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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.

Original publication

DOI

10.1105/tpc.104.028118

Type

Journal article

Journal

Plant Cell

Publication Date

03/2005

Volume

17

Pages

1000 - 1015

Keywords

Amino Acid Sequence, Binding Sites, Conserved Sequence, Genes, Plant, Glycosylation, Leucine, Lycopersicon esculentum, Membrane Glycoproteins, Models, Molecular, Molecular Sequence Data, Mutation, Phenotype, Plant Proteins, Plants, Genetically Modified, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins, Repetitive Sequences, Amino Acid, Sequence Homology, Amino Acid, Tobacco