title: Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity.
abstract: Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N(6)-(∆(2)-isopentenyl)adenine and dimethylated N(6)-(∆(2)-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N(6)-(∆(2)-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N(6)-(∆(2)-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.
authors.affiliation: Plant Productivity Systems Research Group (V.R., N.U., M.K., H.S.) and Environmental Metabolic Analysis Research Team (Y.T., J.K.), RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan;RIKEN BioResource Center, Tsukuba 305-0074, Japan (T.K.); andGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan (H.S.).
authors.forename: Venkatesan
authors.initials: V
authors.lastname: Radhika
authors.affiliation: Plant Productivity Systems Research Group (V.R., N.U., M.K., H.S.) and Environmental Metabolic Analysis Research Team (Y.T., J.K.), RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan;RIKEN BioResource Center, Tsukuba 305-0074, Japan (T.K.); andGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan (H.S.).
authors.forename: Nanae
authors.initials: N
authors.lastname: Ueda
authors.affiliation: Plant Productivity Systems Research Group (V.R., N.U., M.K., H.S.) and Environmental Metabolic Analysis Research Team (Y.T., J.K.), RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan;RIKEN BioResource Center, Tsukuba 305-0074, Japan (T.K.); andGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan (H.S.).
authors.forename: Yuuri
authors.initials: Y
authors.lastname: Tsuboi
authors.affiliation: Plant Productivity Systems Research Group (V.R., N.U., M.K., H.S.) and Environmental Metabolic Analysis Research Team (Y.T., J.K.), RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan;RIKEN BioResource Center, Tsukuba 305-0074, Japan (T.K.); andGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan (H.S.).
authors.forename: Mikiko
authors.initials: M
authors.lastname: Kojima
authors.affiliation: Plant Productivity Systems Research Group (V.R., N.U., M.K., H.S.) and Environmental Metabolic Analysis Research Team (Y.T., J.K.), RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan;RIKEN BioResource Center, Tsukuba 305-0074, Japan (T.K.); andGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan (H.S.).
authors.forename: Jun
authors.initials: J
authors.lastname: Kikuchi
authors.affiliation: Plant Productivity Systems Research Group (V.R., N.U., M.K., H.S.) and Environmental Metabolic Analysis Research Team (Y.T., J.K.), RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan;RIKEN BioResource Center, Tsukuba 305-0074, Japan (T.K.); andGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan (H.S.).
authors.forename: Takuji
authors.initials: T
authors.lastname: Kudo
authors.affiliation: Plant Productivity Systems Research Group (V.R., N.U., M.K., H.S.) and Environmental Metabolic Analysis Research Team (Y.T., J.K.), RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan;RIKEN BioResource Center, Tsukuba 305-0074, Japan (T.K.); andGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan (H.S.) sakaki@riken.jp.
authors.forename: Hitoshi
authors.identifier: http://orcid.org/0000-0001-5449-6492
authors.initials: H
authors.lastname: Sakakibara
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000382:microbiology*; D003583:Cytokinins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D054884:Host-Pathogen Interactions; D007541:Isopentenyladenosine / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D018716:Molecular Mimicry; D015394:Molecular Structure; D018517:Plant Roots / Q000187:drug effects / Q000254:growth & development; D012240:Rhodococcus / Q000378:metabolism / Q000472:pathogenicity*
chemical_list: D003583:Cytokinins; C001478:N(6)-(delta(2)-isopentenyl)adenine; D007541:Isopentenyladenosine