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 AVR _A1, AVR _A7, AVR _A9, AVR _A13, and allelic AVR _A10/AVR _A22, 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 AVR _A6 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 AVR _A1, AVR _A7, AVR _A10/AVR _A22, and AVR _A13, 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 AVR _A1 and AVR _A6 as well as allelic AVR _A10 and AVR _A22 is largely determined by the receptors’ C-terminal leucine-rich repeats (LRRs). The design of avirulence effector hybrids allowed us to identify four specific AVR _A10 and five specific AVR _A22 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 AVR _A 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.

Author summary

Barley powdery mildew caused by the fungus Blumeria graminis forma specialis hordei ( Bgh) can result in annual yield losses of 15% of this cereal crop. Bgh promotes virulence in plants through the secretion of diverse effector molecules, small proteins of which a subset enters into and modifies the immune status and physiology of the host leaf. In response, the host has evolved a multitude of disease resistance genes. The Mildew locus a ( Mla) resistance gene stands out because diversification in the host population has generated numerous Mla variants encoding multi-domain receptors, each of which can directly recognize an isolate-specific Bgh effector, designated as avirulence (AVR _A) effectors. Recognition of AVR _A effectors by MLA triggers plant immune responses, a phenomenon known as isolate-specific resistance, which invariably results in localized host cell death. Here, we identify the powdery mildew effector AVR _A6 and validate its specific interaction with its matching receptor MLA6. Furthermore, through the use of hybrid receptors constructed from MLA1 and MLA6 as well as MLA10 and MLA22 receptors, we provide insights into the specific domains and amino acid residues generally important for AVR _A recognition by MLA receptors. We find that sequence variation in the leucine-rich repeats (LRRs) of multi-allelic MLA receptors determines specific recognition of AVR _A effectors. These effectors are sequence-unrelated, but our analysis indicates that they may be structurally related. This data may assist in the future generation of synthetic immune receptors with pre-defined recognition specificities.