Project

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Structure, function and biosynthesis of syrbactins

Applicant Dudler Robert
Number 134936
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pflanzen- und Mikrobiologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Molecular Biology
Start/End 01.01.2012 - 30.06.2014
Approved amount 418'392.00
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All Disciplines (3)

Discipline
Molecular Biology
Biochemistry
Experimental Microbiology

Keywords (5)

proteasome inhibition; syringolin; plant-pathogen interactions; stomatal immunity; photorhabdus

Lay Summary (English)

Lead
Lay summary

) strains that inhibits the proteasome in host cells by a novel mechanism. Pss(syringae  pv. Pseudomonas syringae Bacterial plant pathogens are able to manipulate physiology and defense mechanisms of their hosts by means of so-called effectors, which often are proteins encoded by the pathogen that are injected into the host cells by a special transport system, the type III secretion system. In addition to type III effector proteins, phytopathogenic bacteria are known to synthesize a variety of small molecules acting as toxins or virulence factors. In previous projects, we have identified such a virulence factor, named syringolin A. Syringolin A is a small peptide derivative secreted by certain

The experiments proposed in this research project aim at the further elucidation of the biology of syringolin A and similar compounds (collectively dubbed syrbactins). To reach its target, syringolin A must enter plant cells. We will try to elucidate how syringolin A is taken up by plant cells and to identify the uptake transport system that is postulated to exist. These experiments will involve functional expression of candidate Arabidopsis peptide/oligopeptide transporter cDNAs, or if necessary, an Arabidopsis cDNA library, in yeast. Other experiments will aim at the identification of the exact mechanisms of how proteasome inhibition benefits the pathogen.

is postulated to produce. The proposed research has the potential to enlarge the syrbactin class of proteasome inhibitors and to predict occurrence, structure and function of syrbactins from genome and metagenome sequence data with high confidence. Because proteasome inhibitors are a promising class of anti-cancer agents, the results of the proposed research may also have medical implication.Photorhabdus luminescens . In the current project, we will  isolate and structurally and biochemically characterize the putative syringolin A analogon which the insect pathogen Photorhabdus asymbioticaand Burkholderia oklahomensis , an insect pathogen, as well as the human pathogens Photorhabdus luminescensa dangerous human pathogen and causal agent of melioidosis, Burkholderia pseudomallei, . We previously have isolated and characterized the gene clusters encoding the syringolin A and glidobactin A synthetases which turned out to exhibit a characteristic gene architecture. Similar gene clusters were found in five taxa among the more than 1500 completely sequenced bacterial genomes, including BurkholderialesSyringolin A defines a novel class of proteasome inhibitors, dubbed syrbactins, that includes glidobactin A, an antifugal and anti-tumor agent produced by a bacterium belonging to the

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Genome and Transcriptome Sequences of Pseudomonas syringae pv. syringae B301D-R.
Dudnik Alexey, Dudler Robert (2014), Genome and Transcriptome Sequences of Pseudomonas syringae pv. syringae B301D-R., in Genome announcements, 2(2), 306.
Genomics-Based Exploration of Virulence Determinants and Host-Specific Adaptations of Pseudomonas syringae Strains Isolated from Grasses
Dudnik Alexey, Dudler Robert (2014), Genomics-Based Exploration of Virulence Determinants and Host-Specific Adaptations of Pseudomonas syringae Strains Isolated from Grasses, in Pathogens, 3(1), 121-148.
Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16.
Dudnik Alexey, Bigler Laurent, Dudler Robert (2014), Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16., in Applied and environmental microbiology, 80(12), 3741-8.
Protein abundance changes and ubiquitylation targets identified after inhibition of the proteasome with syringolin a.
Svozil Julia, Hirsch-Hoffmann Matthias, Dudler Robert, Gruissem Wilhelm, Baerenfaller Katja (2014), Protein abundance changes and ubiquitylation targets identified after inhibition of the proteasome with syringolin a., in Molecular & cellular proteomics : MCP, 13(6), 1523-36.
Arabidopsis YELLOW STRIPE-LIKE7 (YSL7) and YSL8 Transporters Mediate Uptake of Pseudomonas Virulence Factor Syringolin A into Plant Cells
Hofstetter Silvia Schelbert, Dudnik Alexey, Widmer Heidi, Dudler Robert (2013), Arabidopsis YELLOW STRIPE-LIKE7 (YSL7) and YSL8 Transporters Mediate Uptake of Pseudomonas Virulence Factor Syringolin A into Plant Cells, in MOLECULAR PLANT-MICROBE INTERACTIONS, 26(11), 1302-1311.
Heterologous expression of a Photorhabdus luminescens syrbactin-like gene cluster results in production of the potent proteasome inhibitor glidobactin A
Dudnik Alexey, Bigler Laurent, Dudler Robert (2013), Heterologous expression of a Photorhabdus luminescens syrbactin-like gene cluster results in production of the potent proteasome inhibitor glidobactin A, in MICROBIOLOGICAL RESEARCH, 168(2), 73-76.
High-Quality Draft Genome Sequence of Pseudomonas syringae pv. Syringae Strain SM, Isolated from Wheat.
Dudnik Alexey, Dudler Robert (2013), High-Quality Draft Genome Sequence of Pseudomonas syringae pv. Syringae Strain SM, Isolated from Wheat., in Genome announcements, 1(4), 610.
Manipulation of Host Proteasomes as a Virulence Mechanism of Plant Pathogens
Dudler Robert (2013), Manipulation of Host Proteasomes as a Virulence Mechanism of Plant Pathogens, in ANNUAL REVIEW OF PHYTOPATHOLOGY, VOL 51, 51, 521-542.
Non contiguous-finished genome sequence of Pseudomonas syringae pathovar syringae strain B64 isolated from wheat
Dudnik Alexey, Dudler Robert K. (2013), Non contiguous-finished genome sequence of Pseudomonas syringae pathovar syringae strain B64 isolated from wheat, in Standards in Genomic Sciences, 8(3), 2-6.
The role of bacterial phytotoxins in inhibiting the eukaryotic proteasome
Dudler Robert (2013), The role of bacterial phytotoxins in inhibiting the eukaryotic proteasome, in Trends in Microbiology, 22(1), 28-35.
Activity Enhancement of the Synthetic Syrbactin Proteasome Inhibitor Hybrid and Biological Evaluation in Tumor Cells
Archer CR, Groll M, Stein ML, Schellenberg B, Clerc J, Kaiser M, Kondratyuk TP, Pezzuto JM, Dudler R, Bachmann AS (2012), Activity Enhancement of the Synthetic Syrbactin Proteasome Inhibitor Hybrid and Biological Evaluation in Tumor Cells, in BIOCHEMISTRY, 51(34), 6880-6888.
One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor
Stein ML, Beck P, Kaiser M, Dudler R, Becker CFW, Groll M (2012), One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor, in PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 109(45), 18367-18371.
Regulation of Biosynthesis of Syringolin A, a Pseudomonas syringae Virulence Factor Targeting the Host Proteasome
Ramel C, Baechler N, Hildbrand M, Meyer M, Schadeli D, Dudler R (2012), Regulation of Biosynthesis of Syringolin A, a Pseudomonas syringae Virulence Factor Targeting the Host Proteasome, in MOLECULAR PLANT-MICROBE INTERACTIONS, 25(9), 1198-1208.

Collaboration

Group / person Country
Types of collaboration
TU München Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. André Bachmann, Cancer Research Center of Hawaii, University of Hawaii United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. Markus Kaiser, Chemical Genomics Centre of the Max-Planck-Society Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Associated projects

Number Title Start Funding scheme
145020 Plant metabolite analysis by liquid chromatography-mass spectrometry 01.10.2013 R'EQUIP
115970 Regulation and biosynthesis of syringolin and syringolin-like molecules and investigation of their role and mode of action in the interaction of bacteria with plants and other eukaryotes 01.10.2007 Project funding (Div. I-III)

Abstract

Bacterial plant pathogens have evolved sophisticated means such as type III effectors to subvert physiology and defenses of their hosts. In addition to type III effector proteins, phytopathogenic bacteria can synthesize a variety of small molecules acting as toxins or virulence factors. We have previously isolated syringolin A, a small peptide derivative secreted by certain Pseudomonas syringae pv. syringae (Pss) strains, and shown that it is a virulence factor in the interaction of Pss B728a with its host Phaseolus vulgaris (bean) which inhibits the eukaryotic proteasome by a novel mechanism. The experiments proposed in this research application aim at the further elucidation of the biology of syringolin A and similar compounds (collectively dubbed syrbactins).Recent results showed that proteasome inhibition by syringolin A counteracts stomatal immunity in bean and Arabidopsis. In Arabidopsis, stomatal closure triggered by syringolin A-deficient Pss B728a mutants required the salicylic acid (SA)-mediated defense pathway and was dependent on NPR1, the key transcriptional regulator of the SA pathway. The fact that NPR1 requires turnover by the proteasome for proper function provides a likely explanation for syringolin A’s inhibitory effect on stomatal immunity and defense gene transcription. Surprisingly, stomatal closure was independent of ICS1, the isochorismate synthase responsible for most of the SA needed for pathogen defense responses. We propose follow-up experiments to clarify how the SA needed for stomatal immunity is synthesized, taking advantage of Arabidopsis mutants (ics2, pal1, pal2, pal3, pal4, and combinations) affected in genes proposed to play a role in SA synthesis in plants.To reach its proteasome target, syringolin A must be taken up by plant cells, presumably by active transport because of its hydrophilic nature. It is still an unresolved question how uptake works. Experiments are proposed to identify the syringolin A transporters(s) by functional expression of candidate Arabidopsis peptide/oligopeptide transporter cDNAs, or if necessary, an Arabidopsis cDNA library, in yeast.The fact that proteasome inhibition by syringolin A will likely also affect activity of effectors requiring the host proteasome for their action, syringolin A biosynthesis may be expected to be tightly regulated. This is corroborated by the fact that syringolin A production in vitro only occurs in still cultures in a medium mimicking in planta conditions. In experiments with reporter gene constructs, we have identified the promoters driving the syringolin A synthetase structural genes of Pss B301D-R as well as the LuxR-type transcription factor SylA both in vitro and in planta. Activity of the sylA gene depends on the salA gene, itself encoding a LuxR-type transcription factor. All these promoters are essentially inactive under aerobic in vitro conditions but can be activated under microoxic conditions. The experiments proposed should elucidate the complex regulation and shed light on the involvement of the Pss homolog of the E. coli FNR oxygen sensor protein in syringolin A biosynthesis in vitro and in planta.Finally, we propose to isolate and structurally and biochemically characterize the putative syringolin A analogon which the insect pathogen Photorhabdus luminescens (Plu) is postulated to produce. Cloning of the syringolin A and glidobactin A synthetase genes has allowed the identification of homologous genes with slightly different architectures from a number of human and animal pathogens, including Plu. The proposed research has the potential to enlarge the syringolin A class of proteasome inhibitors (dubbed syrbactins) and to predict occurrence, structure and function of syrbactins from genome and metagenome sequence data with high confidence.
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