LETTER TO THE EDITOR FLS2-Mediated Responses to Ax21-Derived Peptides: Response

W In Danna et al. (2011), we reported the surprising result that sulfated synthetic peptide preparations, corresponding to derivatives of the Xanthomonas oryzae pv oryzae axY S 22 peptide, activate a variety of FLAGELLIN-SENSITIVE2 (FLS2)–dependent defense-related responses in Arabidopsis thaliana seedlings and plants. These included defense gene expression, an oxi-dative burst, and protection of seedlings from infection by Pseudomonas syringae. The Mueller et al. (2012) Commentary raises the important concern of potential contamination of synthetic peptide preparations with flg22 or flg22-like peptides. Although we had not considered the possibility that commercially synthesized peptides might be contaminated during synthesis, we were very cognizant of the fact that flg22 contamination in our peptide collection would explain our results. We therefore explicitly addressed the issue of contamination using mass spectrometry (liquid chromatography– electrospray ionization–mass spectrometry) analysis to determine the level of potential flg22 contamination in the most active pep-tide preparation. As explained in more detail below, our biological analysis suggested that contaminating flg22 would have had to be present at levels well over 10 nM to elicit a full protective response in seedlings, yet the liquid chromatography–mass spec-trometry demonstrated that no flg22 was present at the 10 nM level of detection. Thus, we concluded that flg22 contamination was not a likely explanation for our results. What, then, accounts for the experimental differences observed between our data and the data of Mueller et al. (2012)? As explained below, one explanation for the inability of Mueller et al. (2012) to reproduce our results is the clear experimental differences between the two studies. For example , Mueller et al. (2012) did not test the most active Ax21-derived peptide (called axY S 22-A1 or A1 in this report). The A1 peptide was originally synthesized as part of a structure/function analysis of Ax21 to determine which residues were essential for activation of rice (Oryza sativa) XA21-mediated immunity (Lee et al., 2009). A1 carries an Ala-to-Gly mutation in the N-terminal amino acid of the biologically active axY S 22 peptide and retains the sul-fated Tyr at position 22 (Lee et al., 2009). The A1 peptide retains the ability to activate XA21-mediated immunity but does not occur in any of the X. oryzae pv oryzae se-quenced genomes. Rather than testing the A1 peptide, Mueller et al. (2012) tested a variant of the peptide that is weakly active in our experimental system. They also used different methods to assess the plant …