Abstract
In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function.
Highlights
A critical step in the lifestyles of plant pathogens is the secretion of effectors—pathogen-encoded proteins that are translocated into the plant cell, where they manipulate the host and promote pathogen growth [1]
Plants defend themselves against pathogens using specific multi-domain immune receptors, which are able to recognize secreted “effector” proteins from the pathogen, and activate an immune response
We identified mutations in an unrecognized ATR1 allele that lead to allelespecific recognition by RPP1
Summary
A critical step in the lifestyles of plant pathogens is the secretion of effectors—pathogen-encoded proteins that are translocated into the plant cell, where they manipulate the host and promote pathogen growth [1]. Many effectors function to modulate basal immunity, but their presence in the plant cell may betray the pathogen and activate a second layer of effector-triggered immunity (ETI) if recognized by intracellular host immune receptors [2,3,4]. Well-studied plant NLRs, such as RPS2 and RPM1 from Arabidopsis, recognize effectors indirectly [6,7,8]. These NLRs are activated not by association with effectors themselves, but instead by recognizing their biochemical effects in the plant cell, leading to models of NLR activation in which the receptors recognize perturbation of “guarded” host proteins. NLRs including RPP1, L6, Pi-ta, and others interact directly with recognized effector alleles, suggesting a second model in which direct effector-NLR interaction is required for immune activation [11,12,13,14,15]
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