Pathogen pressure on hosts leads to the evolution of genes regulating the innate immune response. By characterizing naturally occurring polymorphisms in immune receptors, we can better understand the molecular determinants of pathogen recognition. Pseudomonas syringae effector protein HopZ1a is recognized by HOPZ-ACTIVATED RESISTANCE 1 (ZAR1), a classical nucleotide-binding-domain-leucine-rich repeat (NBD-LRR) receptor protein (NLR). Recognition of HopZ1a also requires the pseudokinase ZED1 (HOPZ-EFFECTOR-TRIGGERED-IMMUNITY DEFICIENT 1), which is acetylated by HopZ1a. ZED1 is hypothesized to act as a decoy to trap HopZ1a into being recognized by the host. In previous work, we developed a transient expression assay in Nicotiana benthamiana to test for interactions among HopZ1a, ZED1 and ZAR1, and demonstrated that recognition of ZAR1 is conserved from the Brassicaceae to the Solanaceae. We showed that the coiled coil and LRR regions of ZAR1 directly interact with ZED1 and that interactions within the ZAR1 protein control the induction of immunity. To identify additional determinants of ZAR1 or ZED1 function, we performed an in silico screen to identify ecotypes from the 1001 genomes project that were likely to lack HopZ1a recognition. We tested ~300 ecotypes for their ability to recognize HopZ1a and identified ecotypes containing single nucleotide polymorphisms (SNPs) in ZAR1 and ZED1. Most ecotypes displayed SNPs in ZAR1, although one ecotype had a SNP in ZED1 at a site we had previously identified. We tested ZAR1 homologs from each ecotype to assess their ability to complement silencing of the N. benthamiana homolog of ZAR1, and tested for the effect of the SNPs on ZAR1 or ZED1 interactions and the immune response. This work demonstrates the power of natural diversity to define the molecular determinants and interactions necessary to regulate induction of immunity in response to pathogens.
Coauthors: Mael Baudin – UC Berkeley;Eliza Martin – Institute of Biochemistry of the Romanian Academy;Chodon Sass – UC Berkeley;Claire Bendix – USDA;Jana Hassan – UC Berkeley;Rolin Sauceda – UC Berkeley;Chelsea Specht – Cornell University;Andrei Petrescu – Institute of Biochemistry of the Romanian Academy