title: Structure-function analysis of cf-9, a receptor-like protein with extracytoplasmic leucine-rich repeats.
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.
authors.affiliation: Wageningen University, Laboratory of Phytopathology, 6709 PD, Wageningen, The Netherlands.
authors.forename: Renier A L
authors.initials: RA
authors.lastname: van der Hoorn
authors.forename: Brande B H
authors.initials: BB
authors.lastname: Wulff
authors.forename: Susana
authors.initials: S
authors.lastname: Rivas
authors.forename: Marcus C
authors.initials: MC
authors.lastname: Durrant
authors.forename: Anke
authors.initials: A
authors.lastname: van der Ploeg
authors.forename: Pierre J G M
authors.initials: PJ
authors.lastname: de Wit
authors.forename: Jonathan D G
authors.initials: JD
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D017343:Genes, Plant; D006031:Glycosylation; D007930:Leucine / Q000737:chemistry; D018551:Solanum lycopersicum / Q000235:genetics / Q000502:physiology; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D030821:Plants, Genetically Modified; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: C109609:Cf protein, Lycopersicon esculentum; D008562:Membrane Glycoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D007930:Leucine
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