Structural determinants involved in effector recognition by the nematode immune receptor GPA2

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 and how pathogen effectors evade recognition. To investigate the structural determinants involved 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. Evasion of RBP1 recognition by Gpa2 is also dependent on a single amino acid substitution (S/P), which resides in the hypervariable surface of RBP1. Computational modeling of the 3D structure suggests that this substitution causes a change in the loop region, which might explain the observed differences in Gpa2 recognition. In addition, a structure-informed approach was used to investigate the contribution of other structural motifs present in RBP1 to better understand the underlying mechanism of RBP1-mediated activation of Gpa2.