Structure-function analysis of the Arabidopsis EDS1 immune regulatory complex

Activation of plant innate immune responses by intracellular receptors involves dynamic changes in the subcellular localisations, assemblies and activities of signalling complexes. The A. thaliana nucleocytoplasmic protein EDS1, together with its signalling partners PAD4 and SAG101, coordinates basal and TNL receptor-triggered cell defence reprogramming in response to pathogens. EDS1, PAD4 and SAG101 genes exist in all seed plants and form a plant-specific family with a N-terminal lipase-like domain and a unique C-terminal EP (EDS1-PAD4) domain. Functional analysis of the crystal structure of an A. thaliana EDS1-SAG101 heterodimer showed that EDS1 forms molecularly distinct heterodimers with its partners through a large conserved interface. EDS1 heterodimer formation is chiefly driven by the juxtaposed lipase-like domains and is essential for EDS1 disease resistance signalling. The EDS1 lipase-like domain without its EP domain is stable but insufficient for immunity. These features suggested a key role of the EP domain. To gain functional insights to EP domain function, the EDS1-SAG101 heterodimer crystal structure was used to generate EP domain mutants that were defective in resistance signalling but retained a nucleocytoplasmic distribution and direct partner interactions. Functional analysis of the EDS1 EP domain variants revealed two conserved residues (K478 and R493) that are important for A.thaliana basal and TNL immunity. Mutation of K478 to arginine (K478R) or R493 to alanine (R493A) led to a partial and complete loss of TNL resistance, respectively. These mutants were completely compromised in EDS1-mediated basal resistance. Disease susceptibility of K478R and R493A in TNL resistance to bacterial pathogen strains is due to their failure to counteract virulence activity of the phytotoxin coronatine (a jasmonic acid-isoleucine conjugate mimic). EDS1-dependent transcriptional defence reprogramming over a 24 h time course after TNL activation is delayed in R493A and this causes delayed accumulation of the important stress hormone, salicylic acid (SA). Coronatine does not affect the SA accumulation profile of this mutant, although it suppresses SA accumulation in wild-type plants. By contrast, wild-type like SA accumulation in the K478R mutant in TNL resistance suggests two distinct activities of EDS1 EP domain: one is to promote the SA pathway (intact in K478R and defective in R493A), the second is to antagonize coronatine-promoted JA outputs (defective in both mutants) in disease resistance signalling. Further targeted mutational analysis shows that a positively charged residue at R493 is vital for EDS1 immunity functions and correlates with an ability of EDS1 to bind to nucleic acid in situ. Results presented here identify amino acids in the EP domain that are important for two different EDS1 signalling outputs and suggest that EDS1 chromatin association is integral to EDS1 immune function. This protein structure-function study provides tools to dissect the molecular function and interactions of this central plant immune regulatory node