Evolutionary gain and loss of a plant pattern-recognition receptor for HAMP recognition

Abstract

As a first step in innate immunity, pattern recognition receptors (PRRs) recognize distinct pathogen and herbivore-associated molecular patterns and mediate activation of immune responses, but specific steps in the evolution of new PRR sensing functions are not well understood. We employed comparative genomic and functional analyses to define evolutionary events leading to the sensing of the herbivore-associated peptide inceptin (In11) by the PRR Inceptin Receptor (INR) in legume plant species. Existing and de novo genome assemblies revealed that the presence of a functional INR gene corresponded with ability to respond to In11 across ~53 million years (my) of evolution. In11 recognition is unique to the clade of Phaseoloid legumes, and only a single clade of INR homologues from Phaseoloids was functional in a heterologous model. The syntenic loci of several non-Phaseoloid outgroup species nonetheless contain non-functional INR-like homologues, suggesting that an ancestral gene insertion event and diversification preceded the evolution of a specific INR receptor function ~28 mya. Chimeric and ancestrally reconstructed receptors indicated that 16 amino acid differences in the C1 leucine-rich repeat domain and C2 intervening motif mediate gain of In11 recognition. Thus, high PRR diversity was likely followed by a small number of mutations to expand innate immune recognition to a novel peptide elicitor. Analysis of INR evolution provides a model for functional diversification of other germline-encoded PRRs.

Plant Receptor-like proteins (RLPs): Structural features enabling versatile immune recognition

“…Third, an ancestral sequence reconstruction approach applied to functional and non-functional homologues of the LRR-RLP INR pinpointed a single Tyr residue in the NT loopout that is strongly conserved within all identified INR homologues from 11 legume species. An ancestral INR mutant with an AA substitution at this site resulted in a relatively lower ROS burst [24]. In summary, there is evidence for a crucial role of the NT loopout for several LRR-RLPs, and the observed variation across NT loopouts suggests a function in ligand specificity.…”

“…Genetic studies of LRR-RLPs RLP42, INR and RXEG1 provide evidence for the importance of the ID for LRR-RLP function [21,23,24,37]. Chimeric receptors with a non-functional paralog demonstrated a requirement of the ID for maintaining the function of INR [24]. Similarly for Ve1 and RLP42, chimeric receptors showed the requirement of the ID for specificity (Fradin 2014, Zhang 2021).…”

“…The ID is always positioned N-terminal to the last set of exactly four LRRs, termed C3. Genetic studies of LRR-RLPs RLP42, INR and RXEG1 provide evidence for the importance of the ID for LRR-RLP function [21,23,24,37]. Chimeric receptors with a non-functional paralog demonstrated a requirement of the ID for maintaining the function of INR [24].…”

“…Many individual RLP42→RLP40 substitutions located within these LRRs were also sufficient to abolish function. Cowpea INR and INR-like 1 chimeric receptors similarly revealed the necessity of the C1 domain for INR function, and two AA residues in LRR 14 (H404 and R406) were found to be crucial for INR function through an ancestral sequence reconstruction approach [24]. Finally, the RXEG1 cryoEM structure indicates potential interaction between two C1 domain residues, C247 and N674, with the XEG1 ligand, although this was not further analyzed [21].…”

“…LRR-RLPs and their respective ligands have been characterized in diverse species such as Arabidopsis, tomato, wild tobacco, canola and cowpea and detect diverse molecular patterns from short AA peptides [16,17] to specific features of protein structures, for example the active site groove of fungal XEG1 and the tertiary fold of bacterial translation initiation factor-1 (IF1) [21,22]. A recent breakthrough description of the cryo-EM structure of the LRR-RLP RXEG1, as well as recent functional studies of RLP42 and INR function [21,23,24], motivates a new overview of LRR-RLP structure and function.…”