A Structure-guided mutagenesis study identifies one amino acid that is crucial for the recognition specificity of the potato cyst nematode resistance gene Gpa2

The potato cyst nematode resistance gene Gpa2 confers resistance to Globodera pallida. Gpa2 belongs to the class of CC-NB-LRR resistance genes and is located on a complex locus that also harbours the closely related Potato Virus X (PVX) resistance gene Rx1. Rx1 and Gpa2 are 88% identical at the amino acid level, yet they confer resistance to completely unrelated pathogens. For both R proteins, the pathogen elicitor that triggers the resistance response is known. Gpa2 recognizes the secreted Globodera pallida protein RBP1, whereas Rx1 detects the PVX coat protein. This it an excellent model system to investigate how the recognition specificity is determined in NB-LRR proteins. To determine which amino acids may play a role in Gpa2 specificity, we combined sequence information of 35 closely related homologues derived from wild Solanum species, functional data and a consensus in sillico 3D model. Two residues (M1 and M2) were detected that are located in the region that is crucial for elicitor recognition. In the 3D model, they map together on the LRR surface, suggesting a potential role in pathogen recognition. Functional validation was performed by mutagenesis of these candidate amino acids. Only mutation of M2 resulted in a loss of function upon agroinfiltration in N. benthaminana. Western blotting showed that this was not due to protein instability. Mutation of M2 in an auto-active mutant of Gpa2 showed that signal transduction was not inhibited. Therefore, we conclude that M2 is crucial for the recognition specificity of Gpa2.