Abstract
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.
Highlights
Plants have evolved intracellular immune receptors to detect host-translocated pathogen virulence proteins, known as effectors (Dodds and Rathjen, 2010; Jones et al, 2016; Kourelis and van der Hoorn, 2018)
Mu mutagenesis of NRC4 reveals a short 29 amino acid N-terminal region that is sufficient for induction of hypersensitive response (HR) cell death
Introducing the L9E in NRC41-29-yellow fluorescent protein (YFP) greatly reduced puncta formation (Figure 9A,C). This finding directly connects puncta formation to the activity of full length NRC4 given that L9E affects NRC4 cell death activity (Figure 8). These results indicate that both an intact MADA motif and YFP oligomerization are required for the capacity of NRC41-29-YFP to form puncta as well as cause cell death in N. benthamiana leaves
Summary
Plants have evolved intracellular immune receptors to detect host-translocated pathogen virulence proteins, known as effectors (Dodds and Rathjen, 2010; Jones et al, 2016; Kourelis and van der Hoorn, 2018). These receptors, encoded by disease resistance (R) genes, are primarily nucleotidebinding, leucine-rich repeat proteins (NLRs). NLRs have probably 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 (Adachi et al, 2019a). Dozens of NLR proteins have been subject to functional studies since their discovery in the 1990 s, this body of knowledge has not been interpreted through an evolutionary biology framework that combines molecular mechanisms with phylogenetics
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