Identification of Distinct Specificity Determinants in Resistance Protein Cf-4 Allows Construction of a Cf-9 Mutant That Confers Recognition of Avirulence Protein AVR4

Abstract

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9 , respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9 . We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system. INTRODUCTIONBecause plants are subjected to environments that change continuously, they are armed with recognition systems that can sense diverse biotic and abiotic stresses and subsequently mediate the induction of appropriate responses. A recognition system that can deal with various pathogens is especially crucial for the survival of the plant. Specific recognition of invading pathogens frequently is mediated by resistance ( R ) genes. Upon recognition of the matching pathogen-derived avirulence determinant (AVR), various defense responses are triggered, often including a hypersensitive response. The hypersensitive response involves the death of the tissue surrounding the primary infection site and thereby restricts further growth of the invading pathogen (Hammond-Kosack and Jones, 1996). Surprisingly, R genes that confer resistance to different types of pathogens encode very similar proteins, indicating that in plants, flexible recognition systems are used to monitor attacks by a diverse array of pathogens. The largest class of R genes encodes proteins that are likely located in the cytoplasm and contain leucine-rich repeats (LRRs) and a nucleotide binding site (NBS). Members of this NBS-LRR class have been cloned from various plant species and confer race-specific resistance against viruses, bacteria, fungi, oomycetes, nematodes, or insects (reviewed in Van der Biezen and Jones, 199...

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,

“…However, we observed that overexpression of the LRR domain of AtLRX1, despite its ability to sequester the ligand of the native AtLRX1 protein and induce a dominant negative effect in root hairs, had no influence in the rest of the plant where other LRXs are active (Baumberger et al, 2001). This result indicates that the flanking regions of the LRRs might contribute to the specificity of the interaction with the ligand as observed for the LRRs of the tomato and flax (Linum usitatissimum) resistance genes Cf-4, L6, and L7 (Ellis et al, 1999;Van der Hoorn et al, 2001). The existence of family-or paralog/ortholog-specific regions adjacent to the conserved region of the N-termini of the LRX proteins supports this notion.…”

“…In each LRR, a region of the consensus sequence (xxLxLxx) is predicted to form a ␤-strand/ ␤-turn structure in which the variable residues (x) are exposed to the solvent and determine the specificity of the interaction. This region of the LRR is subjected to diversifying selection in plant resistance genes (McDowell et al, 1998;Meyers et al, 1998;Ellis et al, 1999;Van der Hoorn et al, 2001). The observation that among different LRXs, the solvent-exposed amino acids are well conserved or even identical (AtLRX3/4 and AtLRX6/7) suggests that the LRX proteins interact with very similar or identical ligands.…”

Phylogenomic Analysis of the Receptor-Like Proteins of Rice and Arabidopsis

“…In previous functional studies, the C1 region has been implicated in providing the recognition specificity of RLPs. Deletion of LRRs and introduction of point mutations in the C1 region of Cf-9 changes the specificity of Cf-9 to that of Cf-4 ( Van der Hoorn et al, 2001;Wulff et al, 2001). Our analyses reveal the C1 region to be highly variable, particularly in the number of LRRs.…”

“…This domain in BRI1 has now been shown to be involved in direct binding to brassinolide (Kinoshita et al, 2005). On the other hand, the C2 domains of Cf-9 and Cf-4 are identical, and determinants of the distinct recognition achieved by these proteins reside in LRRs 10 to 16 of domain C1 ( Van der Hoorn et al, 2001;Wulff et al, 2001).…”

“…Mutation of one of the N-terminal Cys in the RLK BRI1 leads to a weak brassinosteroid response phenotype (Dievart and Clark, 2003). Domain B is also required for proper function of Cf-4 ( Van der Hoorn et al, 2001;Wulff et al, 2001). Thus, the conserved Cys are expected to be critical either for maintaining Cf-RLP structure or for mediating intermolecular interactions.…”