AtPeps as danger signals in arabidopsis - their release from PROPEP proteins by highly specific metacaspases

Microbial pathogens and herbivores are some of the key drivers of evolutionary adaptations by plants. As sessile organisms plants have to react quickly and strongly with defense responses to repel any invading organism. Besides preformed structures like thick cell walls and long thorns plants can activate innate immune responses that in a complex way lead to the activation of very efficient countermeasures. These include measurable changes on the plants hormone and gene expression levels but also plenty of secondary metabolites can be produced that directly have antimicrobial or herbivore repellent activity. Key to the timely initiation of defense responses is the perception of the invader and its detrimental activity. Plants carry highly specific pattern recognition receptors (PRR) to detect microbial or herbivore specific molecular signatures, so called microbe- or herbivore-associated molecular patterns (MAMP/HAMP). Less specific but equally efficient plant defenses can also be activated by the perception of self-molecules that behave differently once cell damage occurs. So called damage-associated molecular patterns (DAMP) are released passively or actively from damaged cells and serve as strong indicators of an infection or the presence of an herbivore.
In this work the mechanisms around expression, activation and activity of recently described DAMPs, the family of plant elicitor peptides (PEPs), were investigated in more detail. PEPs are perceived by the plant they are released from via specific PEP receptors (PEPRs) and thereby trigger defense responses. PEPs are expressed as larger PROPEPs, and we first investigated the expression of seven formerly known and a newly identified eighth PROPEP and that of the two PEPRs in Arabidopsis tissues using the promoter-GUS fusion technique. We were able to show that expression of PROPEPs 1-3, 5 and 8 mostly overlapped and correlated with the expression of both PEPRs, whilst PROPEP4 and 7 were only weakly expressed in small areas of the roots. In silico analysis unveiled the influences of biotic stresses on the PROPEP expression patterns and showed that PROPEP 1-3 are most strongly regulated by defense-associated mechanisms. To determine the subcellular localization of a selection of PROPEPs we observed PROPEP 1, 3 and 6 fused to Yellow Fluorescent Protein (YFP) within the cells and found PROPEP1 and 6 to be localized to the tonoplast membrane, whilst PROPEP3 showed a cytoplasmic localization. Despite the apparent different expression and localization patterns of PROPEPs, the elicitation activity of the mature PEPs was very similar, even though all eight AtPEPs were perceived by AtPEPR1 while AtPEPR2 was activated exclusively by AtPEP1 and 2.
Even though a lot of research has been already done on the responses induced after PEP elicitation the circumstances and the mechanism leading to PEP genesis from the PROPEP precursor has not been uncovered so far. Here, we observed the rapid formation of Arabidopsis PEP1 from PROPEP1 upon cell damage. Cleavage of PROPEP1 depended on the presence of the conserved arginine 69 and was impaired by chelating Ca2+ ions or addition of a metacaspase-specific inhibitor. This led to the identification of the arginine-specific cysteine protease AtMetacaspase 4 (MC4). MC4 activation correlated with PEP1 formation, MC4 was able to cleave PROPEP1 in vitro, and lack of MC4 impaired PROPEP1 cleavage in vivo. Furthermore, laser ablation experiments revealed damage-induced relocalization of PROPEP1 that was dependent on MC4 activity. Notably, PEPR1 internalization in cells adjacent to the site of laser ablation indicated PEP1 release. Thus MC4 is the bona fide protease for PROPEP1 processing and thereby enables PEP1 relocalization to first the cytosol and, depending on the cellular integrity, the extracellular space.
In a third project we gained knowledge about the conservation of the PROPEP-Pep-PEPR system across the plant kingdom. We identified new PEPs in Brassicaceae, Solanaceae and Poaceae species with elicitor activity being limited to the plant family of their origin. We deduced Brassicaceae, Solanaceae and Poaceae specific amino acid motifs within the respective PEP families that are required for intra-family elicitor activity and seem to explain the interfamily incompatibility. In addition we identified a large number of PEPRs outside Arabidopsis and cloned the coding sequences of Zea mays PEPR and Solanum Lycopersicum PEPR for further characterization. Expression of these newly identified receptors in Nicotiana benthamiana demonstrated their functionality upon perception of the corresponding PEPs. Thus, contrary to PROPEPs, the PEPRs are interspecies compatible.
In summary with this study valuable new data on the characteristics and ubiquity of the PROPEP-PEP-PEPR system in general and the PROPEPs in particular were generated. Importantly, light was shed on the hitherto unknown processing of PROPEPs that not only significantly advanced PEP research but also the work on plant proteases which is struggling to identify in vivo substrates. Finally, this work might soon be recognized as the foundation to define the first plant cytokines.