Molecular and spatial characterisation of Arabidopsis EDS1 defence regulatory complexes

In plants, cellular innate immune responses are indispensable for defence against pathogens. Arabidopsis EDS1 (Enhanced Disease Susceptibility 1) and PAD4 (Phytoalexin Deficient 4) are essential regulators of basal resistance to virulent pathogens, controlling defence amplification and accumulation of the signalling molecule salicylic acid (SA). Also, EDS1 is necessary for Resistance (R) protein-triggered programmed cell death to avirulent pathogen isolates conditioned by the TIR (Toll-Interleukin-1 Receptor) class of nucleotide-binding/leucine-rich-repeat (NB-LRR) immune receptor. Complete loss of TIR-NB-LRR mediated resistance and its associated cell death programme in Arabidopsis eds1 mutants and partial disabling of the same resistances in pad4 suggested a mechanism in which TIR-type NB-LRR proteins engage EDS1 early in the defence cascade that connects the recognition process to basal defences, requiring both EDS1 and PAD4. Consistent with such a cooperative role, EDS1 and PAD4 interact in Arabidopsis soluble leaf extracts. EDS1 and PAD4 have homology to eukaryotic lipases in their N-terminal halves and share a domain of high sequence homology (the EP domain) in their C-termini with one other plant lipase-like protein, SAG101 (Senescence Associated Gene 101) that was recently identified as part of an EDS1 complex in leaf soluble extracts. However, the nature of this association and whether SAG101 signals in plant innate immunity was not known. The work presented here shows that SAG101 interacts directly with EDS1 inside the nucleus of Arabidopsis cells and, together with PAD4, contributes intrinsic and indispensable signalling activity to the EDS1 defence pathway in resistance and programmed cell death triggered by TIR-type R proteins and in expression of basal defences. The EDS1-SAG101 complex is molecularly and spatially distinct from EDS1-EDS1 homomeric interactions that occur in the cytosol but not in the nucleus. SAG101 possesses a defence regulatory function that is partially redundant with PAD4. Loss of SAG101 can be compensated for by the presence of PAD4. Single null sag101 mutant alleles had no effect on plant disease resistance but combining sag101 with a null pad4 mutation disabled resistance as fully as eds1. Restriction of SAG101 to the nucleus may account for its inability to fully complement loss of PAD4 that co-localises with EDS1 in the cytosol and the nucleus. These new findings demonstrate that all three proteins are important regulators of innate immunity and point to a complex nucleo-cytoplasmic dynamic between EDS1 and its signalling partners that may be important for plant defence signal relay

NLR we there yet? Nucleocytoplasmic coordination of NLR-mediated immunity

Plant intracellular nucleotide-binding leucine-rich repeat immune receptors (NLRs) perceive the activity of pathogen-secreted effector molecules that, when undetected, promote colonisation of hosts. Signalling from activated NLRs converges with and potentiates downstream responses from activated pattern recognition receptors (PRRs) that sense microbial signatures at the cell surface. Efficient signalling of both receptor branches relies on the host cell nucleus as an integration point for transcriptional reprogramming, and on the macromolecular transport processes that mediate the communication between cytoplasm and nucleoplasm. Studies on nuclear pore complexes (NPCs), the nucleoporin proteins (NUPs) that compose NPCs, and nuclear transport machinery constituents that control nucleocytoplasmic transport, have revealed that they play important roles in regulating plant immune responses. Here, we discuss the contributions of nucleoporins and nuclear transport receptor (NTR)-mediated signal transduction in plant immunity with an emphasis on NLR immune signalling across the nuclear compartment boundary and within the nucleus. We also highlight and discuss cytoplasmic and nuclear functions of NLRs and their signalling partners and further consider the potential implications of NLR activation and resistosome formation in both cellular compartments for mediating plant pathogen resistance and programmed host cell death

Puncta-localized TRAF domain protein TC1b contributes to the autoimmunity of snc1

Immune receptors play important roles in the perception of pathogens and initiation of immune responses in both plants and animals. Intracellular nucleotide-binding domain leucine-rich repeat (NLR)-type receptors constitute a major class of receptors in vascular plants. In the Arabidopsis thaliana mutant suppressor of npr1-1, constitutive 1 (snc1), a gain-of-function mutation in the NLR gene SNC1 leads to SNC1 overaccumulation and constitutive activation of defense responses. From a CRISPR/Cas9-based reverse genetics screen in the snc1 autoimmune background, we identified that mutations in TRAF CANDIDATE 1b (TC1b), a gene encoding a protein with four tumor necrosis factor receptor-associated factor (TRAF) domains, can suppress snc1 phenotypes. TC1b does not appear to be a general immune regulator as it is not required for defense mediated by other tested immune receptors. TC1b also does not physically associate with SNC1, affect SNC1 accumulation, or affect signaling of the downstream helper NLRs represented by ACTIVATED DISEASE RESISTANCE PROTEIN 1-L2 (ADR1-L2), suggesting that TC1b impacts snc1 autoimmunity in a unique way. TC1b can form oligomers and localizes to punctate structures of unknown function. The puncta localization of TC1b strictly requires its coiled-coil (CC) domain, whereas the functionality of TC1b requires the four TRAF domains in addition to the CC. Overall, we uncovered the TRAF domain protein TC1b as a novel positive contributor to plant immunity

Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells

Arabidopsis SENESCENCE-ASSOCIATED GENE101 Stabilizes and Signals within an ENHANCED DISEASE SUSCEPTIBILITY1 Complex in Plant Innate Immunity

Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1–SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1–PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay