Universität zu Köln

Moser, Daniel ORCID: 0000-0002-9693-235X (2025). Modulation of papain-like cysteine proteases in microbial interactions. PhD thesis, Universität zu Köln.

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Abstract

Plants engage in interactions with a highly diverse range of microorganisms, which must circumvent the plant’s immune system in order to establish an interaction. Plants defend against microbes using a two-branched immune system. Proteases, particularly papain-like cysteine proteases (PLCPs), play a crucial role in this process, as they are essential for pathogen recognition and the regulation of immune responses. In maize, specific PLCPs are activated in leaves and roots by the phytohormone salicylic acid (SA). Consequently, microbes frequently target PLCPs using effectors with the aim of suppressing immunity. However, the role of microbial PLCP inhibitors in non-pathogenic interactions as well as research on root-associated microbes in the rhizosphere remains relatively limited. The interactions between plants and microbes in crops such as maize remain understudied. This study explores how SA-activated root PLCPs and their microbial inhibitors shape microbial interactions on maize roots. This thesis hypothesizes that non-pathogenic bacteria secrete PLCP inhibitors to modulate protease activity promoting root colonization. Bacterial culture supernatants of maize root-colonizing bacteria were screened for PLCP inhibition, resulting in the suppression of PLCP activity by the Pseudomonas putida supernatant. The PLCP inhibitor P. putida PpCip1, which is conserved in all Pseudomonas species and has a chagasin motif required for PLCP inhibition, was identified via a computational search. Furthermore, signal peptide predictions as well as cell fractionation experiments, fluorescence microscopy, and surface protein labeling using Flag- or mCherry-tagged PpCip1 showed that PpCip1 is a surface-localized PLCP inhibitor. Moreover, preliminary results suggest that surface-localized PpCip1 may protect bacterial surface proteins from cleavage by plant PLCPs. Root colonization experiments on A. thaliana demonstrated that PpCip1 facilitates the early colonization of the meristematic, elongation, and transition zones. Supernatants of P. putida mutants lacking PpCip1 were still capable of inhibiting the activity of PLCPs, indicating that P. putida secrets unknown inhibitors different than PpCip1. A diverse range of computational and biochemical approaches were used to identify 15 putative inhibitor candidates, of which two proteins were identified as potential substrate-like inhibitors. This study shows that also non-pathogenic bacteria use PLCP inhibitors to modulate protease activity thereby promoting root colonization and influencing the bacterial root community structure. Moreover, newly secreted substrate-like PLCP inhibitors have been discovered although their role in plant-microbe interactions still needs to be elucidated.

Item Type:	Thesis (PhD thesis)
Translated title:	Title Language Modulation von Papain-ähnlichen Cysteinproteasen in mikrobiellen Interaktionen German
Creators:	Creators Email ORCID ORCID Put Code Moser, Daniel dmoser1@uni-koeln.de https://orcid.org/0000-0002-9693-235X UNSPECIFIED
URN:	urn:nbn:de:hbz:38-784017
Date:	2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Botanical Institute
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language PLCP English inhibitor English Cip1 English Pci1 English Chagasin English proteases English bacterial English Pseudomonas English Pseudomonas putida English maize English papain-like cysteine protease English C14-inhibiting protein 1 English Pseudomonas chagasin-like inhibitor 1 English Mais German
Date of oral exam:	17 March 2025
Referee:	Name Academic Title Döhlemann, Gunther Prof. Dr. Thomma, Bart Prof. Dr.
Funders:	Germany´s Excellence Strategy – EXC-2048/1 – project ID 390686111, DFG INST 216/512/1FUGG
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/78401