Arabidopsis thimet oligopeptidases are redox-sensitive enzymes active in the local and systemic plant immune response

Abstract

Upon pathogen infection, receptors in plants will activate a localized immune response, the effector-triggered immunity (ETI), and a systemic immune response, the systemic acquired response (SAR). Infection also induces oscillations in the redox environment of plant cells, triggering response mechanisms involving sensitive cysteine residues that subsequently alter protein function. Arabidopsis thaliana thimet oligopeptidases TOP1 and TOP2 are required for plant defense against pathogens and the oxidative stress response. Herein, we evaluated the biochemical attributes of TOP isoforms to determine their redox sensitivity using ex vivo Escherichia coli cultures and recombinant proteins. Moreover, we explored the link between their redox regulation and plant immunity in wild-type and mutant Arabidopsis lines. These analyses revealed that redox regulation of TOPs occurs through two mechanisms: (1) oxidative dimerization of full-length TOP1 via intermolecular disulfides engaging cysteines in the N-terminal signal peptide, and (2) oxidative activation of all TOPs via cysteines that are unique and conserved. Further, we detected increased TOP activity in wild-type plants undergoing ETI or SAR following inoculation with Pseudomonas syringae strains. Mutants unable to express the chloroplast NADPH-dependent thioredoxin reductase C (NTRC) showed elevated TOP activity under unstressed conditions and were SAR-incompetent. A top1top2 knockout mutant challenged with P. syringae exhibited misregulation of ROS-induced gene expression in pathogen-inoculated and distal tissues. Furthermore, TOP1 and TOP2 could cleave a peptide derived from the immune component ROC1 with distinct efficiencies at common and specific sites. We propose that Arabidopsis TOPs are thiol-regulated peptidases active in redox-mediated signaling of local and systemic immunity.

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

“…A logical explanation would be that in WT, the NADP(H) pool is converted to NAD, which is used for catabolic energy production (Hashida and Kawai-Yamada, 2019) and, consequently, ATP accumulation. In top1top2 , this rapid reshuffling of metabolic resources is perturbed and possibly delayed, which would be in accordance with its delayed ROS burst during ETI (Al-Mohanna et al, 2021). Indeed, we found a substantial change in peptide accumulations at 30 mpi between the two genotypes ( Figure 2A ), and many of the proteins these peptides belonged to are involved in ATP synthesis ( Figure 3B ).…”

“…We next used the peptides from the in vitro screen described above and previously published substrates (Al-Mohanna et al, 2021; Iannetta et al, 2021) to learn TOPs cleavage patterns. Further, we considered all peptides with a –log 10 (p-value) <0.29 and a |LFC|<0.5 in all three time points as negative examples.…”

“…In comparison, metabolic processes, including ATP synthesis, glycolysis, and translation regulation (ribosome biogenesis and plastid translation), were enriched at 30 mpi. We discriminated TOP-cleaved peptides from the total complement of peptides processed during ETI through a multi-stage process, whereby we used in vitro validated TOP substrates from our previous screens (Al-Mohanna et al, 2021; Iannetta et al, 2021), as well as discovered new TOP substrates and analyzed the cleaved sequence patterns on all confirmed substrates. We used these insights to develop a support vector machine computational method that learned to differentiate cleavage patterns of validated substrates from the background peptidome and validated negative examples.…”

“…Both oligopeptidases are required for plant defense against avirulent strains of P. syringae through the activation of the resistance proteins RPS2 or RPS4 and both are necessary to regulate PCD (Moreau et al, 2013). Indirect evidence supports a role for TOPs in the controlled proteolysis of rotamase cyclophilin 1 (ROC1/CYP18–3), required for AvrRpt2 protease self-cleavage prior to the activation of ETI (Al-Mohanna et al, 2021). In a current model, TOPs are components in an interconnected organelle and cytosol proteolytic pathway that regulates the ETI oxidative burst and pathogen resistance through SA, ROS, and antioxidants (Westlake et al, 2015).…”

Mass Spectrometric Identification of Antimicrobial Peptides from Medicinal Seeds

“…LC-MS/MS data for bottom-up proteomic analysis of L. usitatissimum , T. pratense, and S. indicum seed fractions were acquired using an Acquity M-class UPLC system (Waters, Milford, MA, USA) coupled to a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) as previously described [ 61 ]. Briefly, mobile phase A consisted of water with 0.1% formic acid (Thermo Scientific, Waltham, MA, USA), and mobile phase B was acetonitrile with 0.1% formic acid (Thermo Fisher Scientific, Waltham, MA, USA).…”