High throughput peptidomics elucidates immunoregulatory functions of plant thimet oligopeptidase-directed proteostasis

Targeted proteolysis activities activated during the plant immune response catalyze the synthesis of stable endogenous peptides. Little is known about their biogenesis and biological roles. Herein, we characterize an Arabidopsis thaliana mutant top1top2 in which targeted proteolysis of immune-active peptides is drastically impaired during effector-triggered immunity (ETI). For effective ETI, the redox-sensitive thimet oligopeptidases TOP1 and TOP2 are required. Quantitative mass spectrometry-based peptidomics allowed differential peptidome profiling of wild type (WT) and top1top2 mutant at the early ETI stages. Biological processes of energy-producing and redox homeostasis were enriched, and TOPs were necessary to maintain the dynamics of ATP and NADP(H) accumulation in the plant during ETI. Subsequently, a set of novel TOPs substrates validated in vitro enabled the definition of the TOP-specific cleavage motif and informed an in-silico model of TOP proteolysis to generate bioactive peptide candidates. Several candidates, including those derived from proteins associated with redox metabolism, were confirmed in planta . The top1top2 background rescued WT’s ETI deficiency caused by treatment with peptides derived from targeted proteolysis of the negative immune regulator FBR12, the reductive enzyme APX1, the isoprenoid pathway enzyme DXR, and ATP-subunit β. These results demonstrate TOPs role in orchestrating the production and degradation of phytocytokines.