Abstract
In vivo pulse labeling of suspension-cultured Arabidopsis cells with [32P]orthophosphate allows a systematic analysis of dynamic changes in protein phosphorylation. Here, we use this technique to investigate signal transduction events at the plant plasma membrane triggered upon perception of microbial elicitors of defense responses, using as a model elicitor flg22, a peptide corresponding to the most conserved domain of bacterial flagellin. We demonstrate that two-dimensional gel electrophoresis in conjunction with mass spectrometry is a suitable tool for the identification of intrinsic membrane proteins, and we show that among them a syntaxin, AtSyp122, is phosphorylated rapidly in response to flg22. Although incorporation of radioactive phosphate into the protein only occurs significantly after elicitation, immunoblot analysis after two-dimensional gel separation indicates that the protein is also phosphorylated prior to elicitation. These results indicate that flg22 elicits either an increase in the rate of turnover of phosphate or an additional de novo phosphorylation event. In vitro, phosphorylation of AtSyp122 is calcium-dependent. In vitro phosphorylated peptides separated by two-dimensional thin layer chromatography comigrate with two of the three in vivo phosphopeptides, indicating that this calcium-dependent phosphorylation is biologically relevant. These results indicate a regulatory link between elicitor-induced calcium fluxes and the rapid phosphorylation of a syntaxin. Because syntaxins are known to be important in membrane fusion and exocytosis, we hypothesize that one of the functions of the calcium signal is to stimulate exocytosis of defense-related proteins and compounds.
Highlights
Substantial evidence supports a pivotal role for protein phosphorylation in the transduction of the elicitor signal [4]
We demonstrate that two-dimensional gel electrophoresis in conjunction with mass spectrometry is a suitable tool for the identification of intrinsic membrane proteins, and we show that among them a syntaxin, AtSyp122, is phosphorylated rapidly in response to flg22
Typical preparations were enriched by a factor of 7 to 9 in the plasma membrane (PM) marker, vanadate-sensitive ATPase activity, compared with the starting material consisting of a crude microsomal fraction (Table I)
Summary
Suspension cultures of Arabidopsis thaliana were maintained as described previously [3]. Cells were homogenized in a glass potter, insoluble debris was removed by centrifugation (20 min at 20,000 ϫ g, 4 °C), and the supernatant incubated with anti-Syp122 bound to protein A-Sepharose for 4 h at 4 °C on a spinning wheel. For in vitro kinase assays, His6-tagged proteins were bound to the column in the presence of 8 M urea, washed with two column volumes each of the same buffer containing 6 and 4 M urea, and on-column renatured with a 10-ml gradient (0.5 ml/min; column volume 0.5 ml) from 4 to 0 M urea. In Vitro Kinase Assay—Five micrograms of recombinant renatured AtSyp122-(1–289) and 2 g of crude plant cell extract (prepared as above) were mixed in kinase buffer (50 mM HEPES/KOH, pH 7.5, 20 mM MgCl2, 1 mM dithiothreitol, 10 mM EGTA, and 5.8 to 9.75 mM CaCl2 to give the indicated free calcium concentrations, as calculated with EQCAL) in a total volume of 25 l. MALDI spectra were acquired on a Bruker Reflex III MALDI-TOF mass spectrometer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
