Decision letter: Multiple pairs of allelic MLA immune receptor-powdery mildew AVRA effectors argue for a direct recognition mechanism

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins in plants and animals mediate intracellular pathogen sensing. Plant NLRs typically detect strain-specific pathogen effectors and trigger immune responses often linked to localized host cell death. The barley Mla disease resistance locus has undergone extensive functional diversification in the host population and encodes numerous allelic NLRs each detecting a matching isolate-specific avirulence effector (AVRA) of the fungal pathogen Blumeria graminis f. sp. hordei (Bgh). We report here the isolation of Bgh AVRa7, AVRa9, AVRa10, and AVRa22, which encode small secreted proteins recognized by allelic MLA7, MLA9, MLA10, and MLA22 receptors, respectively. These effectors are sequence-unrelated, except for allelic AVRa10 and AVRa22 that are co-maintained in pathogen populations in the form of a balanced polymorphism. Contrary to numerous examples of indirect recognition of bacterial effectors by plant NLRs, co-expression experiments with matching Mla-AVRa pairs indicate direct detection of the sequence-unrelated fungal effectors by MLA receptors. https://doi.org/10.7554/eLife.44471.001 eLife digest Powdery mildews are fungal diseases that affect many plants, including important crops such as barley. The fungi behind these diseases deliver molecules known as effectors inside plant cells, which manipulate the plants' biology and help the fungus to invade the plants' tissues. In response, some plants have evolved immune receptors encoded by so-called R genes (short for resistance genes) that detect the effectors inside the plant cell and trigger an immune response. The response often kills the plant cell and those nearby to limit the spread of the fungus. Effectors that are recognized by host immune receptors are termed avirulence effectors (or AVRs for short). Scientists tend to assume that most effectors do not bind directly to their immune receptors. Instead, it is thought that the immune receptors are more likely to be detecting a change in some other plant protein that is caused by the effectors' activities. In barley populations, one R gene that protects against powdery mildew encodes an immune receptor known as MLA. Different plants can carry subtly different versions of this R gene meaning that they make similar but different variants of the same receptor. Each MLA variant confers immunity only to strains of powdery mildew that carry the matching AVR effector. A few AVR effectors from powdery mildews have been identified, but most AVR effectors from powdery mildews remain unknown. Saur et al. looked for new AVR effectors from powdery mildew fungi collected in the field, and found four that were recognized by barley plants carrying MLA variants. Two of these new effectors were fairly similar to each other, but they were all unlike those that had been identified previously. When Saur et al. engineered barley cells to make these new AVRs alongside their matching MLA receptors, the cells died – which is consistent with the expected immune response. Similar experiments with distantly related tobacco plants agave the same results. This suggested that the immune receptors did not need any other barley proteins to recognize the effectors, indicating that the interaction between the two may be direct. Indeed, two other techniques that test for direct protein-protein interactions, – one that involved extracts from tobacco leaves, and another that involved yeast, – gave results consistent with a direct interaction between the MLA receptor variants and the fungal effectors. Plant disease is still a major cause of loss of yield in crops. Transferring an R gene from one plant species to another is a potentially powerful approach to help crops resist disease. The discovery that multiple variants of the same resistance gene can bind to dissimilar effectors from a disease-causing fungus in distantly related plant species underlines the potential of this approach. https://doi.org/10.7554/eLife.44471.002 Introduction The NLR family of immune receptors is structurally conserved between animals and plants, perceives non-self and modified-self molecules inside host cells, and mounts potent innate immune responses to terminate microbial pathogenesis (Maekawa et al., 2011a). Animal NLRs are normally activated by conserved microbe- or damage-associated molecular patterns (MAMPs/DAMPs), whereas plant NLRs typically detect strain-specific pathogen effectors, designated avirulence effector proteins (AVRs) (Maekawa et al., 2011a; Jones et al., 2016). Plant NLRs recognize either the effector structure or sense effector-mediated modifications of host proteins (Jones et al., 2016). Single plant NLRs or NLR pairs can also detect the manipulation of effector target mimics, called decoys, and a variation of the latter mechanism involves the direct integration of decoy domains mimicking host targets within NLRs, termed integrated decoy domains (Jones et al., 2016; Cesari et al., 2014; Kroj et al., 2016). The selective forces shaping the evolution of the comparatively small complement of NLRs in vertebrates are incompletely understood (~20 family members, (Lange et al., 2011; Meunier and Broz, 2017)), but in plant species co-evolution with host-adapted pathogens has strongly influenced the expansion and diversification of NLR repertoires (Jacob et al., 2013; Meyers et al., 2005). Plant disease resistance (R) genes to host-adapted pathogens often encode NLRs, are frequently members of large gene families organized in complex clusters of paralogous genes, and can rapidly evolve through a range of natural gene diversification mechanisms (Jacob et al., 2013; Meyers et al., 2005). There are several examples of allelic series of NLR-type R genes known in plants (Ellis et al., 1999; Allen et al., 2004; Srichumpa et al., 2005; Seeholzer et al., 2010; Kanzaki et al., 2012). In these cases, multiple distinct recognition specificities evolved in the host population at a single R gene with each allele detecting a corresponding strain-specific AVR in the pathogen population. Such multi-allelic NLR-type R genes are particularly instructive for studying the underlying co-evolutionary process between host and pathogen. Ascomycete powdery mildews are widespread pathogens of thousands of angiosperm plant species in temperate climates, including economically relevant crops (Glawe, 2008). They are obligate biotrophic pathogens, meaning that their growth and reproduction is entirely dependent on living host cells. The haploid barley powdery mildew pathogen Blumeria graminis forma specialis hordei (Bgh) multiplies mainly clonally and is a member of the species Blumeria graminis that is specialized for its host plant barley (Hordeum vulgare). There are various specialized forms (formae speciales or fs. spp.) of B. graminis, each of which is capable of infecting the respective host plant species belonging to the grass (Poaceae) family, including cereals such as barley and wheat (Wyand and Brown, 2003). Within each forma specialis, numerous isolates (strains) can be distinguished in the pathogen population, based on their respective virulence or avirulence infection phenotypes vis-à-vis particular genotypes of the host population (Lu et al., 2016). The genomes of powdery mildews are characterized by the loss of several, otherwise widely conserved Ascomycete genes with functions related to carbohydrate degradation and primary and secondary metabolism (Spanu et al., 2010; Wicker et al., 2013), and this is believed to explain their strict dependence on living plant cells. Similar to other filamentous phytopathogens, grass-infecting powdery mildew genomes harbor hundreds of candidate secreted effector protein (CSEP)-coding genes, which are assumed to contribute to fungal pathogenesis (Wicker et al., 2013; Pedersen et al., 2012; Guttman et al., 2014). Pathogen effectors often work by subverting innate immune responses, thereby facilitating host colonization and disease (Rovenich et al., 2014). Domesticated barley and wheat contain numerous powdery mildew R gene loci that were often introgressed from their corresponding wild relatives (Jørgensen and Wolfe, 1994; Lutz et al., 1995; Maekawa et al., 2019). In both barley and its close relative wheat, one of these powdery mildew R loci, designated mildew locus a (Mla) and powdery mildew 3 (Pm3), respectively, has been subject to exceptional functional diversification, resulting in large numbers of Mla or Pm3 recognition specificities (Seeholzer et al., 2010; Lutz et al., 1995; Bhullar et al., 2010). Although wheat Pm3 and barley Mla loci each span a cluster of NLR genes, known Pm3 and Mla recognition specificities to the B. graminis pathogen appear to have arisen from allelic diversification of a single NLR gene in the corresponding NLR clusters (Seeholzer et al., 2010; Maekawa et al., 2019; Shen, 2003). Isolate-specific disease resistance to Bgh mediated by MLA receptors is invariably linked to the activation of localized host cell death, and this immune response likely terminates growth of the biotrophic pathogen by shutting off its nutrient supply (Boyd et al., 1995). Of note, the Mla orthologs Sr33 in wheat (Periyannan et al., 2013) and Sr50 in rye (Mago et al., 2015) confer disease resistance to the stem rust pathogen Puccinia graminis f. sp. tritici (Pgt) isolate Ug99, a major threat to global wheat production. Pgt and Bgh belong to the Basidiomycota and Ascomycota phyla, respectively, indicating that MLA receptors can detect the presence of independently evolved avirulence effectors. Barley Mla and wheat Pm3 both encode intracellular NLRs with an N-terminal coiled-coil (CC) domain but lack significant sequence relatedness (Zhou et al., 2001), whereas 23 allelic barley MLA resistance proteins exhibit >91% amino acid (aa) sequence identity (Seeholzer et al., 2010), and 17 deduced allelic wheat Pm3 receptors share >97% aa sequence identity (Bhullar et al., 2010). Diversifying selection among resistance alleles of Mla and Pm3 is largely confined to regions encoding the C-terminal leucine-rich repeats (LRR) of the receptors (Seeholzer et al., 2010; Maekawa et al., 2019; Bhullar et al., 2010). The polymorphic MLA LRR is critical for effector detection as shown by a series of reciprocal domain swaps between MLA1 and MLA6 (Shen, 2003). Barley Mla1 confers race-specific disease resistance to a Bgh isolate carrying the cognate avirulence gene AVRa1 in transgenic Arabidopsis thaliana, suggesting ∼ 150 million years of evolutionary conservation of the underlying immune mechanism and potentially pointing to a direct recognition mechanism of AVRA effectors that is highly conserved between barley and A. thaliana (Maekawa et al., 2012). Recently, the Bgt avirulence gene AvrPm3a2/f2, recognized by the wheat Pm3a and Pm3f alleles, was identified by a map-based cloning approach and found to encode a typical CSEP that belongs to an effector family of 24 members (Bourras et al., 2015). The clonal nature of the haploid Bgh pathogen facilitated the identification of isolate-specific sequence variation in the transcriptomes of a global collection of 17 Bgh strains. These were associated with Bgh pathotypes on Mla1- and Mla13-containing near-isogenic barley lines to identify sequence-unrelated AVRa1- and AVRa13-encoding CSEPs (Lu et al., 2016). However, it remains unclear whether these powdery mildew avirulence effectors bind directly to the corresponding NLR receptors or whether the receptors sense the presence of the pathogen indirectly through effector-mediated modifications of host proteins. To reveal the molecular mechanism underlying the co-evolutionary diversification of barley MLA NLRs with host-adapted Bgh, we first described the natural AVRa gene diversity in a local Bgh population. This allowed us to isolate four additional AVRA effectors by applying a transcriptome-wide association study (TWAS) with high-confidence single nucleotide polymorphisms (SNPs) underlying disease resistance/susceptibility phenotypes in barley. We then employed co-transfection experiments to show that only matching Mla-AVRa pairs activate cell death in barley leaf protoplasts. Agrobacterium tumefaciens-mediated co-delivery of Mla-AVRa pairs in Nicotiana benthamiana leaves demonstrated that their co-expression is sufficient to trigger cell death in this heterologous plant species. The AVRa avirulence effectors encode CSEPs that are sequence-unrelated except for allelic AVRA10 and AVRA22. These findings are inconsistent with a co-evolutionary arms race model describing iterative cycles of MLA receptor and AVRA effector adaptations. Moreover, we failed to detect cell death activity for the previously reported EKA_AVRa10, a member of the EKA gene family that is derived from part of a class-1 LINE retrotransposon (Ridout, 2006; Amselem et al., 2015). Using co-expression experiments with matching Mla-AVRa pairs, including previously reported AVRa13, we present evidence for direct receptor-avirulence effector interactions. Our findings imply that MLA receptors have an exceptional propensity to directly detect sequence-unrelated and likely structurally diverse pathogen effectors and that this feature might have facilitated the functional diversification of the receptor in the host population. Results Natural variation of AVRa genes within a local Bgh population In 2016 we collected 13 Bgh field isolates from leaves of barley plants within the same locality near Cologne, Germany (GPS 5˚57′N, 6˚51′E 5; isolates designated K2, K3, K4, S11, S15, S16, S19, S20, S21, S22, S23, S25 and S26). We purified the isolates by serial propagation of conidiospores collected from single powdery mildew colonies (Materials and methods). Then, we determined their pathotypes (virulence/avirulence profiles) together with a global collection of 17 previously described Bgh isolates (Lu et al., 2016) on a panel of near-isogenic barley lines (NILs) in the cultivar (cv.) Pallas genetic background (Kolster et al., 1986) carrying Mla1 (P01), Mla3 (P02), Mla6 (P03), Mla7 (P04B), Mla9 (P08B), Mla10 (P09), Mla12 (P10), Mla22 (P12), or Mla23 (P13) recognition specificities (Supplementary file 1). To assess infection types (ITs) independently, we additionally employed a panel of NILs in the genetic background of cv. Manchuria (Moseman, 1972) carrying introgressed Mla1 (CI16137), Mla6 (CI16151), Mla7 (CI16147 and CI16153), Mla10 (CI16149), or Mla13 (CI16155) (Supplementary file 1). Similar to previous studies (Seeholzer et al., 2010; Lu et al., 2016), we scored five macroscopically distinguishable ITs at 9 d after conidiospore inoculation on leaves, ranging from 1 to 3 (=avirulent) or 4 to 5 (=virulent). Unambiguous assignment of virulent and avirulent interactions was possible for all isolates on Mla1, Mla6, Mla9, Mla12, Mla13, and Mla22 NILs that displayed only IT 1, 2 or 5. A subset of the Bgh strains displayed IT 3 (avirulent) or 4 (virulent) on Mla3, Mla7, Mla10, and Mla23 NILs, which were in some cases difficult to distinguish from each other and thus potentially complicating the discrimination of avirulent and virulent interactions with our experimental setup (Supplementary file 1). For two of the tested Bgh isolates different avirulent/virulent interactions were recorded between Pallas and Manchuria NILs carrying Mla7 (isolates K4 and S22) and, similarly, two other isolates exhibited different avirulent/virulent interactions on Pallas and Manchuria NILs carrying Mla10 (isolates CC88 and S20; Supplementary file 1). These few discrepancies of avirulent/virulent interactions might be explained by differences in the size of introgressed chromosomal segments from the respective donor lines in Pallas and Manchuria NILs that might contain other R genes besides Mla. Therefore, we omitted the IT data of isolates K4 and S22 on Mla7 NILs and of CC88 and S20 isolates on Mla10 NILs in the TWAS (see below) to identify AVRa7 and AVRa10 candidates, respectively. Among the 13 newly described Bgh isolates collected from the same geographic site, isolates S19 and S21, as well as S23, S25, and S26 exhibited identical avirulence/virulence patterns on the NIL test panel (Supplementary file 1). Thus, 10 out of 13 isolates showed unique avirulece/virulence patterns and each isolate carries at least five different AVRa genes (Supplementary file 1). These findings point to a high level of genetic variation among AVRa genes within the local Bgh population. We obtained deep fungal transcriptomes (RNA-Seq) for the ten Cologne-derived field isolates with unique avirulent/virulent patterns during pathogenesis on susceptible barley leaves at 16 and 48 hr post conidiospore inoculation, resulting in 0.8 to 6 million sequenced and mapped Bgh read pairs (fragments) per sample (Materials and methods). RNA-seq reads of these isolates, combined with RNA-seq reads of the previously characterized global collection of Bgh isolates (Lu et al., 2016) and of reference isolate DH14, were aligned against the recently assembled near chromosome-level DH14 genome supplemented with updated gene models (Frantzeskakis et al., 2018). Subsequently, a collective set of sequence polymorphisms for all isolates was identified from the combined alignment data. To assess the potential influence of population structure among the isolates prior to TWAS, we performed a population structure analysis, for which we extracted a set of high-quality diallelic and synonymous single-nucleotide polymorphisms (SNPs) from the complete set of polymorphisms (Methods). A principal component analysis (PCA) plot and a neighbour-joining tree including all isolates confirmed the Japanese isolate RACE1 to be exceptionally divergent and assigned the three Australian isolates to a distinct clade, whereas the ten new field isolates clustered mostly together with other European, Japanese, and USA isolates (Figure 1A, Figure 1—figure supplement 1). Although all newly described isolates were collected from a single geographic location, three of these (K2, K3, and S16) appear to form a subgroup that is distinct from the other European isolates (Figure 1A, Figure 1—figure supplement 1). Interestingly, the avirulence profiles of isolates K3 and S16 on the 11 tested Mla barley recognition specificities belong to distinct pathotype clusters (Figure 1B), despite an exceptionally high similarity of K3 and S16 at the transcriptome level (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset Population structure and avirulence profiles of Blumeria graminis f. sp. hordei (Bgh) isolates. (A) Principal Component Analysis (PCA) of the indicated European Bgh isolates, including ten newly collected strains from a local pathogen population in Germany, based on a set of 5170 diallelic high-quality synonymous SNPs. (B) Hierarchical clustering (R package 'pheatmap') of avirulence profiles from 14 Bgh isolates collected within the same area near Cologne, Germany (GPS 5˚57'N, 6˚51'E 5). Numbers correspond to infection types (ITs) 1, 2 and 3 = avirulent, red; infection types 4 and 5 = virulent, green. *denotes differences of ITs between cultivars Pallas and Manchuria, yellow. n.t.: not tested, white. https://doi.org/10.7554/eLife.44471.003 Combined TWAS and Bgh genome analysis identified candidates for AVRa7, AVRa9, AVRa10, and AVRa22 Our TWAS analysis (Supplementary file 2 - 4) allowed us to identify several AVRa candidates. Manual re-inspection of these candidates (Lu et al., 2016), revealed that only transcript polymorphisms of the top candidates AVRa7, AVRa9, AVRa10, and AVRa22 exhibit tight linkage to the avirulence phenotypes of our Bgh strain panel on Mla7, Mla9, Mla10, and Mla22 NILs, respectively. Two genes, BLGH_06689 and BLGH_06672, encoding two identical of were identified as candidates for AVRa7 Supplementary file This is consistent with previous in which two AVRa7 loci were based on analysis among the of between Bgh strains et al., Pedersen et al., In the near chromosome-level genome of isolate DH14 (Frantzeskakis et al., the two are by on and, together with four other form a CSEP cluster in this (Figure We designated the two identical in DH14 as candidate gene (Figure of the DH14 CSEPs in the cluster is a which at the protein level aa identity with (Figure and virulent strains on Mla7 NILs carry a in which results in a in the deduced candidate effector and the corresponding variant was thus (Figure and Interestingly, only of the from the virulent isolate harbor a to the in the deduced The other of the harbor a resulting in a in the deduced protein suggesting that in this strain the two of the transcript reads of all three avirulent Australian isolates and harbor a to an in the deduced protein Figure and suggesting that these isolates also contain an of the most divergent Bgh isolate RACE1 is avirulent on Mla7 NILs (Supplementary file and in its transcriptome we found two in all corresponding to and aa in the deduced protein Figure and of the near chromosome-level RACE1 genome (Frantzeskakis et al., revealed two identical gene and by on a single within a CSEP cluster that is largely with the corresponding DH14 CSEP cluster (Figure et al., 2018). RACE1 is on the and is to the in RACE1 a with and in the deduced effector (Figure and TWAS and the genome analysis revealed the potential of multiple encoded by paralogous genes, and two virulent variants. Figure 2 Download asset Open asset of as an AVRa7 candidate by association of avirulence profiles with transcript polymorphisms and integration in the Bgh (A) AVRa7 transcript variants encoded by the indicated Bgh isolates with corresponding avirulence (B) of deduced amino acid with all variants of the chromosomal regions candidate variants with corresponding gene in the genomes of Bgh isolates DH14 and CSEPs are by different infection types between cultivars Pallas and TWAS identified in DH14 and in as the candidate Supplementary file Figure Analysis of the transcriptome reads showed that isolates virulent on Mla9 carry variants with either or (Figure and we identified as the candidate for both AVRa10 and AVRa22 (Supplementary file Figure Bgh isolates avirulent on Mla10 plants encode a variant with an to the deduced effector protein in reference isolate DH14 Figure and all isolates avirulent on Mla22 plants carry a variant with 13 deduced aa and in Figure and DH14 is the only tested isolate that is virulent on both and plants, and we the variant encoded by DH14 belongs to the CSEP family with the only other family member that significant similarity with candidates AVRA10 and and identical aa these data the that and MLA22 each recognize one of the two major natural variants of in the Bgh population. Figure 3 with 1 supplement see all Download asset Open asset of as an candidate and as an AVRa10 and AVRa22 candidate by association of avirulence profiles with transcript polymorphisms as well as integration in the Bgh (A) variants encoded by each Bgh isolate with corresponding avirulence (B) of deduced amino acid with variants variants encoded by each Bgh isolate with corresponding avirulence of and amino acid of the chromosomal regions and candidates with corresponding gene as well as a of the EKA family class-1 retrotransposon and other CSEPs in the genomes of Bgh isolates DH14, and *denotes differences of infection types between cultivars Pallas and n.t.: not and candidates are each genes in both DH14 and RACE1 Bgh genomes (Frantzeskakis et al., 2018). candidates in DH14 and AVRa10 in RACE1 from the of and on the respective chromosomal (Figure The linkage of these candidate avirulence genes is consistent with a tight genetic of the AVRa10 and and their linkage to the locus et al., 2008). In we found the transcript of candidate AVRa22 to to a in the Bgh genome et al., 2013) that is with the candidate in DH14 and candidate AVRa10 in RACE1 (Figure The findings to the that AVRa10 and AVRa22 two alleles of a single Bgh effector gene (Figure supplement 1). AVRa10, which encodes a only 24 from the of the only other family member and in DH14 and RACE1 and from the of one of the EKA class-1 LINE retrotransposon family members (Figure Amselem et al., 2015). of the cluster of sequence-unrelated effector genes in the Bgh genome (Figure the and sequence-unrelated and and another cluster of candidate secreted effector genes in the fungal analysis of AVRa candidates in barley leaf We the of the identified AVRA candidates to trigger cell death co-expression with matching MLA receptors in barley leaf protoplasts. gene were from the together with the gene (Lu et al., 2016), and relative activity was as a for can with of activity in this (Lu et al., 2016), we from of with When to the co-expression of previously reported et al., 2016) with Mla1 activity in a by (Figure and co-expression of with Mla7 the activity by but not with Mla1 (Figure Interestingly, co-expression of Mla7 with either of the two AVRa7 variants present in and in a and of respectively, whereas their co-expression with Mla1 did not activity (Figure Figure supplement 1A). together these data indicate that in the Bgh population in their propensity to activate cell death. of the to in the DH14 genome (Figure in a significant in activity (Figure Figure supplement We a in activity was with Mla7 but not with of with Mla7 did not in activity to co-expression with Mla1 (Figure supplement 1A). Barley the