title: Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways.
abstract: Arabidopsis (Arabidopsis thaliana) penetration (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-D-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes.
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Xunli
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Jan
authors.initials: J
authors.lastname: Dittgen
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Mariola
authors.initials: M
authors.lastname: Piślewska-Bednarek
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Antonio
authors.initials: A
authors.lastname: Molina
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Bernd
authors.initials: B
authors.lastname: Schneider
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Aleš
authors.identifier: http://orcid.org/0000-0003-1032-7288
authors.initials: A
authors.lastname: Svatoš
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Jan
authors.initials: J
authors.lastname: Doubský
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Korbinian
authors.initials: K
authors.lastname: Schneeberger
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.).
authors.forename: Detlef
authors.identifier: http://orcid.org/0000-0002-2114-7963
authors.initials: D
authors.lastname: Weigel
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.) bednarek@ibch.poznan.pl schlef@mpipz.mpg.de.
authors.forename: Paweł
authors.identifier: http://orcid.org/0000-0002-3064-7775
authors.initials: P
authors.lastname: Bednarek
authors.affiliation: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany (X.L., J.Di., M.P.-B., P.B., P.S.-L.);Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland (M.P.-B., P.B.);Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, 28223 Madrid, Spain (A.M.); Research Groups on Biosynthesis/Nuclear Magnetic Resonance (B.S.) and Mass Spectrometry/Proteomics (A.S., J.Do.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (K.S., D.W.) bednarek@ibch.poznan.pl schlef@mpipz.mpg.de.
authors.forename: Paul
authors.initials: P
authors.lastname: Schulze-Lefert
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000222:Adaptation, Physiological / Q000187:drug effects; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D001203:Ascomycota / Q000502:physiology; D004198:Disease Susceptibility; D007211:Indoles / Q000494:pharmacology; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D000069452:Pathogen-Associated Molecular Pattern Molecules / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D018517:Plant Roots / Q000187:drug effects / Q000254:growth & development; D020156:Salicylic Acid / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D007211:Indoles; C510424:PDR8 protein, Arabidopsis; D000069452:Pathogen-Associated Molecular Pattern Molecules; C014612:indolebutyric acid; D014364:Tryptophan; D020156:Salicylic Acid