Group Gust

Research

Novel PAMPs and their perception in Arabidopsis thaliana

Peptidoglycan Perception and Processing

SOBIR1-dependent signalling of receptor-like proteins

Identification of Novel PAMPs

Dr. Andrea A. Gust
ZMBP - Center for Plant Molecular Biology
Plant Biochemistry
University of Tübingen
Auf der Morgenstelle 32
D-72076 Tübingen
Germany
phone: ++49-(0)7071-29-76655
fax: ++49-(0)7071-29-5226
email: andrea.gustspam prevention@zmbp.uni-tuebingen.de

Research

Novel PAMPs and their perception in Arabidopsis thaliana

Innate immunity constitutes the first line of defence against attempted microbial invasion, and is a well-described phenomenon in vertebrates and insects. Recent work has revealed striking similarities between the molecular organization of animal and plant systems for non-self recognition and antimicrobial defence. Like animals, plants have acquired the ability to recognize invariant pathogen-associated molecular patterns (PAMP) that are characteristic of microbial organisms (lipopolysaccharide, flagellin, glucans, chitins, type III effector molecules, such as harpins), but which are not found in potential host plants.

Such structures are often indispensable for the microbial lifestyle and, upon receptor-mediated perception, inevitably betray the invader to the surveillance system. Remarkable similarities have been uncovered in the molecular mode of PAMP perception in animals and plants, including the discovery of the plant LRR-receptor-like kinase FLS2. FLS2 is very similar to the mammalian Toll-like receptor TLR5 and both FLS2 and TLR5 recognise flagellin as a PAMP. Moreover, defence signal transduction in both animals and plants involves changes in cytoplasmic calcium concentrations, the production of signalling molecules such as nitric oxide (NO), reactive oxygen species (ROS) and lipid-based hormones (lipoxines in animals and oxylipins in plants), the activation of mitogen-activated protein kinase (MAPK) cascades and the activation of transcription factors. We would like to elucidate PAMP recognition and signal transduction, mainly focussing on bacteria-derived peptidoglycans (PGN).

Peptidoglycan (PGN) Perception and Processing

Peptidoglycans (PGN) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants (Gust, PLoS Pathog. 2015; Bertsche et al., IJMM 2015). Recently, we described a tripartite PGN recognition system at the plasma membrane of Arabidopsis thaliana comprising Lysin motif (LysM) domain proteins LYM1 and LYM3 for PGN ligand binding and the transmembrane LysM receptor kinase CERK1 for transmembrane signalling (Willmann et al., PNAS 2011). Moreover, we identified an Arabidopsis lysozyme-like hydrolase (lysozyme 1, LYS1), which releases soluble PGN fragments from insoluble bacterial cell walls, which subsequently trigger typical plant immune responses in a LYM1, LYM3 and CERK1-dependent manner (Liu et al., Elife 2014). Importantly, all proteins, LYM1, LYM3, CERK1 and LYS1 are indispensable for PGN perception and immunity to bacterial infection.

Overview of known components of PGN perception in plants. Upon entry of bacteria into the plant apoplast, their presence is most likely sensed either by PGN fragments spontaneously released into the apoplast or via unrelated bacterial MAMPs, such as flagellin or lipopolysaccharides. PGN fragments of yet unknown size bind to GPI-anchored LysM-proteins localized in the plasma membrane (AtLYM1/3 in Arabidopsis, OsLYP4/6 in rice). Upon PGN binding (1), OsLYP4/6 dissociate to form a complex with OsCERK1, which triggers the release of cytoplasmic OsRLCK176 (and possibly OsRLCK185) and subsequent downstream signalling (2). In Arabidopsis, AtLYM1, AtLYM3, and AtCERK1 mediate PGN perception (1); however, whether a PGN-dependent LysM protein complex is formed and whether receptor-like cytoplasmic kinases (RLCKs) such as avrPphB sensitive 1-like proteins (PBLs) are required is still unknown. Downstream signalling events lead to a transcriptional reprogramming of the cell, and defence proteins, such as the lysozyme-like activity LYS1, are produced. LYS1 is secreted into the plant apoplast to generate more immunogenic PGN fragments (3), which results in an amplification of PGN-triggered immune responses. In a successful infection, however, CERK1 is targeted by bacterial effectors such as AvrPtoB (in the Arabidopsis–Pseudomonas syringae interaction) and Xoo1488 (in the rice–Xanthomonas oryzae interaction) to suppress plant immunity (4). (from Gust, PLoS Pathog. 2015)

Our current projects aim at answering the following questions:

What is the minimal PGN structure required for PAMP activity?
Do LYM1, LYM3 and CERK1 form a PGN-recognition complex by physical interaction and is this complex formation PGN-dependent?
Which parts of LYM1/LYM3 are required for PGN binding and CERK1 interaction?
How can different LysM proteins discriminate between N-acetylglucosamine (GlcNAc)-containing ligands?
What happens to PGN after perception by its receptor?
Are there bacterial mechanisms to suppress PGN perception?

Collaborations:

Mari-Anne Newman, Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Naoto Shibuya, Meiji University, Kawasaki, Japan

Gary Stacey, University of Missouri, Columbia, USA

Antonio Molinaro, University of Naples, Italy

Fumiaki Katagiri, University of Minnesota, USA

Friedrich Götz, Department of Microbial Genetics, University of Tübingen

Ulrich Mähringer, Research Center Borsten

Koichi Fukase, Osaka University, Japan

Waldemar Vollmer, University of Newcastle upon Tyne, UK

SOBIR1-dependent signalling of receptor-like proteins

Receptor-like proteins (RLPs) build large protein families in all higher plants. Apart from RLPs with conserved roles in development, an increasing number of RLPs could be associated with functions as immune receptors detecting specific patterns from a variety of pathogens. Recent work showed that functionality of these RLPs, at least those with Leucine Rich Repeats (LRRs) in their extracellular domain, depends on association with the common adaptor kinase SOBIR1 (Suppressor of Brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1)-interacting receptor kinase 1). For instance, SOBIR1 is essential for the function of the receptor-like protein RLP30, which recognizes the novel PAMP SCFE1 from Sclerotinia culture filtrates, and for immunity towards Sclerotinia sclerotiorum and Botrytis cinerea infections in Arabidopsis (Zhang et al., Plant Cell 2013).

We propose that these RLP/adaptor complexes, formed in the absence of ligands, are bimolecular equivalents of genuine receptor kinases (Gust and Felix, Curr. Opin. Plant Sci. 2014). Similar to receptor kinases, activation of these RLP/adaptor complexes seems to require a ligand‐dependent interaction step with co‐receptors like BAK1 or other SERKs (Albert et al., Nature Plants 2015).

Model for the activation process of RLPs and RLKs. A) RLPs lacking a cytoplasmic kinase domain undergo association with adaptor kinases, a process important for proper localization and stability of these receptors. In the case of LRR‐RLKs that associate with SOBIR1 this could occur by interaction of their LRR domains, by ionic interaction of their opposedly charged juxtamembranes and/or by helix‐helix interactions of their transmembrane domains. B) RLP/adaptor complexes function as bimolecular equivalents of RLKs. Upon binding of their ligands the RLP/adaptor complexes, much like RLKs, undergo complex formation with co‐receptors, notably of the BAK1/SERK‐type, that brings the cytoplasmic domains of the complex partners in proximity, allows mutual phosphorylation and activation of cytoplasmic signal output. (from Gust and Felix, Curr. Opin. Plant Sci. 2014)

Current objectives of this project are:

Analysis of the SOBIR1 phosphorylation status
Identification of SOBIR1 phosporylation targets
Identification of the fungal PAMP SCFE1

Collaborations:

Matthieu Joosten, Wageningen University, Wageningen, The Netherlands

Sarah Liljegren, University of Mississippi, Oxford, USA

Erich Glawischnig, Technical University Munich

Identification of novel PAMPs

To identify novel PAMPs for the innate immune response in Arabidopsis we are screening various natural compound libraries comprising fungus- or bacteria-derived carbohydrate, lipid and protein structures for their ability to drive the expression of a beta-glucuronidase (GUS)-reporter gene controlled by a PAMP/pathogen-responsive promoter. This approach will help answer the question whether plants have evolved PAMP perception systems that recognize a structural variety of PAMPs similar to that recognized by their animal counterparts.

PAMP-induced reporter gene activity (GUS) in Arabidopsis

Collaborations:
Frédéric Brunner, ZMBP, University of Tübingen
Pierre de Wit, Wageningen University, The Netherlands
Chris Ridout, JIC, Norwich, UK
Cyril Zipfel, The Sainsbury Laboratory, Norwich, UK
H. P. Fiedler, Department of Microbiology/Biotechnology, University of Tübingen
Eckhard Thines, Institute of Biotechnology and Drug Research Kaiserslautern

PAMP-triggered signalling pathways

In addition to PAMP perception, we are also interested in investigating downstream signaling events eventually leading to cellular immune responses. A major mechanism of cellular signal transduction during various stress responses is the reversible phosphorylation of signalling components which is mediated by protein kinases such as mitogen-activated protein kinases (MAPKs). MAPKs function as key signal integration points for a vast number of external stimuli that affect the life and death of cells and thus must be subject to rigorous regulation to control appropriate intensity and timing of their activation. Phosphorylation is essential for the activation of both MAPKs and their upstream MAPK kinases; however, their activation occurs normally only transiently, indicating that the phosphate groups of the activation loop are quickly removed. Hence protein phosphatases are likely to be important regulators of signalling through MAPK cascades. In collaboration with Irute Meskiene’s lab in Vienna and Roman Ulm’s lab in Freiburg we are investigating the role of selected PP2Cs and dual-specificity protein phosphatases with respect to MAPK regulation during the plant innate immune response.

collaborations:

Irute Meskiene, Max F. Perutz Laboratories, University of Vienna

Roman Ulm, Albert-Ludwigs-University of Freiburg

Funding

SFB 766: The Bacterial Cell Envelope: Structure, Function and Infection Interface

DFG: Characterization of the two receptor-kinases SOBIR1/EVR and BAK1 as co-receptors of the receptor-like protein RLP30 from Arabidopsis thaliana

BMBF - Transnational Plant(Genome)Research (PLANT-KBBE): "PAMP triggered immunity in crops (PATRIC)"

Tübinger Promotionsverbund (Mini-GK): "Of Plants and Men: Principles of Chitin Recognition in Arabidopsis and Humans"

ERA-PG: “Pattern recognition receptors: discovery, function and application in crops for durable disease control” (PRR-CROP)

ERA-CAPS: “Decoding ligand-receptor specificities of LysM-proteins in plant immunity and symbiosis” (SIPIS).

Publications

Yang, Y., Steidele, C.E., Rössner, C. et al. (2023) Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms. Nat Commun 14, 3621.
https://doi.org/10.1038/s41467-023-39208-8

Gust A.A. (2015) Peptidoglycan Perception in Plants. PLoS Pathog., 11(12):e1005275.

Albert I., Böhm H., Albert M., Feiler C.E., Imkampe J., Wallmeroth N., Brancato C., Raaymakers T.M., Oome S., Zhang H., Krol E., Grefen C., Gust A.A., Chai J., Hedrich R. , Van den Ackerveken G., Nürnberger T. (2015) An RLP23–SOBIR1–BAK1 complex mediates NLP-triggered immunity. Nature Plants, doi:10.1038/nplants.2015.140.

Bertsche U., Mayer C., Götz F., Gust A.A. (2015) Peptidoglycan perception – sensing bacteria by their common envelope structure. Int. J. Med. Microbiol., 305, 217-223.

Gust A.A., Felix G. (2014) Receptor like proteins associate with SOBIR1-type of adaptors to form bimolecular receptor kinases. Curr. Opin. Plant Biol. 21, 104-111.

Petutschnig E.K., Stolze M., Lipka U., Kopischke M., Horlacher J., Valerius O., Rozhon W., Gust A.A., Kemmerling B., Poppenberger B., Braus G.H., Nürnberger T., Lipka V. (2014) A novel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) mutant with enhanced pathogen-induced cell death and altered receptor processing. New Phytol., 204, 955-967.

Liu X., Grabherr H.M., Willmann R., Kolb D., Brunner F., Bertsche U., Kühner D., Franz-Wachtel M., Amin B., Felix G., Ongena M., Nürnberger T., Gust A.A. (2014) Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis. Elife, 3. doi: 10.7554/eLife.01990.

Willmann R., Haischer D.J., Gust A.A. (2014) Analysis of MAPK activities using MAPK-specific antibodies. Methods Mol. Biol., 1171, 27-37.

Zhang W., Fraiture M., Kolb D., Löffelhardt B., Desaki Y., Boutrot F.F., Tör M., Zipfel C., Gust A.A., Brunner F. (2013) Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi. Plant Cell, 25, 4227-4241.

Gust A.A., Nürnberger T. (2012) Plant immunology: A life or death switch. Nature, 486,198-199.

Gust A.A., Willmann R., Desaki Y., Grabherr H.M., Nürnberger T. (2012) Plant LysM proteins: modules mediating symbiosis and immunity. Trends Plant Sci., 17, 495-502.

Willmann R., Lajunen H.M., Erbs G., Newman M.A., Kolb D., Tsuda K., Katagiri F., Fliegmann J., Bono J.J., Cullimore J.V., Jehle A.K., Götz F., Kulik A., Molinaro A., Lipka V., Gust A.A., Nürnberger T. (2011). Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. PNAS, 108, 19824-19829.

Lenz H.D., Vierstra R.D., Nürnberger T., Gust A.A. (2011) ATG7 contributes to plant basal immunity towards fungal infection. Plant Sign. Beh. [Epub ahead of print]

Lenz H.D., Haller E., Melzer E., Gust A.A., Nürnberger T. (2011). Autophagy controls plant basal immunity in a pathogenic lifestyle-dependent manner. Autophagy, 7, 773-774.

Lenz H.D., Haller E., Melzer E., Kober K., Wurster K., Stahl M., Bassham D.C., Vierstra R.D., Parker J.E., Bautor J., Molina A., Escudero V., Shindo T., van der Hoorn R.A., Gust A.A., Nürnberger T. (2011) Autophagy differentially controls plant basal immunity to biotrophic and necrotrophic pathogens. Plant J., 66, 818-830.

Brock A.K. , Willmann R., Kolb D., Grefen L., Lajunen H.M., Bethke G., Lee J., Nürnberger T. Gust A.A. (2010). The Arabidopsis thaliana Mitogen-activated protein kinase (MAPK) phosphatase PP2C5 affects seed germination, stomatal aperture and abscisic acid-inducible gene expression. Plant Physiol., 153, 1098-1111.

Gust A.A. Brunner F. Nürnberger T. (2010) Biotechnological concepts for improving plant innate immunity. Curr. Opin. Biotechnol., 21, 204-210.

Bethke, G., Unthan, T., Uhrig, J.F., Pöschl, Y., Gust, A.A., Scheel, D. Lee, J. (2009) Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. PNAS, 106, 8067-8072.

Gust A.A., Biswas R., Lenz H.D., Rauhut T., Ranf S., Kemmerling B., Götz F., Glawischnig E., Lee J., Felix G. Nürnberger T. (2007) Bacteria-derived peptidoglycans constitute pathogen-associated molecular patterns triggering innate immunity in Arabidopsis. J. Biol. Chem., 282, 32338-32348.

Qutob D., Kemmerling B., Brunner F., Küfner I., Engelhardt S., Gust A.A., Luberacki B., Seitz H.U., Stahl D., Rauhut T., Glawischnig E., Schween G., Lacombe B., Watanabe N., Lam E., Schlichting R., Scheel D., Nau K., Dodt G., Hubert D., Gijzen M., Nürnberger T. (2006) Phytotoxicity and innate immune responses induced by Nep1-like proteins. Plant Cell, 18, 3721-3744.

former publications

Teaching

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