No abstract

Biotrophic plant pathogens encounter a postinfection basal resistance layer controlled by the lipase-like protein enhanced disease susceptibility 1 (EDS1) and its sequence-related interaction partners, senescence-associated gene 101 (SAG101) and phytoalexin deficient 4 (PAD4). Maintainance of separate EDS1 family member clades through angiosperm evolution suggests distinct functional attributes. We report the Arabidopsis EDS1-SAG101 heterodimer crystal structure with juxtaposed N-terminal α/β hydrolase and C-terminal α-helical EP domains aligned via a large conserved interface. Mutational analysis of the EDS1-SAG101 heterodimer and a derived EDS1-PAD4 structural model shows that EDS1 signals within mutually exclusive heterocomplexes. Although there is evolutionary conservation of α/β hydrolase topology in all three proteins, a noncatalytic resistance mechanism is indicated. Instead, the respective N-terminal domains appear to facilitate binding of the essential EP domains to create novel interaction surfaces on the heterodimer. Transitions between distinct functional EDS1 heterodimers might explain the central importance and versatility of this regulatory node in plant immunity.

The NRF2 transcription factor regulates a major environmental and oxidative stress response. NRF2 is itself negatively regulated by KEAP1, the adaptor of a Cul3-ubiquitin ligase complex that marks NRF2 for proteasomal degradation by ubiquitination. Electrophilic compounds activate NRF2 primarily by inhibiting KEAP1-dependent NRF2 degradation, through alkylation of specific cysteines. We have examined the impact on KEAP1 of reactive oxygen and nitrogen species, which are also NRF2 inducers. We found that in untreated cells, a fraction of KEAP1 carried a long range disulfide linking Cys 226 and Cys 613 . Exposing cells to hydrogen peroxide, to the nitric oxide donor spermine NONOate, to hypochlorous acid, or to S-nitrosocysteine further increased this disulfide and promoted formation of a disulfide linking two KEAP1 molecules via Cys 151 . None of these oxidants, except S-nitrocysteine, caused KEAP1 S-nitrosylation. A cysteine mutant preventing KEAP1 intermolecular disulfide formation also prevented NRF2 stabilization in response to oxidants, whereas those preventing intramolecular disulfide formation were functionally silent. Further, simultaneously inactivating the thioredoxin and glutathione pathways led both to major constitutive KEAP1 oxidation and NRF2 stabilization. We propose that KEAP1 intermolecular disulfide formation via Cys 151 underlies the activation of NRF2 by reactive oxygen and nitrogen species.The Cap'n'collar bZip transcription factor NRF2 regulates an environmental and oxidative stress response of major physiological importance in mammals. NRF2 is activated by reactive oxygen and nitrogen species, electrophilic xenobiotics, and heavy metals and promotes cytoprotection and survival toward these stresses (for a review, see Refs. 1 and 2). Activation of NRF2 is intricate, engaging controls at the level of subcellular distribution, interaction with other proteins, phosphorylation, and protein stability (reviewed in Ref. 2). Among these, protein stability is a major control determinant, involving KEAP1, the adaptor of a Cul3-ubiquitin ligase complex that ubiquitinates NRF2 and marks it for proteasomal degradation (3-6). Stress signals that activate NRF2, herein named NRF2 inducers, are primarily sensed at the level of KEAP1, causing NRF2 protein stabilization (7-9) by inhibiting KEAP1-mediated NRF2 ubiquitination (10, 11).The large number of NRF2 inducers and their quite different chemical nature have raised the question of how they are specifically sensed by KEAP1. Although NRF2 inducers are chemically very different, they all have electrophilic properties, which has led to the proposal that they must operate by alkylation and/or oxidation of KEAP1 Cys residues (12). The 624-amino acid-long KEAP1 protein has 25 (mouse) or 27 (human) Cys residues and carries a Broad complex, Tramtrack, Bric-à-Brac (BTB) 2 dimerization domain, an intervening region (IVR), and a six-Kelch repeat domain (Kelch) (see Fig. 2). It also binds zinc with a 1:1 stoichiometry, possibly through the IVR residues Cys 254 , Cys 2...