The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4—a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.

NLRs are modular plant and animal proteins that are intracellular sensors of pathogenassociated molecules that trigger a potent broad-spectrum immune reaction known as the hypersensitive response. An emerging paradigm is that plant NLRs form immune signalling networks with varying degrees of complexity. NLRs may have evolved from multifunctional singleton receptors, which combine pathogen detection (sensor activity) and immune signalling (helper or executor activity) into a single protein, to functionally specialized interconnected receptor pairs and networks. In this article, we highlight some of the recent advances in plant NLR biology by discussing models of NLR evolution, NLR complex formation, and how NLR (mis)regulation modulates immunity and autoimmunity. Multidisciplinary approaches are required to dissect the evolution, assembly and regulation of the immune receptor circuitry of plants. Recent progress in understanding plant immunity revealed that immune receptors, including NLRs, can form networks of varying complexity that translate pathogen detection into immune signalling (Wu et al., 2018). However, despite notable advances in understanding how plant NLRs detect pathogen effectors, other aspects of NLR biology, such as mechanisms of NLR activation and how these activities are regulated at multiple levels, remain largely unknown. In this review, we highlight some of the recent advances in plant NLR biology by discussing models of NLR evolution and functional specialization, NLR complex formation, and how NLR regulation modulates immunity and autoimmunity. NLR evolution: from singleton to pairs to network The conceptual basis of host-pathogen interactions was first developed by Harold Henry Flor in his gene-for-gene model. Flor's model postulated that inheritance of resistance and parasitism is determined by matching single genes in plants and pathogens (Flor, 1971). Fifty years after Flor proposed his gene for gene model, the first R-gene in plants were cloned (Kourelis and van der Hoorn, 2018). To date dozens of R genes have been cloned and remarkably the majority of these R genes encode NLR proteins. True to Flor's model, some NLRs indeed function as a single genetic unit for sensing and signalling, referred to here as "singleton NLR" (Figure 1, Table 1). These NLRs sense effectors either directly or indirectly and trigger the so-called hypersensitive immune response in the host. MILDEW LOCUS A (MLA) protein family, found broadly across different barley accessions, is a well-studied NLR locus required for isolate-specific resistance to powdery mildew fungi (Maekawa et al., 2018). MLA recognizes matching avirulence effector, AVRa, in the heterologous Arabidopsis system, suggesting that it probably behaves both as a sensor and as a signal inducer (Lu et al., 2016). Other likely singleton NLRs that sense and trigger an immune response in heterologous plant systems include Sr50, a rye ortholog of MLA, L6, a flax NLR, RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) and HOPZ-ACTIVATED RESISTANCE1 (ZAR1),