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

The family of Peps and their precursors in Arabidopsis: differential expression and localization but similar induction of pattern-triggered immune responses

In Arabidopsis thaliana, the endogenous danger peptides, AtPeps, have been associated with plant defences reminiscent of those induced in pattern-triggered immunity. AtPeps are perceived by two homologous receptor kinases, PEPR1 and PEPR2, and are encoded in the C termini of the PROPEP precursors. Here, we report that, contrary to the seemingly redundant AtPeps, the PROPEPs fall at least into two distinct groups. As revealed by promoter-β-glucuronidase studies, expression patterns of PROPEP1-3, -5, and -8 partially overlapped and correlated with those of the PEPR1 and -2 receptors, whereas those of PROPEP4 and -7 did not share any similarities with the former. Moreover, bi-clustering analysis indicated an association of PROPEP1, -2, and -3 with plant defence, whereas PROPEP5 expression was related to patterns of plant reproduction. In addition, at the protein level, PROPEPs appeared to be distinct. PROPEP3::YFP (fused to yellow fluorescent protein) was present in the cytosol, but, in contrast to previous predictions, PROPEP1::YFP and PROPEP6::YFP localized to the tonoplast. Together with the expression patterns, this could point to potentially non-redundant roles among the members of the PROPEP family. By contrast, their derived AtPeps, including the newly reported AtPep8, when applied exogenously, provoked activation of defence-related responses in a similar manner, suggesting a high level of functional redundancy between the AtPeps. Taken together, our findings reveal an apparent antagonism between AtPep redundancy and PROPEP variability, and indicate new roles for PROPEPs besides plant immunit

Evolutionary divergence of the plant elicitor peptides (Peps) and their receptors: interfamily incompatibility of perception but compatibility of downstream signalling

Plant elicitor peptides (Peps) co-evolved with their receptors, resulting in interfamily incompatibility of Pep recognition. In contrast, operation of defence pathways by Pep receptors is conserved within the flowering plant

Perception of Arabidopsis AtPep peptides, but not bacterial elicitors, accelerates starvation-induced senescence

Members of the AtPep group of Arabidopsis endogenous peptides have frequently been reported to induce pattern-triggered immunity (PTI) and to increase resistance to diverse pathogens by amplifying the innate immune response. Here, we made the surprising observation that dark-induced leaf senescence was accelerated by the presence of Peps. Adult leaves as well as leaf discs of Col-0 wild type plants showed a Pep-triggered early onset of chlorophyll breakdown and leaf yellowing whereas pepr1 pepr2 double mutant plants were insensitive. In addition, this response was dependent on ethylene signaling and inhibited by the addition of cytokinins. Notably, addition of the bacterial elicitors flg22 or elf18, both potent inducers of PTI, did not provoke an early onset of leaf senescence. Continuous darkness leads to energy deprivation and starvation and therewith promotes leaf senescence. We found that continuous darkness also strongly induced PROPEP3 transcription. Moreover, Pep-perception led to a rapid induction of PAO, APG7, and APG8a, genes indispensable for chlorophyll degradation as well as autophagy, respectively, and all three hallmarks of starvation and senescence. Notably, addition of sucrose as a source of energy inhibited the Pep-triggered early onset of senescence. In conclusion, we report that Pep-perception accelerates dark/starvation-induced senescence via an early induction of chlorophyll degradation and autophagy. This represents a novel and unique characteristic of PEPR signaling, unrelated to PTI

Evolutionary divergence of the plant elicitor peptides (Peps) and their receptors : interfamily incompatibility of perception but compatibility of downstream signalling

Plant elicitor peptides (Peps) are potent inducers of pattern-triggered immunity and amplify the immune response against diverse pathogens. Peps have been discovered and studied extensively in Arabidopsis and only recently orthologs in maize were also identified and characterized in more detail. Here, the presence of PROPEPs, the Pep precursors, and PEPRs, the Pep receptors, was investigated within the plant kingdom. PROPEPs and PEPRs were identified in most sequenced species of the angiosperms. The conservation and compatibility of the Pep-PEPR-system was analysed by using plants of two distantly related dicot families, Brassicaceae and Solanaceae, and a representative family of monocot plants, the Poaceae. All three plant families contain important crop plants, including maize, rice, tomato, potato, and canola. Peps were not recognized by species outside of their plant family of origin, apparently because of a divergence of the Pep sequences. Three family-specific Pep motifs were defined and the integration of such a motif into the Pep sequence of an unrelated Pep enabled its perception. Transient transformation of Nicotiana benthamiana with the coding sequences of the AtPEPR1 and ZmPEPR1a led to the recognition of Pep peptides of Brassicaceae or Poaceae origin, respectively, and to the proper activation of downstream signalling. It was concluded that signalling machinery downstream of the PEPRs is highly conserved whereas the leucine-rich repeat domains of the PEPRs co-evolved with the Peps, leading to distinct motifs and, with it, interfamily incompatibility

Damage on plants activates Ca2+-dependent metacaspases for release of immunomodulatory peptides

Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals