The leucine-rich repeats in allelic barley MLA immune receptors define specificity towards sequence-unrelated powdery mildew avirulence effectors with a predicted common RNase-like fold

Abstract

Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) in plants can detect avirulence (AVR) effectors of pathogenic microbes. The Mildew locus a (Mla) NLR gene has been shown to confer resistance against diverse fungal pathogens in cereal crops. In barley, Mla has undergone allelic diversification in the host population and confers isolate-specific immunity against the powdery mildew-causing fungal pathogen Blumeria graminis forma specialis hordei (Bgh). We previously isolated the Bgh effectors AVRA1, AVRA7, AVRA9, AVRA13, and allelic AVRA10/AVRA22, which are recognized by matching MLA1, MLA7, MLA9, MLA13, MLA10 and MLA22, respectively. Here, we extend our knowledge of the Bgh effector repertoire by isolating the AVRA6 effector, which belongs to the family of catalytically inactive RNase-Like Proteins expressed in Haustoria (RALPHs). Using structural prediction, we also identified RNase-like folds in AVRA1, AVRA7, AVRA10/AVRA22, and AVRA13, suggesting that allelic MLA recognition specificities could detect structurally related avirulence effectors. To better understand the mechanism underlying the recognition of effectors by MLAs, we deployed chimeric MLA1 and MLA6, as well as chimeric MLA10 and MLA22 receptors in plant co-expression assays, which showed that the recognition specificity for AVRA1 and AVRA6 as well as allelic AVRA10 and AVRA22 is largely determined by the receptors’ C-terminal leucine-rich repeats (LRRs). The design of avirulence effector hybrids allowed us to identify four specific AVRA10 and five specific AVRA22 aa residues that are necessary to confer MLA10- and MLA22-specific recognition, respectively. This suggests that the MLA LRR mediates isolate-specific recognition of structurally related AVRA effectors. Thus, functional diversification of multi-allelic MLA receptors may be driven by a common structural effector scaffold, which could be facilitated by proliferation of the RALPH effector family in the pathogen genome.

Identification of specificity‐defining amino acids of the wheat immune receptor Pm2 and powdery mildew effector AvrPm2

“…For example, for Pm3a with an amino acid exchange in the C‐terminal LRR27 the Pm3a‐δ5 mutant showed a lower activation threshold than the natural Pm3a and an expanded recognition spectrum towards naturally occurring AvrPm3 A2/F2 variants (Lindner et al ., 2020). The LRR swaps between the barley Mla1 and Mla6 (Shen et al ., 2003; Bauer et al ., 2021), as well as swaps between Mla10 and Mla22 (Bauer et al ., 2021) demonstrated that the effector recognition specificities are determined by the C‐terminal LRRs of the Mla proteins. The LRR swaps between either the flax L6 and L11 or L5 and L6 variants revealed that the last four LRRs of L6 are required for the recognition of the flax rust effector AvrL567‐D (Ellis et al ., 2007; Ravensdale et al ., 2012).…”

“…For instance, a mutagenesis study on AvrL567 using amino acid polymorphisms across the allelic variants led to the identification of three amino acids involved in the recognition by the L proteins and suggested that the specificity is a result of the additive effect of multiple contact points with the L protein (Dodds et al ., 2006; Ravensdale et al ., 2012). Chimeric effectors between Avra10 and Avra22 demonstrated that four (Avra10) and five (Avra22) amino acids are necessary to confer Mla10‐ and Mla22‐specific recognition, respectively (Bauer et al ., 2021). These findings suggest that multiple amino acids on the Avra effector surface determine the recognition specificities of Mla proteins.…”

The Powdery Mildew Effector CSEP0027 Interacts With Barley Catalase to Regulate Host Immunity

“…A large proportion of Bgh CSEPs (c. 25%) are structurally predicted similar to RNase and/or RNA-binding activity, and these CSEPs are termed as RNase Like Proteins expressed in Haustoria (RALPHs) ( Pedersen et al, 2012 ; Spanu, 2017 ). Interestingly, most of the so far identified Bgh AVR A effectors, each recognized by a cognate barley MLA receptor, are also RALPHs with fungal RNase folds but lacking the residues required for RNase activity ( Lu et al, 2016 ; Saur et al, 2019 ; Bauer et al, 2021 ). So far, several Bgh CSEPs/BECs have been functionally characterized with respect to fungal virulence through transient gene expression and host-induced gene silencing (HIGS) approaches ( Bindschedler et al, 2009 ; Godfrey et al, 2009 ; Nowara et al, 2010 ; Spanu et al, 2010 ; Pedersen et al, 2012 ; Pliego et al, 2013 ; Ahmed et al, 2015 , 2016 ; Menardo et al, 2017 ; Frantzeskakis et al, 2018 ; Pennington et al, 2019 ; Li et al, 2021 ).…”

An interolog-based barley interactome as an integration framework for immune signaling

“…In the interaction between Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew, and its cereal host plant, barley (Hordeum vulgare L.), disease is blocked by the action of specific barley R-proteins, designated ML, that respond to corresponding Bgh effectors, designated AVR (Lu et al, 2016;Saur et al, 2019;Bauer et al, 2021;Ridout et al, 2006). Diversification of the Mla NLR (Wei et al, 2002;Seeholzer et al, 2010), has generated up to 30 allelic variants that confer specific recognition to different AVR-associated Bgh isolates (Lu et al, 2016).…”