title: Small Molecule DFPM Derivative-Activated Plant Resistance Protein Signaling in Roots Is Unaffected by EDS1 Subcellular Targeting Signal and Chemical Genetic Isolation of victr R-Protein Mutants.
abstract: The small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione) was recently shown to trigger signal transduction via early effector-triggered immunity signaling genes including EDS1 and PAD4 in Arabidopsis thaliana accession Col-0. Chemical genetic analyses of A. thaliana natural variants identified the plant Resistance protein-like Toll/Interleukin1 Receptor (TIR)-Nucleotide Binding (NB)-Leucine-Rich Repeat (LRR) protein VICTR as required for DFPM-mediated root growth arrest. Here a chemical genetic screen for mutants which disrupt DFPM-mediated root growth arrest in the Col-0 accession identified new mutant alleles of the TIR-NB-LRR gene VICTR. One allele, victr-6, carries a Gly216-to-Asp mutation in the Walker A domain supporting an important function of the VICTR nucleotide binding domain in DFPM responses consistent with VICTR acting as a canonical Resistance protein. The essential nucleo-cytoplasmic regulator of TIR-NB-LRR-mediated effector-triggered immunity, EDS1, was reported to have both nuclear and cytoplasmic actions in pathogen resistance. DFPM was used to investigate the requirements for subcellular EDS1 localization in DFPM-mediated root growth arrest. EDS1-YFP fusions engineered to localize mainly in the cytoplasm or the nucleus by tagging with a nuclear export signal (NES) or a nuclear localization signal (NLS), respectively, were tested. We found that wild-type EDS1-YFP and both the NES and NLS-tagged EDS1 variants were induced by DFPM treatments and fully complemented eds1 mutant plants in root responses to DFPM, suggesting that enrichment of EDS1 in either compartment could confer DFPM-mediated root growth arrest. We further found that a light and O2-dependent modification of DFPM is necessary to mediate DFPM signaling in roots. Chemical analyses including Liquid Chromatography-Mass Spectrometry and High-Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry identified a DFPM modification product that is likely responsible for bioactivity mediating root growth arrest. We propose a chemical structure of this product and a possible reaction mechanism for DFPM modification.
authors.affiliation: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116, United States of America.
authors.forename: Hans-Henning
authors.initials: HH
authors.lastname: Kunz
authors.affiliation: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116, United States of America.
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Park
authors.affiliation: Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, 92093-0212, United States of America.
authors.forename: Emily
authors.initials: E
authors.lastname: Mevers
authors.affiliation: Max-Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, D-50829 Cologne, Germany.
authors.forename: Ana V
authors.initials: AV
authors.lastname: García
authors.affiliation: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116, United States of America.
authors.forename: Samantha
authors.initials: S
authors.lastname: Highhouse
authors.affiliation: Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, 92093-0212, United States of America.|Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, 92093, United States of America.
authors.forename: William H
authors.initials: WH
authors.lastname: Gerwick
authors.affiliation: Max-Planck Institute for Plant Breeding Research, Department of Plant-Microbe Interactions, D-50829 Cologne, Germany.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Parker
authors.affiliation: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116, United States of America.
authors.forename: Julian I
authors.initials: JI
authors.lastname: Schroeder
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D060467:Disease Resistance; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D013058:Mass Spectrometry; D009154:Mutation; D010880:Piperidines / Q000737:chemistry* / Q000494:pharmacology*; D018517:Plant Roots / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D015398:Signal Transduction / Q000187:drug effects; D013871:Thiones / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: C561624:(5-(3,4-dichlorophenyl)furan-2-yl)piperidine-1-ylmethanethione; D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C118837:EDS1 protein, Arabidopsis; D010880:Piperidines; D013871:Thiones