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Antagonistic peptide technology for functional dissection of CLE peptides revisited.

Czyzewicz N, Wildhagen M, Cattaneo P, Stahl Y, Pinto KG, Aalen RB, Butenko MA, Simon R, Hardtke CS, De Smet I - J. Exp. Bot. (2015)

Bottom Line: Based on the analyses, it was concluded that the antagonistic peptide approach is not the ultimate means to overcome redundancy or lack of loss-of-function lines.However, information collected using antagonistic peptide approaches (in the broad sense) can be very useful, but these approaches do not work in all cases and require a deep insight on the interaction between the ligand and its receptor to be successful.This, as well as peptide ligand structure considerations, should be taken into account before ordering a wide range of synthetic peptide variants and/or generating transgenic plants.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK.

No MeSH data available.


IDA peptide treatment. (A) Sequence of synthetic IDA peptides used. (B) N. benthamiana leaf pieces expressing HSL2 were exposed to various concentrations of peptides as indicated. Oxidative burst by the luminol-based assay was monitored over time as relative light units (RLU). Leaf pieces infiltrated with Agrobacterium without HSL2 were exposed to 1 μM of both peptides and used as control. Error bars indicate standard error of n=3 or 4 replicates.
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Figure 5: IDA peptide treatment. (A) Sequence of synthetic IDA peptides used. (B) N. benthamiana leaf pieces expressing HSL2 were exposed to various concentrations of peptides as indicated. Oxidative burst by the luminol-based assay was monitored over time as relative light units (RLU). Leaf pieces infiltrated with Agrobacterium without HSL2 were exposed to 1 μM of both peptides and used as control. Error bars indicate standard error of n=3 or 4 replicates.

Mentions: In addition, the extent the antagonistic peptide technology can be applied to other small signalling peptides was assessed. For this, the IDA and IDA-LIKE (IDL) family were chosen, given their sequence similarity to CLEs (Stenvik et al., 2006). The IDA and IDL1 peptides of 12 amino acids share a common core at positions four to seven [PS(G/A)P] and the C-terminal end [H(N/H)] with CLV3 and some CLE peptides (Figs 5A, 6A). Like CLV3, hydroxylation of the Pro at position seven of the IDA dodecapeptide (IDAp, also referred to as PIPPo) increases the activity of the peptide (Butenko et al., 2014). An oxidative burst response in Nicotiana benthamiana can be employed as readout for the RLK HAESA-LIKE2 (HSL2) activation by exogenously applied synthetic IDA peptides (Butenko et al., 2014). Previous results indicated that IDAp binds to HSL2 with a Kd of 20nM (Butenko et al., 2014). As the wild-type IDA peptide has an Ala at position six corresponding to the Gly at that position in CLV3, and the ida mutant phenotype can be fully rescued by IDL1, which has a Gly at this position (Stenvik et al., 2008) (Fig. 6A); both of these small amino acids are evidently compatible with high signalling activity. It was, however, conceivable that substitution to the larger Thr (mIDAp6Thr) (Fig. 5A) could have an effect on receptor binding and/or activation. Therefore, the activity of mIDAp6Thr in comparison with the activity of synthetic IDAp was assessed in an oxidative burst assay. For all peptide concentrations tested, mIDAp6Thr gave the same response as IDAp in the presence of its receptor HSL2 (Fig. 5B), indicating that the mutated peptide was just as active as its wild-type counterpart. In conclusion, this mutation neither produced a ligand with weaker activity, nor a peptide with antagonistic effect.


Antagonistic peptide technology for functional dissection of CLE peptides revisited.

Czyzewicz N, Wildhagen M, Cattaneo P, Stahl Y, Pinto KG, Aalen RB, Butenko MA, Simon R, Hardtke CS, De Smet I - J. Exp. Bot. (2015)

IDA peptide treatment. (A) Sequence of synthetic IDA peptides used. (B) N. benthamiana leaf pieces expressing HSL2 were exposed to various concentrations of peptides as indicated. Oxidative burst by the luminol-based assay was monitored over time as relative light units (RLU). Leaf pieces infiltrated with Agrobacterium without HSL2 were exposed to 1 μM of both peptides and used as control. Error bars indicate standard error of n=3 or 4 replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526918&req=5

Figure 5: IDA peptide treatment. (A) Sequence of synthetic IDA peptides used. (B) N. benthamiana leaf pieces expressing HSL2 were exposed to various concentrations of peptides as indicated. Oxidative burst by the luminol-based assay was monitored over time as relative light units (RLU). Leaf pieces infiltrated with Agrobacterium without HSL2 were exposed to 1 μM of both peptides and used as control. Error bars indicate standard error of n=3 or 4 replicates.
Mentions: In addition, the extent the antagonistic peptide technology can be applied to other small signalling peptides was assessed. For this, the IDA and IDA-LIKE (IDL) family were chosen, given their sequence similarity to CLEs (Stenvik et al., 2006). The IDA and IDL1 peptides of 12 amino acids share a common core at positions four to seven [PS(G/A)P] and the C-terminal end [H(N/H)] with CLV3 and some CLE peptides (Figs 5A, 6A). Like CLV3, hydroxylation of the Pro at position seven of the IDA dodecapeptide (IDAp, also referred to as PIPPo) increases the activity of the peptide (Butenko et al., 2014). An oxidative burst response in Nicotiana benthamiana can be employed as readout for the RLK HAESA-LIKE2 (HSL2) activation by exogenously applied synthetic IDA peptides (Butenko et al., 2014). Previous results indicated that IDAp binds to HSL2 with a Kd of 20nM (Butenko et al., 2014). As the wild-type IDA peptide has an Ala at position six corresponding to the Gly at that position in CLV3, and the ida mutant phenotype can be fully rescued by IDL1, which has a Gly at this position (Stenvik et al., 2008) (Fig. 6A); both of these small amino acids are evidently compatible with high signalling activity. It was, however, conceivable that substitution to the larger Thr (mIDAp6Thr) (Fig. 5A) could have an effect on receptor binding and/or activation. Therefore, the activity of mIDAp6Thr in comparison with the activity of synthetic IDAp was assessed in an oxidative burst assay. For all peptide concentrations tested, mIDAp6Thr gave the same response as IDAp in the presence of its receptor HSL2 (Fig. 5B), indicating that the mutated peptide was just as active as its wild-type counterpart. In conclusion, this mutation neither produced a ligand with weaker activity, nor a peptide with antagonistic effect.

Bottom Line: Based on the analyses, it was concluded that the antagonistic peptide approach is not the ultimate means to overcome redundancy or lack of loss-of-function lines.However, information collected using antagonistic peptide approaches (in the broad sense) can be very useful, but these approaches do not work in all cases and require a deep insight on the interaction between the ligand and its receptor to be successful.This, as well as peptide ligand structure considerations, should be taken into account before ordering a wide range of synthetic peptide variants and/or generating transgenic plants.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK.

No MeSH data available.