Multiple Avirulence Loci and Allele-Specific Effector Recognition Control the Pm3 Race-Specific Resistance of Wheat to Powdery Mildew

In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3^a2/f2 from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 allele-dependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 ^a2/f2 revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogen-encoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes.     

Single residues in the LRR domain of the wheat PM3A immune receptor can control the strength and the spectrum of the immune response

The development of improved plant nucleotide‐binding, leucine‐rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3^A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3^A2/F2. PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race‐specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.     

Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew

The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology‐based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single‐cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3‐ and Pm8‐like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co‐evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.     

Nucleotide‐binding resistance gene signatures in sugar beet,insights from a new reference genome

Nucleotide‐binding (NB‐ARC), leucine‐rich‐repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long‐read sequencing, are improving resolution of repeat‐rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB‐ARC domain as a model, 231 tentative NB‐ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB‐ARC signatures as well as full‐length sequences. The NB‐ARC‐associated proteins contained NLR resistance gene domains, including TIR, CC and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB‐ARC domains to validated R‐genes from monocots and eudicots suggested extensive Beta vulgaris‐specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR‐like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic‐acid‐based method for NLR prediction in non‐model plant species.     

Pm21 CC domain activity modulated by intramolecular interactions is implicated in cell death and disease resistance

Nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled-coil (CC) NLR that confers broad-spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1–159) domain has cell-death-inducing activity, whereas the CC-containing truncations, including CC-NB and CC-NB-LRR, do not induce cell-death responses. Coimmunoprecipitation (Co-IP) assay showed that the eCC domain self-associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V-mediated full-length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell-death induction. Two mutations (R73Q and E80K) interfered with D491V-mediated Pm21 autoactivation without affecting the cell-death-inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell-death-inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell-death signalling and disease-resistance signalling, which are potentially independent of each other.     

Cell death triggering and effector recognition by Sw‐5 SD‐CNL proteins from resistant and susceptible tomato isolines to Tomato spotted wilt virus

Only a limited number of dominant resistance genes acting against plant viruses have been cloned, and further functional studies of these have been almost entirely limited to the resistance genes Rx against Potato virus X (PVX) and N against Tobacco mosaic virus (TMV). Recently, the cell‐to‐cell movement protein (NS_M) of Tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant (Avr) of Sw‐5b‐mediated resistance, a dominant resistance gene which belongs to the class of SD‐CC‐NB‐LRR (Solanaceae domain‐coiled coil‐nucleotide‐binding‐leucine‐rich repeat, SD‐CNL) resistance genes. On transient expression of the NS_M protein in tomato and transgenic Nicotiana benthamiana harbouring the Sw‐5b gene, a hypersensitive cell death response (HR) is triggered. Here, it is shown that high accumulation of the Sw‐5b protein in N. benthamiana leaves, achieved by co‐expression of the Sw‐5b protein with RNA silencing suppressors (RSSs), leads to auto‐activity in the absence of NS_M. In a similar approach, Sw‐5a, the highest conserved paralogue of Sw‐5b from Solanum peruvianum, also triggered HR by auto‐activation, whereas the highest conserved orthologue from susceptible S. lycopersicum, named Sw‐5a^S, did not. However, neither of the last two homologues was able to trigger an NS_M‐dependent HR. Truncated and mutated versions of these Sw‐5 proteins revealed that the NB‐ARC [nucleotide‐binding adaptor shared by Apaf‐1 (from humans), R proteins and CED‐4 (from nematodes)] domain is sufficient for the triggering of HR and seems to be suppressed by the SD‐CC domain. Furthermore, a single mutation was sufficient to restore auto‐activity within the NB‐ARC domain of Sw‐5a^S. When the latter domain was fused to the Sw‐5b LRR domain, NS_M‐dependent HR triggering was regained, but not in the presence of its own Sw‐5a^S LRR domain. Expression analysis in planta revealed a nucleocytoplasmic localization pattern of Sw‐5b, in which the SD‐CC domain seems to be required for nuclear translocation. Although the Sw‐5 N‐terminal CC domain, in contrast with Rx, contains an additional SD, most findings from this study support a conserved role of domains within NB‐LRR (NLR) proteins against plant viruses.     

Development of functional markers specific for seven Pm3 resistance alleles and their validation in the bread wheat gene pool

In the ideal case, molecular markers used for marker-assisted selection are allele-specific even if the alleles differ only by a few nucleotide polymorphisms within the coding sequence of target genes. Such ‘perfect’ markers are completely correlated with the trait of interest. In hexaploid wheat (Triticum aestivum L.) the Pm3 locus encodes seven alleles (Pm3a–Pm3g) conferring resistance to different races of Blumeria graminis f.sp. tritici, the agent of powdery mildew, a major disease of bread wheat. All Pm3 alleles are known at the molecular level. Here, we generated specific markers for the Pm3 alleles based on nucleotide polymorphisms of coding and adjacent non-coding regions. The specificity of these markers was validated in a collection of 93 modern or historically important cultivars and breeding lines of wheat and spelt (Triticum spelta L.). These markers confirmed the presence of the predicted Pm3 alleles in 31 varieties and lines known to carry Pm3 resistance alleles. In a few varieties, Pm3 alleles different from alleles previously described based on pathogenicity tests or tightly linked markers were observed. In all these cases, the identity of the marker-detected Pm3 alleles was confirmed by DNA sequence analysis. Pm3 markers confirmed the absence of known Pm3 resistance alleles in 54 European wheat and spelt varieties in which Pm3 alleles had not been previously identified. These results indicate that the developed markers are highly diagnostic for specific Pm3 resistance alleles in a wide range of varieties and breeding lines, and will be useful (1) for identifying Pm3 alleles in the wheat gene pool, (2) for efficient marker-assisted selection of these genes, and (3) for combining multiple Pm3 alleles within a single cultivar through transgenic approaches.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus

A large number of resistance specificities to the powdery mildew fungus Blumeria graminis f. sp. hordei map to the barley Mla locus. This complex locus harbors multiple members of three distantly related gene families that encode proteins that contain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR (CT) region. We identified Mla12, which encodes a CC-NB-LRR-CT protein that shares 89 and 92% identical residues with the known proteins MLA1 and MLA6. Slow Mla12-triggered resistance was altered dramatically to a rapid response by overexpression of Mla12. A series of reciprocal domains swaps between MLA1 and MLA6 identified in each protein recognition domain for cognate powdery mildew fungus avirulence genes (AvrMla1 and AvrMla6). These domains were within different but overlapping LRR regions and the CT part. Unexpectedly, MLA chimeras that confer AvrMla6 recognition exhibited markedly different dependence on Rar1, a gene required for the function of some but not all Mla resistance specificities. Furthermore, uncoupling of MLA6-specific function from RAR1 also uncoupled the response from SGT1, a protein known to associate physically with RAR1. Our findings suggest that differences in the degree of RAR1 dependence of different MLA immunity responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of resistance protein-containing recognition complexes.     

Intramolecular Interaction Influences Binding of the Flax L5 and L6 Resistance Proteins to their AvrL567 Ligands

L locus resistance (R) proteins are nucleotide binding (NB-ARC) leucine-rich repeat (LRR) proteins from flax (Linum usitatissimum) that provide race-specific resistance to the causal agent of flax rust disease, Melampsora lini. L5 and L6 are two alleles of the L locus that directly recognize variants of the fungal effector AvrL567. In this study, we have investigated the molecular details of this recognition by site-directed mutagenesis of AvrL567 and construction of chimeric L proteins. Single, double and triple mutations of polymorphic residues in a variety of AvrL567 variants showed additive effects on recognition strength, suggesting that multiple contact points are involved in recognition. Domain-swap experiments between L5 and L6 show that specificity differences are determined by their corresponding LRR regions. Most positively selected amino acid sites occur in the N- and C-terminal LRR units, and polymorphisms in the first seven and last four LRR units contribute to recognition specificity of L5 and L6 respectively. This further confirms that multiple, additive contact points occur between AvrL567 variants and either L5 or L6. However, we also observed that recognition of AvrL567 is affected by co-operative polymorphisms between both adjacent and distant domains of the R protein, including the TIR, ARC and LRR domains, implying that these residues are involved in intramolecular interactions to optimize detection of the pathogen and defense signal activation. We suggest a model where Avr ligand interaction directly competes with intramolecular interactions to cause activation of the R protein.     

The powdery mildew resistance gene Pm8 derived from rye is suppressed by its wheat ortholog Pm3

The powdery mildew resistance gene Pm8 derived from rye is located on a 1BL.1RS chromosome translocation in wheat. However, some wheat lines with this translocation do not show resistance to isolates of the wheat powdery mildew pathogen avirulent to Pm8 due to an unknown genetically dominant suppression mechanism. Here we show that lines with suppressed Pm8 activity contain an intact and expressed Pm8 gene. Therefore, the absence of Pm8 function in certain 1BL.1RS‐containing wheat lines is not the result of gene loss or mutation but is based on suppression. The wheat gene Pm3, an ortholog of rye Pm8, suppressed Pm8‐mediated powdery mildew resistance in lines containing Pm8 in a transient single‐cell expression assay. This result was further confirmed in transgenic lines with combined Pm8 and Pm3 transgenes. Expression analysis revealed that suppression is not the result of gene silencing, either in wheat 1BL.1RS translocation lines carrying Pm8 or in transgenic genotypes with both Pm8 and Pm3 alleles. In addition, a similar abundance of the PM8 and PM3 proteins in single or double homozygous transgenic lines suggested that a post‐translational mechanism is involved in suppression of Pm8. Co‐expression of Pm8 and Pm3 genes in Nicotiana benthamiana leaves followed by co‐immunoprecipitation analysis showed that the two proteins interact. Therefore, the formation of a heteromeric protein complex might result in inefficient or absent signal transmission for the defense reaction. These data provide a molecular explanation for the suppression of resistance genes in certain genetic backgrounds and suggest ways to circumvent it in future plant breeding.     

Geographical distribution of genes for resistance to formae speciales of Erysiphe graminis in common wheat

The geographical distribution of Pm10, Pm11, Pm14, and Pm15 wheat genes for resistance to inappropriate formae speciales of Erysiphe graminis was investigated using gene-for-gene relationships. Pm10 and Pm15 were very common among many indigenous accessions of common wheat collected from various areas in the world. The diversity of genotypes, which consisted of allelic combination at those loci, was high near the center of origin of common wheat and decreased with increasing distance from the center. In Europe, an apparent contrast of predominant genotypes occurred between the south and the north, suggesting that these genes are useful markers for revealing the routes by which common wheat spread in Europe. On a whole, the genes for resistance to inappropriate formae speciales were observed to be widely distributed throughout the world. We suggest that the difference between these genes and the genes for resistance to races of an appropriate forma specialis may only be in their distribution and that of their corresponding avirulence genes.     

Further analysis of gene-for-gene disease resistance specificity in flax

The flax rust resistance gene L , a nucleotide binding site, leucine-rich repeat (NBS-LRR) class of plant resistance gene, has 12 characterized alleles with different gene-for-gene resistance specificities. Here the specificities of presumptive L1 , L5, L8 and L11 genomic clones are confirmed by transgenic expression. L6 and L11 differ by 33 amino acids, 32 in the LRR region and one in the C-terminal non-LRR region, and recognize unrelated avirulence proteins, AvrL567 and AvrL11, respectively. To analyse the specificity differences, 13 L6L11 recombinant genes were constructed in vitro and tested in transgenic flax for resistance to F2 progeny of rust strain CH5, in which the unlinked avirulence genes AvrL567 and AvrL11 segregate. The data show that the single C-terminal non-LRR region polymorphism is not involved in L6–L11 specificity differences, that polymorphisms necessary for specificity are spread throughout the LRR region and that some polymorphisms essential for L11 are not essential for L6. Seven 'null' recombinants expressed no resistance when tested with CH5-derived rusts. These were tested for new resistance specificities by inoculation with a strain of rust, Bs-1, which is distantly related to CH5 and which potentially carries a different range of avirulence specificities. The 'null' recombinant L6L11RV , which differs from L6 and L11 by its susceptibility to CH5, was resistant to strain Bs-1. The specificity difference is due to a reduction in the number of AvrL567 variants recognized by L6L11RV compared with L6 and not due to recognition of an unrelated Avr gene product in strain Bs-1.     

Pyramiding of transgenic <Emphasis Type="Italic">Pm3</Emphasis> alleles in wheat results in improved powdery mildew resistance in the field

Key message

The combined effects of enhanced total transgene expression level and allele-specificity combination in transgenic allele-pyramided Pm3 wheat lines result in improved powdery mildew field resistance without negative pleiotropic effects.

Abstract

Allelic Pm3 resistance genes of wheat confer race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and encode nucleotide-binding domain, leucine-rich repeat (NLR) receptors. Transgenic wheat lines overexpressing alleles Pm3a, b, c, d, f, and g have previously been generated by transformation of cultivar Bobwhite and tested in field trials, revealing varying degrees of powdery mildew resistance conferred by the transgenes. Here, we tested four transgenic lines each carrying two pyramided Pm3 alleles, which were generated by crossbreeding of lines transformed with single Pm3 alleles. All four allele-pyramided lines showed strongly improved powdery mildew resistance in the field compared to their parental lines. The improved resistance results from the two effects of enhanced total transgene expression levels and allele-specificity combinations. In contrast to leaf segment tests on greenhouse-grown seedlings, no allelic suppression was observed in the field. Plant development and yield scores of the pyramided lines were similar to the mean scores of the corresponding parental lines, and thus, the allele pyramiding did not cause any negative effects. On the contrary, in pyramided line, Pm3b × Pm3f normal plant development was restored compared to the delayed development and reduced seed set of parental line Pm3f. Allele-specific RT qPCR revealed additive transgene expression levels of the two Pm3 alleles in the pyramided lines. A positive correlation between total transgene expression level and powdery mildew field resistance was observed. In summary, allele pyramiding of Pm3 transgenes proved to be successful in enhancing powdery mildew field resistance.

     

Isolation of resistance gene analogues to powdery mildew resistance sequences in hexaploid wheat

This paper reports the characterization of the powdery mildew resistance homologous genes family of Triticum aestivum. Using degenerate primer pair for wheat resistance genes, we have cloned seven 3′ truncated powdery mildew resistance gene homologous fragments Tpc5a, Tp25a, Tp25b, Tp3a5a, Tp3a5b, Tp4b5a and Tp4b5b. These fragments were sequenced. The deduced amino acid sequences showed that six of them have premature stop codons. All these sequences had a very high level of similarity to known Pm resistance genes such as Pm3a, Pm3b, Pm3d and pm3f in hexaploid wheat. By ignoring the stop codons in the sequences, their deduced protein sequences were of coiled-coil (CC)-nucleotide binding site (NBS)-leucine repeat rich (LRR) structure. These results suggest that there are many powdery mildew resistance gene analogues in both resistant and susceptible wheat. Among them, small insertion/deletion events and point mutations can result in the diversity of wheat Pm resistance homologous genes.     

Two different CC‐NBS‐LRR genes are required for Lr10‐mediated leaf rust resistance in tetraploid and hexaploid wheat

Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC‐NBS‐LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety ‘Altar’ that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus‐induced gene silencing in tetraploid wheat lines. In contrast to most described NBS‐LRR proteins, the N‐terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS‐LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10‐based resistance is highly unusual both in its dependence on two, only distantly, related CC‐NBS‐LRR proteins, as well as in the pattern of diversifying selection in the N‐terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.     

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 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.     

Isolation and Evolution Mode Analysis of NBS–LRR Resistance Gene Analogs from Hexaploid Wheat

Based on conserved regions of a sequence of a previously isolated powdery mildew (Pm) resistance gene, Pm3b, from hexaploid wheat (Triticum aestivum L.), a pair of primers was designed, and 11 resistance gene analogs (RGAs) were obtained using a polymerase chain reaction-based cloning approach. Three RGAs were deemed as pseudogenes, while the remaining eight corresponded to protein-encoding genes. At the nucleotide level, all these RGAs shared a sequence identity of 99% and showed 86% sequence identity with Pm3b. Phylogenetic analysis revealed that the eight protein-encoding genes were paraphyletic with Pm3 alleles. Positively selected sites were identified using the Selecton 2.1 program, and these were located on secondary structures. Based on these findings, the following two inferences could be made on the mode of evolution of the nucleotide-binding site and leucine-rich repeat (LRR) class of resistance genes. First, the majority of evolution events must have occurred primarily in the LRR domain, and this might have contributed to an increase in the proportion of evolution events in other domains. Second, these evolutionary events in the LRR domain must have occurred initially in secondary structures and then in the β-sheet. The crystalline structure models of RGAs were constructed. De-Shun Feng and Yan Li contributed equally to this paper. Sequence data of 2Q2, 2Q3, 2Q4, 2Q7, 2Q9, 2Q11, 2Q12, 12Q11, 15Q1, and 15Q7 from this article have been deposited at GenBank under accession numbers EF157980, EF157981, EF157982, EF157983, EF157984, EF157985, EF157986, EF157987, EF157988, EF157989, and EF157990.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

Arms race co‐evolution of Magnaporthe oryzae AVR‐Pik and rice Pik genes driven by their physical interactions

Attack and counter‐attack impose strong reciprocal selection on pathogens and hosts, leading to development of arms race evolutionary dynamics. Here we show that Magnaporthe oryzae avirulence gene AVR‐Pik and the cognate rice resistance (R) gene Pik are highly variable, with multiple alleles in which DNA replacements cause amino acid changes. There is tight recognition specificity of the AVR‐Pik alleles by the various Pik alleles. We found that AVR‐Pik physically binds the N‐terminal coiled‐coil domain of Pik in a yeast two‐hybrid assay as well as in an in planta co‐immunoprecipitation assay. This binding specificity correlates with the recognition specificity between AVR and R genes. We propose that AVR‐Pik and Pik are locked into arms race co‐evolution driven by their direct physical interactions.