In plants and animals acquired immunity to biotic and abiotic stress is associated with priming of cells for faster and stronger activation of defense responses. It has been hypothesized that cell priming involves accumulation of latent signaling components that are inactive until challenge exposure to stress. However, the identity of such signaling components has remained elusive. Here I show using various chemical compounds or inoculation with avirulent bacterial pathogens, that induction of the primed state of Arabidopsis thaliana, requires accumulation of mRNA and inactive protein of mitogen-activated protein kinases (MPK) 3 and MPK6. Only upon challenge exposure to biotic or abiotic stress, MPK3/MPK6 enzymes were strongly activated in primed plants, resulting in enhanced defense gene expression and manifestation of acquired immunity. Strong elicitation of stress-induced MPK3/MPK6 activity is also seen in the constitutive priming mutant edr1, while it was attenuated in the priming-deficient npr1 mutant. Moreover, priming of defense gene expression and acquired immunity were lost in mpk3 and mpk6 mutant plants. My findings unveiled for the first time that pre-stress deposition of the signaling components MPK3 and MPK6 is a critical step in priming plants for potentiated defense responses. In addition, I conducted and validated a proteome-wide analysis of phosphorylated proteins to identify novel candidate proteins involved in the priming mechanism in Arabidopsis. A thorough understanding of the molecular mechanism of priming in Arabidopsis will contribute to modern pest management in the field and likely lead to new discoveries in non-plant research