Structure-Function Analysis of Cf-9, a Receptor-Like Protein with Extracytoplasmic Leucine-Rich Repeats

R.A.L. van der Hoorn, B.B.H. Wulff, S. Rivas, M.C. Durrant, A. van der Ploeg, P.J.G.M. de Wit, J.D.G. Jones

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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 -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 ß-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 languageEnglish
Pages (from-to)1000-1015
Number of pages15
JournalThe Plant Cell
Volume17
Issue number3
DOIs
Publication statusPublished - 2005

Keywords

  • polygalacturonase-inhibiting protein
  • kinase-like protein
  • gene encodes
  • arabidopsis-thaliana
  • crystal-structure
  • resistance gene
  • ribonuclease inhibitor
  • cladosporium-fulvum
  • signal-transduction
  • phaseolus-vulgaris

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