Biochemical Basis of Autoactivity of a Pair of Plant NLR Immune Receptors

Abstract

An immune system enables organisms to defend themselves against a myriad of pathogens and diseases. Plants, which can rely only on innate immunity, have evolved different types of receptors to detect pathogens. Transmembrane receptors can recognize pathogenic conserved structures called pathogen- associated molecular patterns (PAMPs) such as flagellin, leading to PAMP-triggered immunity (PTI). Intracellular receptors recognize specific molecules delivered by pathogens into plant cells, known as effectors, and activate effector-triggered immunity (ETI) when PTI is overcome. Most of the ETI-mediating intracellular receptors belong to the NLR protein family characterized by a nucleotide-binding domain and leucine-rich repeats. NLRs perceive matching effectors, through either physical binding to the effector (direct recognition) or by sensing effector-induced biochemical modifications of a host target (indirect recognition). NLR-dependent signaling often leads to a hypersensitive response (HR), featured by localized cell death at the site of the infection, which stops pathogen spread and disease development. Mis-regulation of NLRs in the absence of pathogens can lead to inappropriate responses of the immune system, known as autoimmunity, causing spontaneous cell death, necrotic lesions and developmental defects. Autoimmunity can occasionally be observed in hybrid plants. This type of hybrid weakness can result from deleterious epistatic interactions between NLR genes from the two parents. In this thesis, I investigated the biochemical mechanism of autoimmunity in hybrids of two natural Arabidopsis thaliana accessions from Umkirch (Southwestern Germany). The two causal genes involved, DM1 (DANGEROUS MIX 1) from Uk-3 and DM2 from Uk-1, both encode NLRs. The causal DM2 variant is located in a multi-gene cluster with diverse NLR members, while DM1 is a single-gene NLR locus. In this study,I showed that signaling mediated by DM1 and DM2 uses the same pathway that other plant NLRs deploy upon non-self recognition. Cell death signaling induced by DM1 and DM2 involves heteromeric association of both proteins through their N-terminal regions including TIR domains, with DM1 forming inactive homo-oligomers in the absence of DM2. Mutations in the P-loop of either DM1 or DM2 suppressed HR, indicating the 1 contribution of both proteins to signaling. The contributions of the two NLRs to downstream signaling are, however, not symmetrical. Mutations in an NLR signature motif that are likely to affect conformation around the ATP binding pocket greatly change the activity of only DM2. Taken together, my results suggest that DM1 acts primarily as a signal transducer, and DM2 as a signal trigger. Autoimmunity triggered by joint action of this NLR pair thus suggests that the activity of the signaling complex depends on the sum of the complementary activities of the partner NLRs. Knowledge of the biochemical basis of autoactivity induced by plant NLR pairs will help us to understand how plant autoimmunity arises through NLR interaction, and how NLR activity is regulated to avoid inappropriate activation to minimize the plant fitness cost in the absence of pathogens.