Random mutagenesis of the nucleotide-binding region of NRC1, a down-stream NB-LRR from tomato, identifies gain-of-function mutations in the GLPL-motif

P391 - Plant disease resistance (R) proteins confer immunity to pathogens possessing the corresponding avirulence (Avr) proteins. The majority of R proteins belong to the Nucleotide-Binding, Leucine-Rich Repeat (NB-LRR) class. Activation of NB-LRRs is often associated with induction of the Hypersensitive Response (HR), a form of programmed cell death. NRC1 (NB-LRR required for HR-associated Cell death-1) is an NB-LRR from tomato that participates in the down-stream signalling cascade leading to resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae. Silencing in Nicotiana benthamiana using the tomato NRC1 coding sequence diminishes the HR triggered by various R/Avr combinations. To identify mutations in NRC1 that cause a higher signalling activity we generated a random mutant library restricted to the NB-ARC domain of this protein and the mutants were screened for their capacity to induce an elicitor-independent HR upon transient expression in N. tabacum. The screening of 1920 clones retrieved 12 mutants that showed auto-activity. In nine mutants a single amino acid substitution was responsible for this gain-of-function phenotype. Interestingly, five of these substitutions were located at the GLPL motif of the NB-ARC domain, suggesting that this motif is involved in modulating the signalling activity of NRC1. We also identified a mutation in the highly conserved argin ine residue (R) of the RNBS-B motif. 3D modelling of the NB-ARC domain of NRC1 revealed that all nine mutations are centred around the bound nucleotide. Our mutational approach provides novel gain-of-function mutations in the NB-ARC domain of NRC1, giving insight into how the activity of this NB-LRR may be regulated.