Gerold J.M. Beckers - Signaling In Plant Defense Priming: A Phosphoproteomics Approach

In plants and animals, induced resistance (IR) to biotic and abiotic stress is associated with priming of cells for faster and stronger activation of defense responses. Cell priming involves accumulation of latent signaling components that are not used until challenge exposure to stress. During development of induced resistance in Arabidopsis thaliana, priming is associated with accumulation of mRNA and inactive proteins of mitogen-activated protein kinases (MPKs), MPK3 and MPK6. Upon challenge exposure to biotic or abiotic stress, these two enzymes are more strongly activated in primed plants than in nonprimed plants. This elevated activation is linked to enhanced defense gene expression and development of IR. Because the identity of most in vivo MPK target proteins remained elusive, we developed a robust and powerful method to identify and quantify site-specific phosphorylation of low-abundant MPK substrates on a large scale. Our approach represents a two-step chromatographic procedure combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography (MOAC), tryptic digest of enriched phosphoproteins, and TiO2-based MOAC to enrich phosphopeptides. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the tandem-MOAC approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many so far unknown, MPK substrates in a single experiment.