title: RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins.
abstract: The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.
authors.affiliation: From the Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Fujiwara
authors.affiliation: From the Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Kawazoe
authors.affiliation: the Research Institute of Molecular Genetics, Kochi University, Nankoku 783-0093, Japan.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Ohnishi
authors.affiliation: the Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Kitagawa
authors.affiliation: the Departament de Genètica, Universitat de Barcelona and Centre for Research in Agricultural Genomics, Bellaterra, Catalonia, Spain.
authors.forename: Crina
authors.initials: C
authors.lastname: Popa
authors.affiliation: the Departament de Genètica, Universitat de Barcelona and Centre for Research in Agricultural Genomics, Bellaterra, Catalonia, Spain.
authors.forename: Marc
authors.initials: M
authors.lastname: Valls
authors.affiliation: INRA-CNRS, Laboratoire des Interactions Plantes-Microoganismes, UMR441-2594, Castanet-Tolosan, France, and.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Genin
authors.affiliation: the Center of Clinical Laboratories, Tokuyama Medical Association Hospital, Shunan 745-0846, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Nakamura
authors.affiliation: the Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kuramitsu
authors.affiliation: From the Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan.
authors.forename: Naotaka
authors.initials: N
authors.lastname: Tanaka
authors.affiliation: From the Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan, mtabuchi@ag.kagawa-u.ac.jp.
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Tabuchi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D053263:Gene Regulatory Networks; D005978:Glutathione / Q000378:metabolism; D054884:Host-Pathogen Interactions; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010944:Plants / Q000382:microbiology; D017434:Protein Structure, Tertiary; D043368:Ralstonia solanacearum / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D000069296:Type III Secretion Systems / Q000235:genetics* / Q000378:metabolism; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005722:gamma-Glutamylcyclotransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C103243:TRX1 protein, S cerevisiae; C516110:TRX2 protein, S cerevisiae; D000069296:Type III Secretion Systems; D037521:Virulence Factors; D013879:Thioredoxins; D054464:Peroxiredoxins; D005722:gamma-Glutamylcyclotransferase; D005978:Glutathione
keywords: Saccharomyces cerevisiae; plant defense; redox regulation; thioredoxin; type III secretion system (T3SS)
references.citation: Mol Microbiol. 2004 Jul;53(1):115-28
references.pmid: 15225308
references.citation: Plant Physiol. 2004 Mar;134(3):1006-16
references.pmid: 14976236
references.citation: J Bacteriol. 1983 Jan;153(1):163-8
references.pmid: 6336730
references.citation: Nucleic Acids Res. 1991 May 25;19(10):2785
references.pmid: 1645866
references.citation: Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2320-4
references.pmid: 8460141
references.citation: J Biol Chem. 1993 May 25;268(15):11296-303
references.pmid: 8496184
references.citation: Cell Mol Life Sci. 2005 Jan;62(1):24-35
references.pmid: 15619004
references.citation: Science. 2005 Apr 22;308(5721):548-50
references.pmid: 15746386
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14620-5
references.pmid: 16983093
references.citation: Plant J. 2007 Jan;49(1):159-72
references.pmid: 17144898
references.citation: Nat Protoc. 2006;1(6):3159-65
references.pmid: 17406579
references.citation: Nature. 2007 Oct 18;449(7164):827-34
references.pmid: 17943119
references.citation: Cell Host Microbe. 2008 Jul 17;4(1):8-15
references.pmid: 18621006
references.citation: J Biol Chem. 2008 Aug 8;283(32):22031-42
references.pmid: 18515354
references.citation: Science. 2008 Aug 15;321(5891):952-6
references.pmid: 18635760
references.citation: Curr Opin Microbiol. 2009 Feb;12(1):44-52
references.pmid: 19144559
references.citation: Cell Host Microbe. 2009 Jun 18;5(6):571-9
references.pmid: 19527884
references.citation: Biosci Biotechnol Biochem. 2009 Oct;73(10):2261-7
references.pmid: 19809180
references.citation: Sci Signal. 2009;2(95):ra71
references.pmid: 19887681
references.citation: Annu Rev Genet. 2009;43:335-67
references.pmid: 19691428
references.citation: Proteomics. 2009 Nov;9(22):5078-89
references.pmid: 19771552
references.citation: Mol Plant Microbe Interact. 2010 Mar;23(3):251-62
references.pmid: 20121447
references.citation: Plant Physiol. 2010 Jul;153(3):1144-60
references.pmid: 20488891
references.citation: New Phytol. 2010 Sep;187(4):920-8
references.pmid: 20673287
references.citation: Planta. 2011 May;233(5):895-910
references.pmid: 21234598
references.citation: EMBO J. 2011 May 18;30(10):2044-56
references.pmid: 21478822
references.citation: Curr Opin Plant Biol. 2011 Aug;14(4):358-64
references.pmid: 21454121
references.citation: FEMS Microbiol Rev. 2011 Nov;35(6):1100-25
references.pmid: 21517912
references.citation: Mol Microbiol. 2011 Dec;82(6):1454-67
references.pmid: 22040088
references.citation: Plant Signal Behav. 2012 Feb;7(2):210-2
references.pmid: 22353869
references.citation: Annu Rev Phytopathol. 2012;50:67-89
references.pmid: 22559068
references.citation: Trends Plant Sci. 2012 Nov;17(11):644-55
references.pmid: 22796464
references.citation: EMBO Rep. 2012 Dec;13(12):1095-101
references.pmid: 23070364
references.citation: Microb Biotechnol. 2013 Nov;6(6):614-20
references.pmid: 23617636
references.citation: Mol Biol Evol. 2013 Dec;30(12):2725-9
references.pmid: 24132122
references.citation: BMC Genomics. 2013;14:859
references.pmid: 24314259
references.citation: Proc Natl Acad Sci U S A. 2014 Mar 25;111(12):4560-5
references.pmid: 24616501
references.citation: Cell Rep. 2014 May 8;7(3):681-8
references.pmid: 24767995
references.citation: Curr Opin Plant Biol. 2014 Aug;20:110-7
references.pmid: 24880553
references.citation: MBio. 2014;5(6). pii: e02065-14. doi: 10.1128/mBio.02065-14
references.pmid: 25538193
references.citation: Biochem J. 2015 May 15;468(1):73-85
references.pmid: 25716890
references.citation: Cell. 2015 May 21;161(5):1074-88
references.pmid: 26000483
references.citation: J Biol Chem. 2000 May 5;275(18):13259-65
references.pmid: 10788431
references.citation: EMBO J. 2001 Apr 17;20(8):1840-9
references.pmid: 11296218
references.citation: Nature. 2002 Jan 31;415(6871):497-502
references.pmid: 11823852
references.citation: Mol Plant Microbe Interact. 2003 May;16(5):447-55
references.pmid: 12744516
references.citation: Eukaryot Cell. 2004 Aug;3(4):827-34
references.pmid: 15302815