Chromosomal barcodes for simultaneous tracking of near-isogenic bacterial strains in plant microbiota.

DNA-amplicon-based microbiota profiling can estimate species diversity and abundance but cannot resolve genetic differences within individuals of the same species. Here we report the development of modular bacterial tags (MoBacTags) encoding DNA barcodes that enable tracking of near-isogenic bacterial commensals in an array of complex microbiome communities. Chromosomally integrated DNA barcodes are then co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We use this approach to assess the contributions of individual bacterial genes to Arabidopsis thaliana root microbiota establishment with synthetic communities that include MoBacTag-labelled strains of Pseudomonas capeferrum. Results show reduced root colonization for certain mutant strains with defects in gluconic-acid-mediated host immunosuppression, which would not be detected with traditional amplicon sequencing. Our work illustrates how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in the plant microbiota.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Commensal lifestyle regulated by a negative feedback loop between Arabidopsis ROS and the bacterial T2SS.

Despite the plant health-promoting effects of plant microbiota, these assemblages also comprise potentially detrimental microbes. How plant immunity controls its microbiota to promote plant health under these conditions remains largely unknown. We find that commensal bacteria isolated from healthy Arabidopsis plants trigger diverse patterns of reactive oxygen species (ROS) production dependent on the immune receptors and completely on the NADPH oxidase RBOHD that selectively inhibited specific commensals, notably Xanthomonas L148. Through random mutagenesis, we find that L148 gspE, encoding a type II secretion system (T2SS) component, is required for the damaging effects of Xanthomonas L148 on rbohD mutant plants. In planta bacterial transcriptomics reveals that RBOHD suppresses most T2SS gene expression including gspE. L148 colonization protected plants against a bacterial pathogen, when gspE was inhibited by ROS or mutation. Thus, a negative feedback loop between Arabidopsis ROS and the bacterial T2SS tames a potentially detrimental leaf commensal and turns it into a microbe beneficial to the host.

A dominant-negative avirulence effector of the barley powdery mildew fungus provides mechanistic insight into barley MLA immune receptor activation.

Nucleotide-binding leucine-rich repeat receptors (NLRs) recognize pathogen effectors to mediate plant disease resistance often involving host cell death. Effectors escape NLR recognition through polymorphisms, allowing the pathogen to proliferate on previously resistant host plants. The powdery mildew effector AVRA13-1 is recognized by the barley NLR MLA13 and activates host cell death. We demonstrate here that a virulent form of AVRA13, called AVRA13-V2, escapes MLA13 recognition by substituting a serine for a leucine residue at the C-terminus. Counterintuitively, this substitution in AVRA13-V2 resulted in an enhanced MLA13 association and prevented the detection of AVRA13-1 by MLA13. Therefore, AVRA13-V2 is a dominant-negative form of AVRA13 and has probably contributed to the breakdown of Mla13 resistance. Despite this dominant-negative activity, AVRA13-V2 failed to suppress host cell death mediated by the MLA13 autoactive MHD variant. Neither AVRA13-1 nor AVRA13-V2 interacted with the MLA13 autoactive variant, implying that the binding moiety in MLA13 that mediates association with AVRA13-1 is altered after receptor activation. We also show that mutations in the MLA13 coiled-coil domain, which were thought to impair Ca2+ channel activity and NLR function, instead resulted in MLA13 autoactive cell death. Our results constitute an important step to define intermediate receptor conformations during NLR activation.

Structural polymorphisms within a common powdery mildew effector scaffold as a driver of coevolution with cereal immune receptors.

In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7, and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.

Cofunctioning of bacterial exometabolites drives root microbiota establishment.

Soil-dwelling microbes are the principal inoculum for the root microbiota, but our understanding of microbe-microbe interactions in microbiota establishment remains fragmentary. We tested 39,204 binary interbacterial interactions for inhibitory activities in vitro, allowing us to identify taxonomic signatures in bacterial inhibition profiles. Using genetic and metabolomic approaches, we identified the antimicrobial 2,4-diacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites whose combined functions explain most of the inhibitory activity of the strongly antagonistic Pseudomonas brassicacearum R401. Microbiota reconstitution with a core of Arabidopsis thaliana root commensals in the presence of wild-type or mutant strains revealed a root niche-specific cofunction of these exometabolites as root competence determinants and drivers of predictable changes in the root-associated community. In natural environments, both the corresponding biosynthetic operons are enriched in roots, a pattern likely linked to their role as iron sinks, indicating that these cofunctioning exometabolites are adaptive traits contributing to pseudomonad pervasiveness throughout the root microbiota.

Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two.

BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated. RESULTS: We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions. CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

A wheat resistosome defines common principles of immune receptor channels.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.

Barley Ror1 encodes a class XI myosin required for mlo-based broad-spectrum resistance to the fungal powdery mildew pathogen.

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites.

TIR domains of plant immune receptors are 2',3'-cAMP/cGMP synthetases mediating cell death.

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Maize Field Study Reveals Covaried Microbiota and Metabolic Changes in Roots over Plant Growth.

Plant roots are colonized by microorganisms from the surrounding soil that belong to different kingdoms and form a multikingdom microbial community called the root microbiota. Despite their importance for plant growth, the relationship between soil management, the root microbiota, and plant performance remains unknown. Here, we characterize the maize root-associated bacterial, fungal, and oomycetal communities during the vegetative and reproductive growth stages of four maize inbred lines and the pht1;6 phosphate transporter mutant. These plants were grown in two long-term experimental fields under four contrasting soil managements, including phosphate-deficient and -sufficient conditions. We showed that the maize root-associated microbiota is influenced by soil management and changes during host growth stages. We identified stable bacterial and fungal root-associated taxa that persist throughout the host life cycle. These taxa were accompanied by dynamic members that covary with changes in root metabolites. We observed an inverse stable-to-dynamic ratio between root-associated bacterial and fungal communities. We also found a host footprint on the soil biota, characterized by a convergence between soil, rhizosphere, and root bacterial communities during reproductive maize growth. Our study reveals the spatiotemporal dynamics of the maize root-associated microbiota and suggests that the fungal assemblage is less responsive to changes in root metabolites than the bacterial community. IMPORTANCE Plant roots are inhabited by microbial communities called the root microbiota, which supports plant growth and health. We show in a maize field study that the root microbiota consists of stable and dynamic members. The dynamics of the microbial community appear to be driven by changes in the metabolic state of the roots over the life cycle of maize.

Gnotobiotic Plant Systems for Reconstitution and Functional Studies of the Root Microbiota.

Healthy plants host a multi-kingdom community of microbes, which is known as the plant microbiota. Amplicon sequencing technologies for microbial genomic markers were a milestone in revealing the taxonomic composition of the microbiota and its variation associated with a plant host in natural environments. However, this method alone does not allow conclusions to be drawn about functions of these microbial assemblages for the plant. The development of culture collections, which recapitulate natural microbial communities in their diversity, and multiple gnotobiotic plant systems therefore represent a breakthrough in plant-microbiota research such that plants can be inoculated with defined communities to study proposed microbiota functions. These systems provided, for the root microbiota, first insights into mechanisms underlying microbial community establishment and contributions of its microbial members to indirect pathogen protection and mineral nutrition of the host. We argue that the choice of a gnotobiotic system for microbiota reconstitution and subsequent functional analysis depends on the particular plant trait that is influenced by the microbiota. We start by discussing the advantages and limitations of using individual gnotobiotic systems and then describe the general procedures for preparing bacterial cultures from the Arabidopsis thaliana At-R-SPHERE culture collection for inoculation and cocultivation in two gnotobiotic plant growth systems using agar and perlite matrix. Additionally, a protocol for inoculation of plants with opportunistic Pseudomonas pathogens is provided. Lastly, we describe a high-throughput system for visual assessment of roots after inoculation with individual mutants of a transposon library generated from a root-derived bacterial commensal. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of bacterial cultures from At-R-SPHERE Support Protocol 1: Validation of strains by sequencing hypervariable regions of the 16S rRNA gene Basic Protocol 2: Coinoculation of plants grown on an agar matrix with microbial elicitor and a defined microbial community Alternate Protocol: Inoculation of plants cultivated in a perlite-based growth system Support Protocol 2: Surface sterilization of Arabidopsis thaliana seeds Basic Protocol 3: Inoculation using a Pseudomonas opportunistic pathogen Basic Protocol 4: Assessment of commensal-mediated root phenotypes using phytostrips.

A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota.

Powdery mildew is a foliar disease caused by epiphytically growing obligate biotrophic ascomycete fungi. How powdery mildew colonization affects host resident microbial communities locally and systemically remains poorly explored. We performed powdery mildew (Golovinomyces orontii) infection experiments with Arabidopsis thaliana grown in either natural soil or a gnotobiotic system and studied the influence of pathogen invasion into standing natural multi-kingdom or synthetic bacterial communities (SynComs). We found that after infection of soil-grown plants, G. orontii outcompeted numerous resident leaf-associated fungi while fungal community structure in roots remained unaltered. We further detected a significant shift in foliar but not root-associated bacterial communities in this setup. Pre-colonization of germ-free A. thaliana leaves with a bacterial leaf-derived SynCom, followed by G. orontii invasion, induced an overall similar shift in the foliar bacterial microbiota and minor changes in the root-associated bacterial assemblage. However, a standing root-derived SynCom in root samples remained robust against foliar infection with G. orontii. Although pathogen growth was unaffected by the leaf SynCom, fungal infection caused a twofold increase in leaf bacterial load. Our findings indicate that G. orontii infection affects mainly microbial communities in local plant tissue, possibly driven by pathogen-induced changes in source-sink relationships and host immune status.

Host preference and invasiveness of commensal bacteria in the Lotus and Arabidopsis root microbiota.

Roots of different plant species are colonized by bacterial communities, that are distinct even when hosts share the same habitat. It remains unclear to what extent the host actively selects these communities and whether commensals are adapted to a specific plant species. To address this question, we assembled a sequence-indexed bacterial culture collection from roots and nodules of Lotus japonicus that contains representatives of most species previously identified using metagenomics. We analysed taxonomically paired synthetic communities from L. japonicus and Arabidopsis thaliana in a multi-species gnotobiotic system and detected signatures of host preference among commensal bacteria in a community context, but not in mono-associations. Sequential inoculation experiments revealed priority effects during root microbiota assembly, where established communities are resilient to invasion by latecomers, and that host preference of commensal bacteria confers a competitive advantage in their cognate host. Our findings show that host preference in commensal bacteria from diverse taxonomic groups is associated with their invasiveness into standing root-associated communities.

Differential Impact of Plant Secondary Metabolites on the Soil Microbiota.

Plant metabolites can shape the microbial community composition in the soil. Two indole metabolites, benzoxazolinone (BOA) and gramine, produced by different Gramineae species, and quercetin, a flavonoid synthesized by many dicot species, were studied for their impacts on the community structure of field soil bacteria. The three plant metabolites were directly added to agricultural soil over a period of 28 days. Alterations in bacterial composition were monitored by next generation sequencing of 16S rRNA gene PCR products and phospholipid fatty acid analysis. Treatment of the soil with the plant metabolites altered the community composition from phylum to amplicon sequence variant (ASV) level. Alpha diversity was significantly reduced by BOA or quercetin, but not by gramine. BOA treatment caused a decrease of the relative abundance of 11 ASVs, while only 10 ASVs were increased. Gramine or quercetin treatment resulted in the increase in relative abundance of many more ASVs (33 or 38, respectively), most of them belonging to the Proteobacteria. Isolation and characterization of cultivable bacteria indicated an enrichment in Pseudarthrobacter or Pseudomonas strains under BOA/quercetin or BOA/gramine treatments, respectively. Therefore, the effects of the treatments on soil bacteria were characteristic for each metabolite, with BOA exerting a predominantly inhibitory effect, with only few genera being able to proliferate, while gramine and quercetin caused the proliferation of many potentially beneficial strains. As a consequence, BOA or gramine biosynthesis, which have evolved in different barley species, is accompanied with the association of distinct bacterial communities in the soil, presumably after mutual adaptation during evolution.

Coordination of microbe-host homeostasis by crosstalk with plant innate immunity.

Plants grown in natural soil are colonized by phylogenetically structured communities of microbes known as the microbiota. Individual microbes can activate microbe-associated molecular pattern (MAMP)-triggered immunity (MTI), which limits pathogen proliferation but curtails plant growth, a phenomenon known as the growth-defence trade-off. Here, we report that, in monoassociations, 41% (62 out of 151) of taxonomically diverse root bacterial commensals suppress Arabidopsis thaliana root growth inhibition (RGI) triggered by immune-stimulating MAMPs or damage-associated molecular patterns. Amplicon sequencing of bacterial 16S rRNA genes reveals that immune activation alters the profile of synthetic communities (SynComs) comprising RGI-non-suppressive strains, whereas the presence of RGI-suppressive strains attenuates this effect. Root colonization by SynComs with different complexities and RGI-suppressive activities alters the expression of 174 core host genes, with functions related to root development and nutrient transport. Furthermore, RGI-suppressive SynComs specifically downregulate a subset of immune-related genes. Precolonization of plants with RGI-suppressive SynComs, or mutation of one commensal-downregulated transcription factor, MYB15, renders the plants more susceptible to opportunistic Pseudomonas pathogens. Our results suggest that RGI-non-suppressive and RGI-suppressive root commensals modulate host susceptibility to pathogens by either eliciting or dampening MTI responses, respectively. This interplay buffers the plant immune system against pathogen perturbation and defence-associated growth inhibition, ultimately leading to commensal-host homeostasis.

Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research.

The complex structure and function of a plant microbiome are driven by many variables, including the environment, microbe-microbe interactions and host factors. Likewise, resident microbiota can influence many host phenotypes. Gnotobiotic growth systems and controlled environments empower researchers to isolate these variables, and standardized methods equip a global research community to harmonize protocols, replicate experiments and collaborate broadly. We developed two easily constructed peat-based gnotobiotic growth platforms: the FlowPot system and the GnotoPot system. Sterile peat is amenable to colonization by microbiota and supports growth of the model plant Arabidopsis thaliana in the presence or absence of microorganisms. The FlowPot system uniquely allows one to flush the substrate with water, nutrients and/or suspensions of microbiota via an irrigation port, and a mesh retainer allows for the inversion of plants for dip or vacuum infiltration protocols. The irrigation port also facilitates passive drainage, preventing root anoxia. In contrast, the GnotoPot system utilizes a compressed peat pellet, widely used in the horticultural industry. GnotoPot construction has fewer steps and requires less user handling, thereby reducing the risk of contamination. Both protocols take up to 4 d to complete with 4-5 h of hands-on time, including substrate and seed sterilization. In this protocol, we provide detailed assembly and inoculation procedures for the two systems. Both systems are modular, do not require a sterile growth chamber, and cost less than US$2 per vessel.

Gene expression evolution in pattern-triggered immunity within Arabidopsis thaliana and across Brassicaceae species.

Plants recognize surrounding microbes by sensing microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). Despite their significance for microbial control, the evolution of PTI responses remains largely uncharacterized. Here, by employing comparative transcriptomics of six Arabidopsis thaliana accessions and three additional Brassicaceae species to investigate PTI responses, we identified a set of genes that commonly respond to the MAMP flg22 and genes that exhibit species-specific expression signatures. Variation in flg22-triggered transcriptome responses across Brassicaceae species was incongruent with their phylogeny, while expression changes were strongly conserved within A. thaliana. We found the enrichment of WRKY transcription factor binding sites in the 5'-regulatory regions of conserved and species-specific responsive genes, linking the emergence of WRKY-binding sites with the evolution of gene expression patterns during PTI. Our findings advance our understanding of the evolution of the transcriptome during biotic stress.

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.

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.