiana benthamina epidermal leaf cells, analyzed by confocal laser scanning microscopy. (A) DAPI staining of cells expressing NLS-deleted EIN2479−1294(ΔNLS)-GFP. Nuclei are indicated by white arrows. (B) DAPI staining of cells expressing EIN2479−1294-GFP. Nuclei are indicated by white arrows. Yellow arrows indicate putative ER localization. (C) Co-localization of EIN2479−1294-GFP and ER-mCherry marker protein at the ER membrane. (D) Cytosolic localization of EIN2479−1294(ΔNLS)-GFP co-expressed with ER-mCherry marker protein. Scale bars in (A–D) represent 5 μm.
(E and F) The NLS domain in EIN2 is essential for EIN2-ETR1 interaction. (E) FRET efficiencies calculated from transient expression of GFP-tagged EIN2(ΔNLS) and mCherry-tagged ETR1 in tobacco leaf cells underline that EIN2 lacking the NLS domain is not able to form a tight complex with the ETR1 receptor. (F) Tryptophan fluorescence-based in vitro protein–protein interaction assay between ETR1 and EIN2479−1294(ΔNLS) indicates unspecific interaction of EIN2 lacking the NLS domain (EIN2479−1294(ΔWΔNLS)) with ethylene receptor ETR1.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2
NLS-based Peptide NOP-1 Inhibits Ethylene Responses In Vitro and In Vivo.
(A) Kd values of the EIN2-ETR1 complex obtained by titration of ETR1 with EIN2479−1294(ΔW) as a function of synthetic octapeptides NOP-1 and ROP-1, respectively. NOP-1 strongly decreases complex formation of EIN2-ETR1 in a concentration-dependent manner; ROP-1 had no effect.
(B) FRET-based in planta interaction assay performed in the presence of the synthetic octapeptides. NOP-1 prevents EIN2-ETR1 complex formation in tobacco cells, ROP-1 was uninfluential. EIN2-ETR1 complex formation in the absence of any peptides was used as positive control.
(C) Effect of 200 μM octapeptides on the triple response of dark-grown wild-type Arabidopsis (ecotype Columbia) seedlings. NOP-1 leads to a less pronounced triple response. ROP-1 had no such effect.
(D) Synthetic peptide NOP-1 inhibits ethylene response (hypocotyl length) in Arabidopsis in a concentration-dependent manner. Asterisks represent statistical significance (*P < 0.05 compared with the positive control [no NOP-1]).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3
FRET-Based In Vitro Assay to Monitor the Inhibition of EIN2-ETR1 Interaction.
(A) Overlapping emission and excitation spectra of Alexa dye-labeled recombinant proteins ETR1 and EIN2479−1294.
(B) Inhibitory peptide NOP-1 (100 μM) leads to decreased energy transfer from ETR1-Alexa 488 to EIN2479−1294-Alexa 568, as shown by reduced acceptor fluorescence. Control peptide ROP-1 (100 μM) was ineffective.
(C) Fluorophore control measurements emphasize that NOP-1 had no effect on isolated dyes.
(D) Calculation of the IC50 value of NOP-1 for inhibition of EIN2-ETR1 complex formation, determined via the FRET-based in vitro assay. All data represent the mean of three independent measurements ± SD.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4
Structure Activity Analysis of the Inhibitory Effect of the NLS Motif on EIN2-ETR1 Complex Formation.
(A and B) Impact of single alanine substitutions in inhibitory NLS peptide NOP-1 on EIN2-ETR1 interaction. (A) List of sequences and physicochemical characteristics of alanine substitution peptides of NOP-1. Alanine substitutions are highlighted in red. (B) Inhibitory effect of 100 μM substitution peptides on EIN2-ETR1 complex formation measured by FRET-based in vitro assay. Complex formation is affected to a similar extent as the control peptide NOP-1.
(C and D) Impact of NLS peptide length on EIN2-ETR1 interaction. (C) List of sequences and physicochemical characteristics of synthetic peptides corresponding to the NLS motif in EIN2 extended by residues adjacent to this motif. The NLS motif is highlighted in bold; the adjacent N-terminal residues are illustrated in italics. (D) Inhibitory effect of 100 μM peptides on EIN2-ETR1 complex formation measured by FRET-based in vitro assay. Inhibition correlates with sequence length. EIN2-ETR1 interaction is more efficiently blocked with increasing peptide length. Control peptide PNOP-1 corresponding to residues adjacent to the NLS motif in EIN2 has no effect on the EIN2-ETR1 complex.
(E and F) Impact of peptide variants derived from plant NLS motifs on EIN2-ETR1 interaction. (E) List of sequences and physicochemical characteristics of synthetic peptide variants derived from plant importin recognition sequences. (F) Inhibitory effect of peptide variants on EIN2-ETR1 complex formation measured by FRET-based in vitro assay. In general, all variants inhibit complex formation. However, synthetic peptides YSOP-1 and YSOP-2 show a more pronounced inhibition of the EIN2-ETR1 interaction than the NOP-1 peptide, corresponding to the endogenous NLS motif of EIN2. Peptides SHOP-1, SHOP-2, and YSOP-4 are less efficient. Control peptide AOP-1 consisting essentially of acidic residues was ineffective. All data represent the mean of three independent measurements ± SD.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

6. Figure 5 Impact of NLS Peptides on Tomato Fruit Ripening.
(A) NLS-based peptide NOP-1 leads to a delayed ethylene response in living plants. The impact of synthetic peptides NOP-1 and ROP-1 on fruit ripening of climacteric tomato fruits is given in the left panel. Graphical summary of the fruit ripening process of climacteric tomato fruits treated with synthetic peptides is illustrated in the right panel. Green represents an immature fruit, red a mature fruit.
(B) Graphical summary of tomato ripening of surface-treated fruits with alanine substitution variants (APs) of NLS peptide NOP-1. All APs showed an effect on fruit ripening.
(C) Graphical summary of the ripening process of tomato fruits treated with NLS peptides of increasing length. Maturation of tomato fruits is less delayed with increasing length of the NLS peptide applied. Control peptide PNOP-1 corresponding to residues adjacent to the NLS motif in EIN2 had no effect on maturation.
(D) Graphical summary of tomato fruit maturation of fruits surface treated with peptide variants derived from plant NLS motifs. Control peptide AOP-1 consisting essentially of acidic residues had no effect on fruit ripening.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions