A coevolved EDS1-SAG101-NRG1 module mediates cell death signaling by TIR-domain immune receptors

Abstract

~200 words) 28 Plant intracellular nucleotide-binding/leucine-rich repeat (NLR) immune receptors are 29 activated by pathogen effectors to trigger host defenses and cell death. Toll-30 Interleukin1-receptor (TIR)-domain NLRs (TNLs) converge on the Enhanced Disease 31 Susceptibility1 (EDS1) family of lipase-like proteins for all resistance outputs. In 32 Arabidopsis TNL immunity, AtEDS1 heterodimers with Phytoalexin Deficient4 33 (AtPAD4) transcriptionally boost basal defense pathways. AtEDS1 uses the same 34 surface to interact with PAD4-related Senescence-Associated Gene101 (AtSAG101), 35 but the role of AtEDS1-AtSAG101 heterodimers was unclear. We show that AtEDS1-36 AtSAG101 function together with AtNRG1 coiled-coil domain helper NLRs as a 37 coevolved TNL cell death signaling module. AtEDS1-AtSAG101-AtNRG1 cell death 38 activity is transferable to the solanaceous species, Nicotiana benthamiana, and 39 cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with 40 endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and 41 heterodimer structure-guided phenotyping of AtEDS1 variants or AtPAD4-AtSAG101 42 chimeras identify closely aligned ɑ-helical coil surfaces in the AtEDS1-AtSAG101 43 partner C-terminal domains that are necessary for TNL cell death signaling. Our data 44 suggest that TNL-triggered cell death and pathogen growth restriction are determined 45 by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these 46 signaling machineries coevolved with further components within plant species or 47 clades to regulate downstream pathways in TNL immunity. 48 49

Estradiol-inducible AvrRps4 expression reveals distinct properties of TIR-NLR-mediated effector-triggered immunity

“…NRC family proteins in Solanaceae species required for many NLRs, and NRG1/ADR1s in Arabidopsis required for TIR-NLRs and ADR1s for some CC-NLRs (Bonardi et al , 2011; Dong et al , 2016; Wu et al , 2017, 2019; Castel et al , 2019). NRG1s and ADR1s seem to function downstream of EDS1 and may function distinctly with SAG101 and PAD4, respectively (Lapin et al , 2019). SETI lines carry heterologously expressed RRS1-R/RPS4 and also endogenous RRS1-S/RPS4, RRS1B/RPS4B, which together provide three redundant copies of NLR pairs that can recognize AvrRps4.…”

“…Another major question regarding the signaling pathways is that SAG101 and PAD4 seems to be redundant but functionally equivalent to EDS1 (Wagner et al , 2013; Lapin et al , 2019). They also have been shown to be genetically linked to helper NLRs NRG1s and/or ADR1s to function (Castel et al , 2019; Wu et al , 2019).…”

Plant mixed lineage kinase domain-like proteins limit biotrophic pathogen growth

“…Sequence similarity between the animal and plant MLKL families was established by Hidden Markov Model (HMM)-to-HMM comparison using the HHSEARCH package ( 10 ). The 99,696 orthogroups (OGs) among 52 plant species have been established recently ( 11 ). An OG containing At MLKLs was used for the neighbor-net analysis of codon-aligned nucleotide sequence as descried previously ( 12 ).…”

“…108 coding sequences (CDS) of plant MLKLs belong to the same orthogroups ( 11 ). A CDS derived from Arabidopsis halleri is incomplete due to the ambiguous assembly of the corresponding genomic region.…”

Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

“…The RFP‐Trap beads were similarly treated but the bound proteins were directly subjected to on‐bead trypsin digestion prior LC‐MS/MS analysis. As controls, three biological replicates of protein extracts were prepared from wild type (Col‐0) and 35S:StrepII‐3xHA‐YFP (Lapin et al ., ) expressing plants of the same age, harvested and processed in parallel with the CDKD;2–GFP‐, CYCH–mCherry‐ and HISTONE H3.1‐expressing plants.…”