A single amino acid substitution in the LRR domain is sufficient to compromise effector recognition by the nematode immune receptor GPA2

A SINGLE AMINO ACID SUBSTITUTION IN THE LRR DOMAIN IS SUFFICIENT TO COMPROMISE EFFECTOR RECOGNITION BY THE NEMATODE IMMUNE RECEPTOR GPA2 Bakker, EH1, Roosien, J1, Slootweg, EJ1, Butterbach, PBE1, Spiridon, LN2, Smant, G1, Petrescu, AJ2, Bakker, J1 and Goverse, A1 1Laboratory of Nematology, Department of Plant Sciences, Wageningen University, 6708 PB Wageningen, The Netherlands 2Institute of Biochemistry of the Romanian Academy, 060031 Bucharest, Romania The potato cyst nematode resistance gene Gpa2 confers resistance to Globodera pallida and is located on a complex locus together with the closely related Potato Virus X (PVX) resistance gene Rx1. They encode NB-LRR immune receptors, which are 88% identical at the amino acid level. Yet, they confer resistance to completely unrelated pathogens. Gpa2 recognizes the secreted nematode effector RBP1, whereas Rx1 detects the PVX coat protein. This makes Gpa2 and Rx1 an excellent model system to investigate how the recognition specificity is determined in NB-LRR proteins. To investigate which amino acids play a role in RBP1 recognition by Gpa2, an integrated approach was used combining bioinformatics, remote homology modeling and functional assays. First, Gpa2-specific residues were identified in a sequence alignment of 35 closely related Rx1/Gpa2 homologues derived from wild Solanum species. Six residues were located in the C-terminal region of the LRR domain of Gpa2 involved in RBP1 induced activation of the protein. Next, systematic exchange of these polymorphic residues between Gpa2 and Rx1 revealed that a single amino acid residue in the C-terminal end of the LRR domain is required for RBP1 recognition. This mutant was still signalling competent, as it was able to induce a constitutive cell death response in trans when combined with the D460V mutation in the CC-NB-ARC domain. The fact that this residue maps on the LRR surface and is subject to diversifying selection further supports its role as specificity determinant of Gpa2.