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

Plant Cell. 2005 Mar;17(3):1000-15. doi: 10.1105/tpc.104.028118. Epub 2005 Feb 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • Conserved Sequence
  • Genes, Plant
  • Glycosylation
  • Leucine / chemistry
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / physiology*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Nicotiana / genetics
  • Nicotiana / metabolism
  • Phenotype
  • Plant Proteins / chemistry*
  • Plant Proteins / genetics
  • Plant Proteins / physiology*
  • Plants, Genetically Modified
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Repetitive Sequences, Amino Acid
  • Sequence Homology, Amino Acid
  • Solanum lycopersicum / genetics
  • Solanum lycopersicum / physiology

Substances

  • Cf protein, Lycopersicon esculentum
  • Membrane Glycoproteins
  • Plant Proteins
  • Recombinant Proteins
  • Leucine