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PMID: 15722474 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Structure-function analysis of cf-9, a receptor-like protein with extracytoplasmic leucine-rich repeats.

The Plant cell ·Vol. 17 ·No. 3 ·2005-03-00 ·Pages 1000-15

van der Hoorn RA, Wulff BB, Rivas S, Durrant MC, van der Ploeg A, de Wit PJ, Jones JD

Abstract

The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring recognition of various pathogen molecules and plant hormones. We report here a large-scale structure-function analysis of an eLRR protein. A total of 66 site-directed mutants of Cf-9 were analyzed for activity in Avr9 recognition and for protein stability and the results interpreted with the help of a homology model of the Cf-9 structure. Conserved Trp and Cys pairs in the N-terminal LRR-flanking domain appear to be important for Cf-9 activity and are probably exposed at the putative concave inner surface of the Cf-9 protein, where recognition specificity also resides. Removal of each of the 22 putative N-linked glycosylation sites (PGS) revealed that many PGSs contribute to Cf-9 activity and that the PGSs in the putative alpha-helices of the LRR modules are essential. Immunoblot analysis and mass spectrometry showed that all but one of the PGSs are N-glycosylated. Introduction of glycosylation at the putative concave beta-sheet surface blocks Cf-9 activity, in some cases probably by disturbing specific recognition, and in another case by steric hindrance with existing N-glycans. The glycosylation pattern and several other features are conserved in other eLRR proteins, where similar mutations show similar phenotypes.

MeSH Terms
Amino Acid Sequence Binding Sites/genetics Conserved Sequence Genes, Plant Glycosylation Leucine/chemistry Lycopersicon esculentum/genetics,physiology Membrane Glycoproteins/chemistry,genetics,physiology Models, Molecular Molecular Sequence Data Mutation Phenotype Plant Proteins/chemistry,genetics,physiology Plants, Genetically Modified Protein Conformation Protein Structure, Secondary Protein Structure, Tertiary Recombinant Proteins/chemistry,genetics,metabolism Repetitive Sequences, Amino Acid Sequence Homology, Amino Acid Tobacco/genetics,metabolism
Chemicals
Cf protein, Lycopersicon esculentum Membrane Glycoproteins Plant Proteins Recombinant Proteins Leucine
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
van der Hoorn Renier A L
Wageningen University, Laboratory of Phytopathology, 6709 PD, Wageningen, The Netherlands.
Wulff Brande B H
Rivas Susana
Durrant Marcus C
van der Ploeg Anke
de Wit Pierre J G M
Jones Jonathan D G
References (83)
83 references, click to expand
  1. Antisense phenotypes reveal a role for SHY, a pollen-specific leucine-rich repeat protein, in pollen tube growth.
    Plant J. 2004 Aug;39(4):643-54 PMID: 15272880
  2. The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required for root hair morphogenesis in Arabidopsis thaliana.
    Genes Dev. 2001 May 1;15(9):1128-39 PMID: 11331608
  3. Cell signaling by receptor tyrosine kinases.
    Cell. 2000 Oct 13;103(2):211-25 PMID: 11057895
  4. Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats.
    Nature. 1993 Dec 23-30;366(6457):751-6 PMID: 8264799
  5. The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability.
    EMBO J. 1999 May 4;18(9):2352-63 PMID: 10228150
  6. A cysteine-rich extracellular protein, LAT52, interacts with the extracellular domain of the pollen receptor kinase LePRK2.
    Plant Cell. 2002 Sep;14(9):2277-87 PMID: 12215520
  7. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  8. Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection.
    Plant Cell. 2003 Jan;15(1):93-106 PMID: 12509524
  9. BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana.
    Proc Natl Acad Sci U S A. 2001 May 8;98(10):5916-21 PMID: 11320207
  10. The Protein Data Bank.
    Nucleic Acids Res. 2000 Jan 1;28(1):235-42 PMID: 10592235
  11. The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency.
    J Biol Chem. 1996 Mar 15;271(11):6363-6 PMID: 8626433
  12. Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A.
    J Mol Biol. 1996 Dec 20;264(5):1028-43 PMID: 9000628
  13. The receptor kinases LePRK1 and LePRK2 associate in pollen and when expressed in yeast, but dissociate in the presence of style extract.
    Proc Natl Acad Sci U S A. 2003 May 27;100(11):6860-5 PMID: 12748390
  14. Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase.
    Science. 2003 Jan 3;299(5603):109-12 PMID: 12411574
  15. The LKA gene is a BRASSINOSTEROID INSENSITIVE 1 homolog of pea.
    Plant J. 2003 Nov;36(3):291-300 PMID: 14617087
  16. Structural diversity of leucine-rich repeat proteins.
    J Mol Biol. 1998 Apr 3;277(3):519-27 PMID: 9533877
  17. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling.
    Cell. 2002 Jul 26;110(2):203-12 PMID: 12150928
  18. Tomato Ve disease resistance genes encode cell surface-like receptors.
    Proc Natl Acad Sci U S A. 2001 May 22;98(11):6511-5 PMID: 11331751
  19. Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade.
    Plant Physiol. 2003 Mar;131(3):1313-26 PMID: 12644681
  20. The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency.
    Biochemistry. 1998 May 12;37(19):6833-7 PMID: 9578569
  21. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.
    Plant J. 2003 Mar;33(5):949-56 PMID: 12609035
  22. The C-terminal dilysine motif confers endoplasmic reticulum localization to type I membrane proteins in plants.
    Plant Cell. 2000 Jul;12(7):1179-201 PMID: 10899983
  23. The functions of the human insulin receptor are affected in different ways by mutation of each of the four N-glycosylation sites in the beta subunit.
    J Biol Chem. 1994 Jul 8;269(27):18062-71 PMID: 8027066
  24. The excess microsporocytes1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther.
    Genes Dev. 2002 Aug 1;16(15):2021-31 PMID: 12154130
  25. Glycosylation of ovalbumin nascent chains. The spatial relationship between translation and glycosylation.
    J Biol Chem. 1980 Oct 10;255(19):9236-42 PMID: 7410422
  26. Receptor-like protein kinase genes of Arabidopsis thaliana.
    Plant J. 1993 Mar;3(3):451-6 PMID: 8220453
  27. EXS, a putative LRR receptor kinase, regulates male germline cell number and tapetal identity and promotes seed development in Arabidopsis.
    Curr Biol. 2002 Oct 15;12(20):1718-27 PMID: 12401166
  28. The Cf-9 disease resistance protein is present in an approximately 420-kilodalton heteromultimeric membrane-associated complex at one molecule per complex.
    Plant Cell. 2002 Mar;14(3):689-702 PMID: 11910014
  29. The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis.
    Cell. 1997 May 16;89(4):575-85 PMID: 9160749
  30. A carrot leucine-rich-repeat protein that inhibits ice recrystallization.
    Science. 1998 Oct 2;282(5386):115-7 PMID: 9756474
  31. HAR1 mediates systemic regulation of symbiotic organ development.
    Nature. 2002 Nov 28;420(6914):426-9 PMID: 12442172
  32. Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L.
    Plant J. 1992 May;2(3):367-73 PMID: 1303801
  33. Genetic variation at the tomato Cf-4/Cf-9 locus induced by EMS mutagenesis and intralocus recombination.
    Genetics. 2004 May;167(1):459-70 PMID: 15166169
  34. Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging.
    Science. 1994 Nov 4;266(5186):789-93 PMID: 7973631
  35. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.
    Plant Cell. 1996 Apr;8(4):735-46 PMID: 8624444
  36. Comparison of the structures of the cubic and tetragonal forms of horse-spleen apoferritin.
    Acta Crystallogr D Biol Crystallogr. 1997 Sep 1;53(Pt 5):580-7 PMID: 15299889
  37. Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato.
    Cell. 1997 Dec 12;91(6):821-32 PMID: 9413991
  38. Rapid migration in gel filtration of the Cf-4 and Cf-9 resistance proteins is an intrinsic property of Cf proteins and not because of their association with high-molecular-weight proteins.
    Plant J. 2003 Aug;35(3):305-15 PMID: 12887582
  39. HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission.
    Genes Dev. 2000 Jan 1;14(1):108-17 PMID: 10640280
  40. Apple contains receptor-like genes homologous to the Cladosporium fulvum resistance gene family of tomato with a cluster of genes cosegregating with Vf apple scab resistance.
    Mol Plant Microbe Interact. 2001 Apr;14(4):508-15 PMID: 11310738
  41. An LRR receptor kinase involved in perception of a peptide plant hormone, phytosulfokine.
    Science. 2002 May 24;296(5572):1470-2 PMID: 12029134
  42. A receptor kinase gene regulating symbiotic nodule development.
    Nature. 2002 Jun 27;417(6892):962-6 PMID: 12087406
  43. Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase.
    Plant Physiol. 2000 Aug;123(4):1247-56 PMID: 10938344
  44. Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein Avr4.
    Plant Cell. 2001 Feb;13(2):273-85 PMID: 11226185
  45. The fasciated ear2 gene encodes a leucine-rich repeat receptor-like protein that regulates shoot meristem proliferation in maize.
    Genes Dev. 2001 Oct 15;15(20):2755-66 PMID: 11641280
  46. Both the extracellular leucine-rich repeat domain and the kinase activity of FSL2 are required for flagellin binding and signaling in Arabidopsis.
    Plant Cell. 2001 May;13(5):1155-63 PMID: 11340188
  47. Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein.
    Plant J. 2004 Feb;37(4):517-27 PMID: 14756760
  48. Using mutant alleles to determine the structure and function of leucine-rich repeat receptor-like kinases.
    Curr Opin Plant Biol. 2003 Oct;6(5):507-16 PMID: 12972053
  49. VH1, a provascular cell-specific receptor kinase that influences leaf cell patterns in Arabidopsis.
    Plant Cell. 2002 Nov;14(11):2707-22 PMID: 12417696
  50. Control of stomatal distribution on the Arabidopsis leaf surface.
    Science. 2002 May 31;296(5573):1697-700 PMID: 12040198
  51. Structures of glycoprotein Ibalpha and its complex with von Willebrand factor A1 domain.
    Science. 2002 Aug 16;297(5584):1176-9 PMID: 12183630
  52. The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signaling complex that includes KAPP and a Rho-related protein.
    Plant Cell. 1999 Mar;11(3):393-406 PMID: 10072399
  53. Shoot control of root development and nodulation is mediated by a receptor-like kinase.
    Nature. 2002 Nov 28;420(6914):422-6 PMID: 12442170
  54. Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2.
    Plant Cell. 1998 Mar;10(3):319-30 PMID: 9501107
  55. Disulfide bonds as switches for protein function.
    Trends Biochem Sci. 2003 Apr;28(4):210-4 PMID: 12713905
  56. BRI1 is a critical component of a plasma-membrane receptor for plant steroids.
    Nature. 2001 Mar 15;410(6826):380-3 PMID: 11268216
  57. CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development.
    Plant Cell. 2003 May;15(5):1198-211 PMID: 12724544
  58. Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation.
    Mol Gen Genet. 1990 Jan;220(2):245-50 PMID: 2325623
  59. Secondary structure and post-translational modifications of the leucine-rich repeat protein PGIP (polygalacturonase-inhibiting protein) from Phaseolus vulgaris.
    Biochemistry. 2001 Jan 16;40(2):569-76 PMID: 11148052
  60. Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins.
    Plant Cell. 2001 Feb;13(2):255-72 PMID: 11226184
  61. The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase.
    Plant Cell. 1999 Oct;11(10):1925-34 PMID: 10521522
  62. The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato.
    Plant Cell. 2004 Jun;16(6):1604-15 PMID: 15155877
  63. Granzyme B proteolysis of a neuronal glutamate receptor generates an autoantigen and is modulated by glycosylation.
    J Immunol. 2001 Feb 1;166(3):1433-8 PMID: 11160179
  64. FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis.
    Mol Cell. 2000 Jun;5(6):1003-11 PMID: 10911994
  65. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.
    Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14440-5 PMID: 8962070
  66. A plant receptor-like kinase required for both bacterial and fungal symbiosis.
    Nature. 2002 Jun 27;417(6892):959-62 PMID: 12087405
  67. Raster3D: photorealistic molecular graphics.
    Methods Enzymol. 1997;277:505-24 PMID: 18488322
  68. Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis.
    Mol Plant Microbe Interact. 2000 Apr;13(4):439-46 PMID: 10755307
  69. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction.
    Cell. 1997 Sep 5;90(5):929-38 PMID: 9298904
  70. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling.
    Cell. 2002 Jul 26;110(2):213-22 PMID: 12150929
  71. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.
    Nature. 2000 Dec 14;408(6814):796-815 PMID: 11130711
  72. Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint.
    Plant Cell. 2000 Sep;12(9):1591-606 PMID: 11006334
  73. The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense.
    Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):10124-8 PMID: 12904578
  74. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21.
    Science. 1995 Dec 15;270(5243):1804-6 PMID: 8525370
  75. Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated.
    Plant J. 2000 Mar;21(6):529-36 PMID: 10758503
  76. The tomato Cf-2 disease resistance locus comprises two functional genes encoding leucine-rich repeat proteins.
    Cell. 1996 Feb 9;84(3):451-9 PMID: 8608599
  77. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes.
    Cell. 2003 Jun 27;113(7):935-44 PMID: 12837250
  78. The systemin receptor SR160 from Lycopersicon peruvianum is a member of the LRR receptor kinase family.
    Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9585-90 PMID: 12060717
  79. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases.
    Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10763-8 PMID: 11526204
  80. Arabidopsis downy mildew resistance gene RPP27 encodes a receptor-like protein similar to CLAVATA2 and tomato Cf-9.
    Plant Physiol. 2004 Jun;135(2):1100-12 PMID: 15155873
  81. The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety.
    Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):886-90 PMID: 14715897
  82. Mechanism of integrin activation by disulfide bond reduction.
    Biochemistry. 2001 Jul 31;40(30):8861-7 PMID: 11467947
  83. Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin.
    Cell. 2002 Dec 13;111(6):825-36 PMID: 12526809
Article Info
Journal
The Plant cell
Abbr.
Plant Cell
ISSN
1040-4651
DOI
10.1105/tpc.104.028118
Published
2005-03-00
Epub
2005-00-18
Pages
1000-15
Language
English
Region
England
NLM ID
9208688
PMCID
PMC1069714
Subset
IM
External Links
PubMed source DOI: 10.1105/tpc.104.028118 PMC Full Text View journal details
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