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. 2022 Nov 9;4(2):100472. doi: 10.1016/j.xplc.2022.100472

Orthologous genes Pm12 and Pm21 from two wild relatives of wheat show evolutionary conservation but divergent powdery mildew resistance

Shanying Zhu 1,2,8, Cheng Liu 3,8, Shuangjun Gong 4,8, Zhaozhao Chen 2, Rong Chen 5, Tianlei Liu 2, Renkang Liu 2, Haonan Du 2, Rui Guo 2, Genying Li 3, Miaomiao Li 6, Renchun Fan 6, Zhiyong Liu 6, Qian-Hua Shen 6, Anli Gao 5,∗, Pengtao Ma 7,∗∗, Huagang He 2,∗∗∗
PMCID: PMC10030366  PMID: 36352792

Abstract

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease that threatens wheat production worldwide. Pm12, which originated from Aegilops speltoides, a wild relative of wheat, confers strong resistance to powdery mildew and therefore has potential use in wheat breeding. Using susceptible mutants induced by gamma irradiation, we physically mapped and isolated Pm12 and showed it to be orthologous to Pm21 from Dasypyrum villosum, also a wild relative of wheat. The resistance function of Pm12 was validated via ethyl methanesulfonate mutagenesis, virus-induced gene silencing, and stable genetic transformation. Evolutionary analysis indicates that the Pm12/Pm21 loci in wheat species are relatively conserved but dynamic. Here, we demonstrated that the two orthologous genes, Pm12 and Pm21, possess differential resistance against the same set of Bgt isolates. Overexpression of the coiled-coil domains of both PM12 and PM21 induces cell death in Nicotiana benthamiana leaves. However, their full-length forms display different cell death-inducing activities caused by their distinct intramolecular interactions. Cloning of Pm12 will facilitate its application in wheat breeding programs. This study also gives new insight into two orthologous resistance genes, Pm12 and Pm21, which show different race specificities and intramolecular interaction patterns.

Key words: Pm12, Pm21, powdery mildew resistance, evolutionary conservation, race specificity, intramolecular interaction

This study reports the physical mapping and cloning of an Aegilops speltoides-derived powdery mildew resistance gene, Pm12, and provides evidence that Pm12 is orthologous to Pm21 from Dasypyrum villosum. Comparative analyses reveal that Pm12 and Pm21 possess different recognition specificities to wheat powdery mildew pathogens, and their proteins show different cell death-inducing activities caused by distinct intramolecular interactions.

Introduction

Wheat powdery mildew, caused by the obligate biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a devastating disease that threatens wheat production and yield worldwide. Incorporation of powdery mildew resistance (Pm) genes into susceptible wheat cultivars is the most effective, economical, and environmentally friendly means for controlling this disease. Traditional breeding methods take many years to introduce resistance, whereas cloned resistance genes could be transferred directly through a transgenic approach. Currently, more than 100 Pm genes/alleles have been identified from common wheat and its cultivated or wild relatives (McIntosh et al., 2017; He et al., 2021a; 2021b). Only a few of these Pm genes have been cloned, such as Pm1–Pm5 (Yahiaoui et al., 2004; Sánchez-Martín et al., 2016, 2021; Xie et al., 2020; Hewitt et al., 2021), Pm8 (Hurni et al., 2013), Pm17 (Singh et al., 2018), Pm21 (He et al., 2018), Pm24 (Lu et al., 2020), Pm38/Yr18/Lr34/Sr57 (Krattinger et al., 2009), Pm41 (Li et al. 2020), Pm46/Yr46/Lr67/Sr55 (Moore et al. 2015), Pm60 (Zou et al., 2018), and WTK4 (Gaurav et al., 2022). Pm3, Pm5, Pm21, Pm24, Pm38/Yr18/Lr34/Sr57, Pm41, Pm46/Yr46/Lr67/Sr55, and Pm60 were cloned by a map-based strategy. Pm1, Pm2, and Pm4 were isolated based on a mutant sequencing strategy. WTK4 was identified by genome-wide association study (GWAS). Pm8 and Pm17 were both isolated using a homology-guided approach based on the previously cloned Pm3.

The powdery mildew resistance gene Pm21, which originates from Dasypyrum villosum (2n = 2x = 14, VV), a wild diploid relative of wheat, confers immunity to all tested Bgt isolates and has been widely utilized in wheat breeding (Chen et al., 2013). However, it is difficult to clone Pm21 on 6VS of D. villosum owing to the restriction of exchange between the alien and wheat homoeologous chromosomal arms (Qi et al., 1998). Using the gene chip technique, Cao et al. (2011) identified a candidate gene, Stpk-V, from the Pm21 region, which encoded a serine-threonine kinase that enhanced powdery mildew resistance when overexpressed in common wheat. He et al. (2017) identified susceptible D. villosum accessions from a natural population and performed fine genetic analysis of Pm21 using a mapping population derived from a cross between resistant and susceptible D. villosum accessions. Two nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) genes, but not Stpk-V, were located in the Pm21 locus. Further functional analysis revealed that DvRGA2, one of the two NLR genes, is Pm21 (He et al., 2018). Another study also verified the same NLR gene as Pm21 via RenSeq-PacBio (Xing et al., 2018).

Pm12 also confers highly effective resistance to powdery mildew, as reported by several researchers (Niewoehner and Leath 1998; An et al., 2019; Zhang et al., 2019; Wan et al., 2021). Pm12 was originally transferred from Aegilops speltoides (2n = 2x = 14, SS) into wheat cv. Wembley, generating Line #31 (Wembley Line #31, WL31). WL31 harbors the translocated chromosome 6BS-6SS.6SL (Miller et al., 1988; Jia et al., 1996). Subsequently, the introgression line 1338-8, which contains only a partial short arm of chromosome 6S (6SS), was generated from WL31. However, linkage drag, including delayed maturation and yield depression, could not be eliminated because of severe recombination suppression between chromosomes 6SS and 6BS (Song et al., 2007). Therefore, the powdery mildew resistance conferred by Pm12 has rarely been used in breeding. Recombination suppression also greatly impeded the isolation of Pm12 using traditional map-based cloning in a wheat background.

As an alternative to genetic analysis, chromosomal deletion analysis is also an efficient method for finely mapping a gene of interest (Zhu et al., 2018). Here, using chromosome deletion lines induced by irradiation that are susceptible to powdery mildew, we quickly revealed that the Pm12 locus corresponds to the Pm21 orthologous region of Ae. speltoides based on molecular marker analysis combined with comparative genomics analysis. We now report the isolation and functional validation of Pm12 via ethyl methanesulfonate (EMS)-induced mutagenesis, virus-induced gene silencing (VIGS), and stable wheat transformation. We demonstrate a significant difference between the two orthologous genes Pm12 and Pm21 through comparative analyses of their resistance spectra and intramolecular interaction patterns. We also reveal the evolutionary dynamics of orthologous Pm12/Pm21 loci in Triticeae species.

Results

Chromosomal deletion analysis suggests that Pm12 might be orthologous to Pm21

Ae. speltoides Pm12 (6SS) and D. villosum Pm21 (6VS) are both located on the short arms of the homoeologous group 6 chromosomes. To evaluate the collinearity relationship, we used a set of Pm21-associated 6VS markers reported previously (He et al., 2016; Zhu et al., 2018) to detect the Pm12 and Pm21 regions. The results revealed that 9 of 46 6VS markers showed polymorphisms between Pm12-carrying materials (WL31 and 1338-8) and Pm12-null common wheat YM9. Another four polymorphic markers (6SS-03, 6SS-04, 6SS-05, and 6SS-07) were newly developed in this study. Among the 13 polymorphic markers, 10 could simultaneously distinguish the alien chromosomes 6SS and 6VS in a wheat background (Supplemental Figure 1, Supplemental Table 1).

Seeds of line 1338-8 carrying Pm12 were treated with 60Co-γ-ray irradiation. From the 316 M2 families, four susceptible mutants (1338-8-S1 to 1338-8-S4) were obtained (Figure 1A). The 13 6SS-specific markers described above were then used to detect whether there were alien chromosomal deletions in the four susceptible mutants. The result showed that the mutant lines 1338-8-S2 and 1338-8-S3 contained alien chromosomal deletions flanked by markers 6SS-03 and 6SS-05, and 6SS-03 and 6SS-06, respectively (Figure 1B). Thus, the Pm12 locus could be narrowed to a relatively small physical region flanked by markers 6SS-03 and 6SS-05, corresponding to TraesCS6B02G146300 and TraesCS6B02G151600, respectively, in the B subgenome of the Chinese Spring reference genome, RefSeq v1.1 (International Wheat Genome Sequencing Consortium et al., 2018). Because most disease resistance genes belong to the NLR family, we checked the Chinese Spring genome sequence spanning TraesCS6B02G146300 to TraesCS6B02G151600 and found only one annotated NLR gene, TraesCS6B02G148400 (Figure 1B). BLAST analysis showed that this gene was highly homologous to the recently cloned powdery mildew resistance gene Pm21 from D. villosum (88% nucleotide identity; E value = 0). Further analysis showed that TraesCS6B02G148400 fused two NLR genes corresponding to the two adjacent genes DvRGA1 and DvRGA2 (Pm21). In the recently published genome of Ae. speltoides accession AEG-9674-1 (Avni et al., 2022), a gene (AE.SPELTOIDES.r1.6BG0531060) highly homologous to Pm21 was also found (94% identity; E value = 0). Molecular detection showed that marker 6SS-04 corresponding to AE.SPELTOIDES.r1.6BG0531060 had no product in the susceptible mutants 1338-8-S2 and 1338-8-S3. Therefore, AE.SPELTOIDES.r1.6BG0531060, the ortholog of Pm21, was considered to be a candidate for Pm12. In lines 1338-8-S1 and 1338-8-S4, the specific bands of marker 6SS-04 were present. We suggest that the candidate gene may contain minor change(s) that require further analysis.

Figure 1.

Figure 1

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Physical mapping of Pm12 and characteristics of the candidate gene Pm12-can.

(A) Powdery mildew-susceptible mutants obtained after 60Co-γ-ray irradiation.

(B) Physical mapping of Pm12 using chromosomal deletion mutant lines and comparative mapping using the reference genomes of Ae. speltoides accession AEG-9674-1 and wheat cv. Chinese Spring.

(C) Gene structures of Pm12 and Pm21. Genes in the Pm12 and Pm21 loci are indicated by arrows that are broken by introns.

(D) Sequence comparison of the LRR domains encoded by Pm12 and Pm21. Both LRR domains of PM12 and PM21 are divided into 16 LRR motifs (LRR1 to LRR16). LxxLxLxx motifs are marked below the corresponding sequences. L represents a leucine or another aliphatic amino acid residue, and x represents the predicted solvent-exposed LRR residue. Green amino acids or – indicate insertions/deletions, and red amino acids differ between PM12 and PM21. Arrows represent solvent-exposed LRR residues.

The Pm12 candidate, Pm12-can, shares high identity and the same exon–intron structure with Pm21

In the Pm21 locus, two NLR genes, DvRGA1 and DvRGA2 (Pm21), with typical coiled-coil (CC), nucleotide-binding site (NBS), and leucine-rich repeat (LRR) domains are paralogous and closely linked. According to their conserved sequences, we designed three primer pairs to isolate Pm12-related DNA fragments from the genomic DNA of line WL31. The downstream sequence of the candidate gene was obtained using the TAIL–PCR method (Supplemental Figure 2). Finally, a 9355-bp genomic sequence from WL31 was assembled. This sequence contained two NLR genes, AesRGA1 (DvRGA1-like gene) and Pm12-can (the candidate for Pm12, DvRGA2-like gene). The Pm12-can sequence was identical to that in the recently published genome of Ae. speltoides accession AEG-9674-1 (Avni et al., 2022) and differed in only one base from the genome sequence of Ae. speltoides accession TS01 (Li et al., 2022), leading to the amino acid change R769Q. We concluded that the sequence we obtained originated from Ae. speltoides chromatin introgressed into wheat. Through comparing sequences of the above genomic DNA and its corresponding cDNA obtained by RT–PCR, we found that Pm12-can and Pm21 have the same gene structure, consisting of three exons and two introns (Figure 1C). The transcription of both Pm-can and Pm21 was slightly activated when challenged with Bgt01 (Supplemental Figure 3).

The coding sequence of Pm12-can shares 89.9% and 72.8% identity with those of Pm21 and its paralog DvRGA1, respectively. In addition to five InDels, there were 268 SNPs, including 133 synonymous SNPs and 135 non-synonymous SNPs, between Pm12 and Pm21 (Supplemental Table 2). Sixty-three of 135 amino acid changes appeared in the LRR domain, 16 of which were located in the solvent-exposed LRR residues (Figure 1D) that may alter recognition specificity to the powdery mildew pathogen (Wulff et al., 2009). The putative protein of Pm12-can showed the highest identity with PM21 (84.9%), relatively lower identity with DvRGA1 (64.2%), and low identities with the powdery mildew resistance proteins PM1a, PM2, PM3b, PM5e, PM8, PM17, PM41, and PM60 and other disease resistance proteins (<36%).

Pm12-can is necessary for resistance, as shown by mutational and VIGS analyses

To further examine whether Pm12-can is responsible for disease resistance, we obtained its genomic sequences from two susceptible mutant lines, 1338-8-S1 and 1338-8-S4, by PCR followed by Sanger sequencing. Line 1338-8-S1 contained a 1-bp deletion in the CC domain of Pm12-can that led to a frameshift and the formation of a truncated protein. Line 1338-8-S4 contained a missense mutation in the Kinase-2 motif of the NBS domain of Pm12-can. We also carried out mutation analysis of 15 independent susceptible mutant lines (1338-8-S5 to 1338-8-S19) identified from 1206 EMS-induced M2 families (Figure 2A). Each mutant contained a single base change in the coding region of Pm12-can. Two mutations caused premature stop codons, and the other 13 mutations led to amino acid changes in the CC domain (1), the linker between the CC and NBS domains (1), the Kinase-3a (RNBS-B) motif (1), the RNBS-C motif (2), the region close to the GLPL motif (1) of the NBS domain, and the LRR domain (7) (Figure 2B). We also amplified and sequenced the DvRGA1-like gene, AesRGA1, in each mutant line and found no mutations. The fact that mutations were found only in Pm12-can strongly supports the notion that Pm12-can is actually the gene that controls Pm12-mediated resistance.

Figure 2.

Figure 2

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Functional validation of Pm12.

(A) Responses of EMS-induced mutants to Bgt01 at the seedling stage, using 1338-8 and YM9 as the resistant and susceptible controls, respectively, whose images can be found in Figure 1A.

(B) Gene structure showing mutation sites identified in susceptible mutants. The green, brown, and blue boxes are the CC, NBS, and LRR domains, respectively.

(C) Macroscopic phenotypes of the resistant wheat WL31 (carrying Pm12) after treatment with BSMV:Pm21as. BSMV:Pm21as-infected YM18 (carrying Pm21) and BSMV:00 (with empty vector)-infected WL31 were used as the positive and negative controls, respectively.

(D) Silencing efficiencies of Pm12 in WL31 leaves and Pm21 in YM18 leaves measured by qPCR. Error bars represent the standard error of the mean of three independent experiments. Statistical significance of differences was assessed using Student’s t-test. ∗P < 0.001.

(E) Responses of T2 transgenic lines expressing Pm12 to four Bgt isolates at the one-leaf stage, using Pm12 donors WL31 and 1338-8 as the resistant controls and the recipient Fielder as the susceptible control. The responses of Pm12 plants were also compared with those of YM18, the transgenic wheat line (Pm21 Line 1) carrying Pm21, and KN199 without any powdery mildew resistance gene.

The VIGS approach was also used to test whether Pm12-can was required for Pm12 resistance. In our previous work, the recombinant virus BSMV:Pm21as carrying a 362-bp LRR-encoding sequence that shares 94.2% nucleotide identity with that of Pm12-can effectively blocked Pm21 resistance (He et al., 2018). Here, we found that infection of the resistant line WL31 with the BSMV:Pm21as construct compromised its Pm12 resistance and allowed the development of Bgt colonies with disease symptoms and fungal sporulation on the leaves (Figure 2C and 2D). Taken together, these data indicate that the candidate gene Pm12-can is required for Pm12-mediated resistance to wheat powdery mildew.

Pm12-can confers highly effective resistance to powdery mildew in transgenic wheat lines

To validate the resistance function of Pm12-can, a construct harboring Pm12-can cDNA driven by the maize ubi promoter was transformed into the susceptible wheat cv. Fielder using Agrobacterium-mediated transformation. Three of the four regenerated T0 plants were confirmed to carry Pm12-can (Supplemental Figure 4). The T2 plants derived from these three lines showed strong resistance to 122 Bgt isolates with infection type (IT) 0 or 1, with the exception of isolate WH-1. The responses of the transgenic plants from each line to the same isolate were consistent with those of the donors WL31 and 1338-8 (Figure 2E, Supplemental Table 3). Therefore, we concluded that Pm12-can is indeed Pm12. As a comparison, we also determined the disease responses of YM18 carrying Pm21 and Pm21 T5 Line 1 (transgenic wheat line expressing Pm21) to the same set of Bgt isolates. The results showed that Pm21 conferred immunity to all 123 isolates tested. This finding suggested that there is a difference in race specificity between Pm12 and Pm21, which might be associated with the sequence variations in their LRR domains.

PM12 and PM21 differ in cell death-inducing activity caused by their different intramolecular structures

Cell death in Nicotiana benthamiana induced by overexpression of NLR proteins, such as MLA10, SR33, SR35, Rp1-D21, RGA4, and PM60, has been widely reported (Bai et al., 2012; Cesari et al., 2014, 2016; Wang et al., 2015; Zou et al., 2018). However, several studies have shown that different NLR-type powdery mildew resistance proteins identified from wheat crops may have different cell death-inducing activities (Bourras et al., 2015; Hewitt et al., 2021; Müller et al., 2022; Praz et al., 2016; Zou et al., 2018). To assess the cell death-inducing activities of PM12 and PM21, we infiltrated Pm12 and Pm21 into N. benthamiana leaves; overexpression of PM12 resulted in strong cell death, but overexpression of PM21 did not. We then detected the cell death-inducing activities of their CC, NBS, and LRR domains as well as the fragments CC-NBS and NBS-LRR. The PM12 and PM21 CC domains were able to induce cell death, whereas the other single domains were not (Figure 3A and 3B), indicating that the CC domains of both PM12 and PM21 are sufficient to trigger cell death. Interestingly, the PM12 CC-NBS fragment elicited a strong cell death response, whereas the PM21 CC-NBS fragment did not induce cell death. These findings suggest that the activities of the CC domains of PM12 and PM21 are intramolecularly regulated in different ways. Different domains of an NLR protein usually interact with each other, and cell death activity may be regulated by other domains in the same protein (Moffett et al., 2002; Rairdan et al., 2008; Wang et al., 2015, 2019a, 2019b; Bernoux et al., 2016; El Kasmi et al., 2017; Zhao et al., 2022). The above results prompted us to speculate that differences in cell death caused by full-length PM12 and PM21 might be caused by differences in regulation and interactions of the domains in the two proteins.

Figure 3.

Figure 3

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Cell death assay and Co-IP analysis in N. benthamiana leaves.

(A) Comparative analyses of cell death-inducing activities of the single or combined domain(s) encoded by Pm12 and Pm21. N. benthamiana leaves were infiltrated with agrobacteria carrying different constructs. Cell death was observed by trypan blue staining at 48 h post infiltration.

(B) Western blotting detection of different domains or proteins expressed in N. benthamiana leaves. EV: empty vector. FL: full-length proteins. Tagged proteins were detected by western blotting with anti-GFP antibodies (α-GFP).

(C) Comparative analyses of interactions of different domains encoded by Pm12 and Pm21. Different domains fused to GFP or Myc tags were transiently co-expressed in N. benthamiana leaves in the indicated combinations (+, agro-infiltrated construct; –, non-infiltrated construct), and total proteins were extracted at 20 h post infiltration. Tagged proteins were detected by western blotting in the input and after immunoprecipitation with anti-Myc antibodies (α-Myc). Actin was used as the protein loading control. Asterisk indicates non-specific signals.

To test this hypothesis, co-immunoprecipitation (Co-IP) assays were performed on N. benthamiana leaves by co-expressing either NBS-GFP or LRR-GFP with CC-Myc of PM12 or PM21, followed by pull-down experiments with anti-GFP antibody (αGFP). The results showed that PM21 CC-Myc co-immunoprecipitated with both PM21 NBS-GFP and PM21 LRR-GFP, whereas PM12 CC-Myc did not co-immunoprecipitate with either PM12 NBS-GFP or PM12 LRR-GFP. Co-IP assays also revealed that both PM12 and PM21 NBS domains interacted with their LRR domains (Figure 3C). Combining the cell death assays and Co-IP data, we suggest that differences in cell death-inducing activities of PM12 and PM21 likely resulted from differences in their intramolecular domain interactions.

Genomic data reveal the origin and evolutionary dynamics of the Pm12/Pm21 loci in Triticeae species

In D. villosum, both Pm21 (DvRGA2) and the immediately adjacent gene, DvRGA1, encode NLR proteins. Similarly, Pm12 is also linked to the DvRGA1-like gene AesRGA1 in Ae. speltoides. Using BLAST searches, we found frequent occurrences of this NLR gene pair in the genomes of Triticeae species, including common wheat, durum wheat, wild emmer, T. urartu, Ae. tauschii, and Ae. speltoides (Figure 4). Based on their location proximity and their high sequence similarity, the two genes in the pair are likely to be paralogs derived from a tandem duplication event in the Triticeae genomes. In the syntenic region of the Brachypodium distachyon genome, there are three tandemly duplicated copies of DvRGA1-like genes (Bradi3g03874, Bradi3g03878, and Bradi3g03882) that share 71.4%–72.9% identity and the same exon–intron organization as DvRGA1. However, no DvRGA1-like gene appears in rice (Oryza sativa), maize (Zea mays), and sorghum (Sorghum bicolor). This suggests that DvRGA1-like genes existed before the separation of Brachypodium and the Triticeae lineage. Furthermore, there is no DvRGA2-like gene near the DvRGA1-like gene or elsewhere in the Brachypodium genome, indicating that the Pm21/Pm12 orthologs originated after the divergence of wheat species and Brachypodium but before the divergence of D. villosum and the common ancestor of the three wheat donor species T. urartu (A subgenome), Ae. speltoides (B subgenome), and Ae. tauschii (D subgenome).

Figure 4.

Figure 4

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Phylogenetic tree of PM12 (PM12-can) and relevant NLR proteins from wheat species, rice, and Arabidopsis.

The phylogenetic tree was constructed based on full-length NLR proteins by the neighbor-joining method with the Poisson model in MEGA7 software. Arabidopsis RPP13 was used to root the tree. The scale bar indicates an evolutionary distance of 0.20 amino acid substitutions per site. Red and green dots indicate DvRGA2-like (including PM21 and PM12) and DvRGA1-like NLR proteins, respectively, from D. villosum and Ae. speltoides. Aet: Ae. tauschii. Aes: Ae. speltoides. Dv: D. villosum. Ta: Triticum aestivum (common wheat). Tas: T. aestivum ssp. spelta (spelta wheat). Tdi: T. dicoccoides (wild emmer). Tdu: T. durum (durum wheat). Tu: T. urartu. Wheat cultivars or related accessions are shown in brackets.

Sequence analysis showed that the DvRGA1 orthologs shared 96.4%–100% identity with each other; however, the transcribed or predicted coding sequences of Pm12/Pm21 (DvRGA2-like) orthologs had only 72.0%–100% identity. This indicates that DvRGA1-like genes are relatively conserved in wheat and its related species, whereas Pm12/Pm21 orthologs are more evolutionarily dynamic. The DvRGA1-like gene has a single intron (250–355 bp) that lies at the immediate N terminus of the sequence encoding the Kinase-2 motif in the NBS domain. Pm12/Pm21 orthologs have three exons and two introns. The first intron is very short (only 96–101 bp) and lies upstream of the sequence encoding the NBS domain. The second intron lies in the same location as that of the DvRGA1-like genes. Compared with Pm21 and Pm12, the second introns of the orthologs TaRGA2-6A, TaRGA2-6B, and TaRGA2-6D, derived from the wheat A, B, and D subgenomes, respectively, have clearly expanded, probably through independent insertion events of different transposable elements (TEs) and/or other unknown repetitive elements (REs). Further analysis at the subgenome level showed that most of the intron expansion events in the Pm12/Pm21 orthologs of common wheat cv. Chinese Spring could be clearly traced back to its tetraploid and diploid ancestral species, including durum wheat, wild emmer, T. urartu, and Ae. tauschii (Figure 5A).

Figure 5.

Figure 5

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Evolutionary, transcriptional, and transient expression analyses of Pm12/Pm21 orthologs from wheat species.

(A) Gene structures of Pm12/Pm21 orthologs in common wheat and its diploid and tetraploid ancestral species. Triangles with different colors represent different transposable elements (TEs) and repetitive elements (REs). The Pm12/Pm21 orthologs TaRGA2-6A, TaRGA2-6B, and TaRGA2-6D were identified from wheat cv. Chinese Spring, TduRGA2-6A and TduRGA2-6B from T. durum (durum wheat), TdiRGA2-6A and TdiRGA2-6B from T. dicoccoides (wild emmer), TuRGA2-6A from T. urartu, and TduRGA2-6D from Ae. tauschii.

(B) qPCR analysis of Pm12/Pm21 orthologs TaRGA2-6A, TaRGA2-6B, and TaRGA2-6D in Chinese Spring leaves. The leaves were inoculated with Bgt01 and sampled at 0, 12, and 24 h post inoculation (hpi). The relative expression levels of TaRGA2-6A, TaRGA2-6B, and TaRGA2-6D were compared with those of Pm12 and Pm21.

(C) Single-cell transient expression assay of TaRGA2-6A, TaRGA2-6B, and TaRGA2-6D in wheat leaves. The vector expressing Pm12 and the corresponding empty vector pUBI were used as the positive and negative controls. Statistical significance of differences was assessed using Student’s t-test. ∗P < 0.01.

The expressed Pm12/Pm21 orthologs in Chinese Spring do not confer resistance to Bgt

The insertion of multiple TEs in the second intron was originally thought to disrupt the expression of Pm12/Pm21 genes in Chinese Spring wheat. However, a search of the wheat expression database (http://www.wheat-expression.com) revealed that TaRGA2-6A, TaRGA2-6B, and TaRGA2-6D are all transcribed, and their full-length open reading frame (ORF) sequences were identified as TraesCS6A02G120000.1, TraesCS6B02G148400.1, and TraesCS6D02G110000.1, respectively. qPCR analysis confirmed that, despite the large expansion of the second intron, the three orthologs could still be transcribed in Chinese Spring. However, their transcript levels were significantly lower than those of Pm12 and Pm21 during invasion by the Bgt isolate Bgt01 (YZ01) (Figure 5B). Transcriptional analysis also indicated that TaRGA2-6A and TaRGA2-6B were spliced in a similar manner to Pm12 and Pm21. We also performed single-cell transient assays to test whether overexpression of Pm12/Pm21 orthologs provides resistance functions. The haustorium indexes of TaRGA2-6A, TaRGA2-6B, and TaRGA2-6D were 81.8%, 75.4%, and 78.2%, respectively, significantly greater than that of Pm12 (42.3%) and similar to that of the negative control (80.3%) (Figure 5C). These results indicated that overexpression of the three Chinese Spring orthologs could not confer Bgt resistance, suggesting that accumulated variations in the coding regions likely led to loss of function.

Discussion

Alien disease resistance genes from wild relatives usually confer high resistance to wheat diseases. However, after transfer into cultivated wheat, most of them have not been well utilized in breeding programs because of linkage drag that is difficult to overcome. The best way to use such genes is to clone them and transfer them directly into elite wheat lines using transgenic technology (Wulff and Moscou, 2014; Sánchez-Martín and Keller, 2019). However, owing to suppressed recombination between wheat and alien chromatins, it is difficult to isolate the alien genes in a wheat background using a positional cloning strategy based on genetic mapping. Here, we demonstrate an efficient strategy for physical mapping and identification of the alien powdery mildew resistance gene Pm12 using mutants obtained by irradiation. EMS treatment is useful for creating mutations, but most mutations involve single-base variations rather than deletions within chromosomes (He et al., 2018). By contrast, we found that irradiation is a powerful tool for the development of new genetic stocks with significant chromosomal aberrations (Zhang et al., 2015). Consequently, we proposed that irradiation would create more deletion mutations at the chromosome level. Such chromosomal deletions could easily be detected using traditional marker analysis and used for physical mapping of genes of interest. In the present study, two of four susceptible mutants of Pm12 derived from the irradiated population were shown to have lost large chromosomal fragments, which enabled precise mapping of Pm12. Subsequently, another two irradiated susceptible mutants without chromosomal deletion in the target region were used to identify the candidate gene for Pm12. Thus, we successfully isolated the alien gene Pm12. The above strategy, based on irradiation-induced mutants, is fast and efficient for mapping and cloning of alien genes in a wheat background. The efficient cloning of effective alien genes can not only accelerate their application in breeding but also enable understanding of their functions and mechanisms.

In the Pm21 locus of D. villosum, only one of the two paralogous NLR genes DvRGA2 (Pm21) and DvRGA1 participates in powdery mildew resistance (He et al., 2018). Here, we showed that Pm12, the orthologous gene of Pm21 in Ae. speltoides, is also sufficient for resistance, independent of the adjacent gene AesRGA1. Interestingly, this NLR pair exists not only in D. villosum and Ae. speltoides but also in other published Triticeae genomes, including those of common wheat, wild emmer (T. dicoccoides), durum wheat (T. durum), T. urartu, and Ae. tauschii. The NLR pair does not appear in Brachypodium, rice, maize, and sorghum; however, DvRGA1-like genes can be found within a cluster in the orthologous region of Brachypodium. We suggest that DvRGA1-like NLRs appeared in the common ancestor of wheat and Brachypodium, whereas DvRGA2-like (Pm12/Pm21-like) NLRs might be duplicated from non-functional DvRGA1-like NLRs after the divergence of wheat and Brachypodium. Subsequently, the duplicated copies might have experienced more rapid evolution and gained resistance against powdery mildew diseases.

In the three Pm12/Pm21 orthologs of the Chinese Spring genome, independent insertions of TEs and other REs occurred in the second intron. The insertions in the A and D subgenomes of wheat can be traced back directly to their diploid progenitors, T. urartu and Ae. tauschii, respectively, indicating that these insertion events occurred after the divergence of the two diploid progenitors. In the B subgenome of Chinese Spring, there are two insertions in the Pm12/Pm21 ortholog. The first insertion (Stowaway MITE) is also found in durum wheat but not in wild emmer, whereas the other (RE 1) is not found in either durum wheat or wild emmer. Ae. speltoides is considered to be the donor of the wheat B subgenome, and Pm12, which originated from Ae. speltoides, does not have a large insertion in its second intron. A number of TE insertion events that appeared to decrease the transcript levels of the corresponding genes were reported in intronic regions of the Ae. tauschii genome (Zhao et al. 2017). Here, we also showed that insertions of large fragments significantly decreased the transcript levels of the three Pm12/Pm21 orthologs in Chinese Spring, which were only 13.4%–25.6% of those of Pm12 and Pm21. In the VIGS experiment, transcript levels of Pm12 and Pm21 were reduced to 29.6% and 24.7%, respectively, making resistant plants susceptible to powdery mildew. Therefore, the reduced transcript levels of the orthologs caused by insertion events may have undermined resistance. Furthermore, transient overexpression revealed that the three orthologs from susceptible wheat cv. Chinese Spring did not provide resistance to powdery mildew. Hence, we conclude that the loss of function of the three Chinese Spring orthologs is most likely caused by both insertion events and sequence variations accumulated in their coding regions.

The functional Pm12 and Pm21 resistance genes identified in the two diploid wild species, Ae. speltoides and D. villosum, respectively, have undoubtedly undergone independent evolution in different environments since divergence from their common ancestral gene. The two orthologous resistance genes now share 89.9% nucleotide sequence identity and 84.9% amino acid sequence identity. A total of 135 amino acid variations were found in different domains of the two proteins, 16 of which involve the solvent-exposed residues of the LRR motifs. We observed that Pm12 and Pm21 showed different responses to isolate WH-1, suggesting that there are differences in race specificity between the two orthologous genes. We propose that this difference may result from the variations in the solvent-exposed residues of the LRR motifs, as they are considered to be responsible for perception of the pathogen effector (Wulff et al., 2009; He et al., 2020). From another point of view, a key effector in WH-1 might have obtained new variation(s), leading it to escape recognition by PM12 but still be perceived by PM21. It would be interesting to investigate whether the two orthologous NLR proteins perceive the same effector and how the isolate WH-1 escapes from PM12 but not from PM21.

In general, NLRs must be strictly regulated in the absence of their cognate pathogen effectors to avoid autoimmunity that would be damaging to the host. Previous studies have shown that some NLRs, including SR33, SR50, Rp1-D21, and RGA4, can induce pathogen effector-independent cell death when transiently overexpressed in N. benthamiana (Cesari et al., 2014, 2016; Wang et al., 2015), suggesting that these NLRs are in an autoactive state. However, other NLRs, such as Rx, RPM1, and ZAR1 (Bendahmane et al., 2002; Baudin et al., 2017; El Kasmi et al., 2017), do not lead to cell death, indicating that they are in an autoinhibited state. In the present study, the full-length forms of PM12 and PM21 showed obvious differences in cell death activity. This result suggests that in the absence of a pathogen effector, PM21 is present in an autoinhibited state, whereas PM12 is autoactive.

Previous transient overexpression assays have demonstrated the complexity of the autoinhibited/autoactive states of NLR-type powdery mildew resistance proteins of wheat crops. For instance, T. urartu PM60 can induce cell death (Zou et al., 2018), whereas wheat PM1, PM2, PM3, and PM17 cannot (Bourras et al., 2015; Praz et al., 2016; Hewitt et al., 2021; Müller et al., 2022). Furthermore, some NLRs encoded by alleles with high sequence identity also have different cell death-inducing activity. For instance, overexpression of MLA10 from barley (Hordeum vulgare) can lead to cell death, whereas overexpression of MLA1 and MLA6 does not (Bai et al., 2012; Bauer et al., 2021). Confusingly, MLA10 showed no cell death activity in the report of Bauer et al. (2021). Intramolecular interactions in NLRs commonly play crucial roles in stabilizing active or autoinhibited protein conformations (Rairdan et al., 2008; Wang et al., 2015, 2019a; El Kasmi et al., 2017; Zhao et al., 2022). Co-IP assays revealed that interaction occurs between the PM21 CC domain and its NBS and LRR domains but not between the PM12 CC domain and its NBS or LRR domains.

Because PM12 and PM21 show a relatively high number of amino acid differences, we believe that differences in their intramolecular interactions must be generated by their sequence variations. These sequence variations may also alter their effector recognition. It will be very interesting to determine which of the amino acid differences influence recognition and which influence activity leading to altered cell death signaling. Future work involving structural characterization and functional experiments in wheat protoplasts will further our understanding of these molecular mechanisms.

Methods

Plant materials and growth conditions

The common wheat–Ae. speltoides translocation Wembley Line #31 (WL31, 6BS-6SS.6SL), originally generated at the John Innes Centre, Norwich, UK, is the donor of Pm12 (Miller et al., 1988; Jia et al., 1996). The resistant introgression line 1338-8 was derived from WL31 (Song et al., 2007). Wheat cv. Chinese Spring was provided by the Germplasm Resources Information Network (GRIN). The resistant wheat cv. Yangmai 18 (YM18) carrying Pm21 on the translocated chromosome T6AL.6VS and the susceptible cv. Yangmai 9 (YM9) were both developed at Yangzhou Academy of Agricultural Sciences, Yangzhou, China. The susceptible cv. Fielder was used as the recipient for genetic transformation. Pm21 T5 Line 1, a transgenic wheat line expressing Pm21, was originally developed in our previous work (He et al., 2018). Kenong 199 (KN199), the recipient for Pm21 transformation, is a highly susceptible wheat variety without any Pm genes. Nicotiana benthamiana was used for transient expression assays. Plants were grown in a greenhouse with LED lighting under long-day conditions (16 h light/8 h dark) at 24°C.

Evaluation of powdery mildew resistance

Three seedlings of each line at the one-leaf stage were inoculated with Bgt isolates by dusting fungal conidiospores onto leaves. The inoculated plants were grown under a daily cycle of 16 h light/8 h dark at 24°C in a greenhouse. Responses to powdery mildew were evaluated at 7–10 days after inoculation. The responses of transgenic wheat to Bgt pathogens were assessed using two sets of Bgt isolates collected from different regions of China. The first set contained 22 isolates maintained by Dr. Pengtao Ma at Yantai University (He et al., 2021b), and the second set included 100 isolates held by the Institute of Plant Protection and Soil Science, Hubei Academy of Agricultural Sciences (Bourras et al., 2019). Isolate WH-1 was newly collected from Wuhan, China. Infection types were scored on a 0 to 4 scale as described by An et al. (2013), where ITs 0, 1, and 2 were regarded as resistant and ITs 3 and 4 as susceptible.

Genomic DNA isolation and molecular marker analysis

Genomic DNA was isolated from the leaves of seedlings using the TE-boiling method (He et al., 2017). A total of 50 markers, including 46 D. villosum 6VS-specific markers reported previously (Song et al., 2009; Qi et al., 2010; Bie et al., 2015a, 2015b; He et al., 2016, 2017; Zhu et al., 2018) and four new markers developed in this study using the CISP (conserved-intron scanning primers) strategy (He et al., 2013) based on the potential collinearity relationship between Ae. speltoides 6SS and D. villosum 6VS, were used to screen for polymorphisms among YM9, WL31, and 1338-8. The diagnostic molecular marker 6SS-04 was designed based on the promoter sequences of Pm12 and Pm21. PCR products were separated on an 8% non-denaturing polyacrylamide gel, followed by silver staining. All polymorphic DNA markers for 6SS are listed in Supplemental Table 1. In the physical map, the order of molecular markers was adopted from the reference genome of Ae. speltoides accession AEG-9674-1 (Avni et al., 2022).

Isolation of the candidate gene for Pm12 (Pm12-can)

Three primer pairs were used to isolate the genes or fragments at the Pm12 locus. The first pair, Pm12-I-1, was designed according to D. villosum DvRGA1 and used to isolate a DvRGA1-like gene from genomic DNA of the line WL31. The second pair, Pm12-I-2, was designed based on the conserved sequences of Pm21 alleles reported previously (He et al., 2020) and used to amplify the DNA fragment that covered most of the coding sequence of Pm12-can, the candidate for Pm12. The third primer pair, Pm12-I-3, was designed based on the terminus of DvRGA1 and the head of Pm21 and was used for isolating the intergenic region between the two genes. Subsequently, the downstream sequence of Pm12-can was obtained by thermal-asymmetric-interlaced (TAIL) PCR (Liu and Huang, 1998). The genomic sequence of the Pm12 locus was then assembled, and the corresponding cDNA sequences of the two genes were confirmed by reverse transcriptase (RT)–PCR. The high fidelity PrimeSTAR Max Premix (TaKaRa, Japan) was used for all PCR amplification. All primers used in this study are listed in Supplemental Table 4.

RT–PCR and quantitative RT–PCR analyses

Total RNA was extracted from the first fully expanded leaves of seedlings from lines WL31, 1338-8, Yangmai 18, and Chinese Spring, with or without Bgt01 inoculation, using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Two micrograms of total RNA were used for synthesis of first-strand cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan). qPCR was performed on an ABI 7300 Real Time PCR System (Life Technologies, Carlsbad, CA, USA) as previously described (He et al., 2016). The wheat actin gene was used as the reference gene (Bahrini et al., 2011). When performing qPCR analysis, three independent biological replicates of each line were used. Statistical significance of differences was assessed using Student’s t-test.

Sequence analysis

Pm12 was used to perform BLAST searches against the genomes of durum wheat cv. Svevo (Maccaferri et al., 2019), wild emmer accession Zavitan (Avni et al., 2017), T. urartu accession G1812 (Ling et al., 2018), Ae. tauschii accession AL8/78 (Luo et al., 2017), Ae. speltoides accessions AEG-9674-1 and TS01 (Avni et al., 2022; Li et al., 2022), and multiple wheat cultivars (International Wheat Genome Sequencing Consortium et al., 2018; Guo et al., 2020; Walkowiak et al., 2020; Sato et al., 2021). The matched sequences with the highest identities from homeologous group 6 of wheat species were considered to be Pm12/Pm21 orthologs whose exon–intron structures were predicted according to those of Pm12 and Pm21. The DvRGA1-like and DvRGA2-like sequences were also obtained from the above genomes after BLAST analysis and annotated based on the expression data of DvRGA1 and DvRGA2 from D. villosum (He et al., 2018). TEs were predicted by comparison with the TREP database (https://trep-db.uzh.ch). Other DNA REs were found by BLAST searches against the reference genome of wheat cv. Chinese Spring (RefSeq v1.1). Nucleotide and amino acid sequence identities were calculated with DNASTAR software (Burland 2000). Protein domain prediction and multiple sequence alignment were performed with SMART (Letunic et al., 2015) and CLUSTAL W (Thompson et al., 1994), respectively. Annotation of LRR motifs was performed as described in our previous work (He et al., 2020). The phylogenetic tree was constructed based on the (putative) full-length proteins by the neighbor-joining method with the Poisson model in MEGA7 software (Kumar et al., 2016).

Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) analysis

BSMV-VIGS was used to perform a transient assay to investigate the potential functional relationship between Pm12 and Pm21. The recombinant virus BSMV:Pm21as (syn. BSMV:DvRGA2as) harboring the DNA fragment of the Pm21 gene was used to infect the leaves of WL31 carrying Pm12. BSMV:Pm21as-infected YM18 (harboring Pm21) and BSMV:00 (harboring empty vector)-infected WL31 were used as positive and negative controls, respectively. At 6 days, the virus-infected leaves were challenged with the avirulent isolate Bgt01. At 5 days after Bgt inoculation, the powdery mildew reactions were observed. Details of the silencing test are described in our previous work (He et al., 2016, 2018).

Mutation analysis

About 500 dry seeds of the powdery mildew-resistant line 1338-8 that were harvested in the most recent season and had a high germination rate were exposed to 60Co-γ-ray irradiation with 200 Gy at a dosage rate of 1.0 Gy/min (Zhang et al., 2015). After irradiation, the seeds were sown immediately in the field, and 316 M2 families were generated. Independently, ∼2000 seeds of 1338-8 were treated with 0.6% EMS, resulting in 1206 M2 families. About 100 individuals of each M2 family were used to screen for susceptible mutants by inoculation with Bgt isolate Bgt01, which is avirulent to Pm12. The resistant line 1338-8 and the susceptible line YM9 were used as the controls. Susceptible mutants obtained by irradiation were then used for molecular marker analysis to test for potential chromosomal deletion. The candidate gene Pm12-can in each mutant line was amplified from its genomic DNA using the primer pair Pm12-GT and subjected to Sanger sequencing. Each mutated Pm12-can gene was verified independently by sequencing the PCR product harboring the candidate mutation site. The DvRGA1-like gene in each mutant was also amplified by PCR and sequenced.

Plasmid construction

For wheat transformation, cDNA with the entire ORF of Pm12-can was cloned into the binary vector pLGY02 at the SmaI and SpeI sites. The resulting construct was designated pLGY02-Pm12-can and expressed the candidate gene under the control of a maize ubiquitin promoter. For single-cell transient assays in wheat, the Pm12/Pm21 orthologs Pm21-CS6A, Pm21-CS6B, and Pm21-CS6D were amplified from wheat cv. Chinese Spring cDNA and individually cloned into the destination vector pUbi-GW using gene-specific primers with Gateway technology (Shen et al., 2007). As a positive control, Pm12 was also inserted into the same vector. For transient assays in N. benthamiana, the coding sequences of Pm12 and Pm21 were amplified and inserted into the pS1300-GFP-Nos or pS1300-myc-Nos vector to create fusions with GFP or Myc tags at the C terminus. Genes on the constructs were driven by a constitutive promoter (Dong et al., 2018; Gao et al., 2020).

Wheat transformation

The construct pLGY02-Pm12-can was used to transform the susceptible wheat cv. Fielder by Agrobacterium tumefaciens (agrobacterium)-mediated transformation (Zhang et al., 2018). Both T0 and T1 plants were tested for the presence of the transgene by PCR amplification with primer pair Pm12-MA, and cv. Fielder was used as the negative control. Powdery mildew responses of three plants from each T2 line were tested at the one-leaf stage after inoculation with different Bgt isolates.

Single-cell transient assay in wheat leaves

Single-cell transient expression assays were performed as described previously (Shen et al., 2007). In brief, a mixture of the construct expressing an individual Pm12/Pm21 ortholog and the pUBI:GUS reporter construct was introduced into leaves of the susceptible wheat cv. KN199 by biolistic bombardment. After 4 h, the leaves were inoculated with Bgt isolate E18 (held by the lab of Dr. Qian-Hua Shen, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China) for 48 h. Two days after inoculation, the leaves were GUS stained, and fungal structures were examined under a microscope. The haustorium index (i.e., the percentage of GUS-stained cells with haustoria among all GUS-stained cells invaded by Bgt) was used to assess whether the tested genes conferred resistance to powdery mildew. Constructs carrying Pm12 or the empty vector were used as positive and negative controls, respectively.

Agroinfiltration of N. benthamiana leaves

Agrobacterium-mediated transient expression (agroinfiltration) was conducted as described in Liu et al. (2010) with minor modifications. In brief, the appropriate plasmid construct was transformed into Agrobacterium strain GV3101. The Agrobacterium strains were then grown to an optical OD600 of about 1.0. The cultures were harvested by centrifugation, resuspended in infiltration medium containing 2% sucrose, 0.5% MS, 10 mM MES (pH 5.6), and 200 μM acetosyringone, adjusted to an OD600 from 0.5 to 1, and incubated for 2–4 h at room temperature before infiltration. N. benthamiana leaves were used for infiltration. For co-expression of multiple proteins, agrobacteria carrying different constructs were mixed in a 1:1 ratio and used for infiltration. After infiltration, plants were maintained in growth chambers for subsequent assays.

Cell death assay on N. benthamiana leaves

The cell death assay was performed as described by Bai et al. (2012) and Gao et al. (2020). In brief, each Agrobacterium strain carrying a plasmid construct was infiltrated into a 1-cm circular region on five different leaves for three independent experiments. Leaves were photographed at 24–48 h after infiltration, soaked in trypan blue solution (10 ml lactic acid, 10 ml glycerol, 10 g phenol, 10 mg trypan blue, and 50 ml ethanol dissolved in 30 ml distilled water), and heated in a boiling water bath for 5 min. Leaves were then destained in 2.5 g ml−1 chloral hydrate solution until the background was clear.

Protein extraction, immunoblotting, and co-immunoprecipitation assay

Agrobacterium strain GV3101 carrying constructs expressing GFP- or Myc-fused proteins were mixed and infiltrated into N. benthamiana leaves as described above. Before cell death was visible (at about 20 h post infiltration), the total leaf proteins were extracted in 0.5 ml plant extraction buffer (CWBIO, Beijing, China), followed by centrifugation at 12 000 g for 20 min. Portions of the supernatants were saved as input samples for later SDS–PAGE and immunoblot analysis. For the Co-IP assay, the remaining supernatant was incubated with 4 μg of anti-Myc monoclonal antibody (Proteintech, Chicago, IL, USA) at 4°C for 2–4 h. The antibody–protein complexes were captured by incubation with 18 μl of agarose resin conjugated with protein A/G for 2–4 h at 4°C. After washing six times with IP buffer, the immunocomplexes were heated at 95°C for 5 min in 50 μl 1× SDS loading buffer. The supernatant after centrifugation was used for immunoblot analysis (Gao et al., 2020).

Funding

This study was supported by grants from the National Natural Science Foundation of China (32171990, 32072053, 31971874, 31872009, and U1604116), the Key Research and Development Program of Zhenjiang (NY2021001), the State Key Laboratory of Plant Cell and Chromosome Engineering (PCCE-KF-2021-05, PCCE-KF-2022-07), the State Key Laboratory of Crop Biology in Shandong Agricultural University (2021KF01), the Taishan Scholars Project (tsqn201812123), and the Key Research and Development Program of Yantai (2019YT06000470).

Author contributions

H.H., A.G., and P.M. conceived and designed the research. S.Z., C.L., S.G., Z.C., R.C., T.L., RL., H.D., R.G., G.L., M.L., R.F., and H.Z. performed experiments. S.Z., S.G., Z.L., Q.S., and H.H. analyzed the data. S.Z., C.L., S.G., A.G., P.M., and H.H. wrote the manuscript. All authors read and approved the final manuscript. S.Z., C.L., and S.G. contributed equally to this work.

Acknowledgments

The authors thank Dr. Jie Zhang (Institute of Biotechnology and Nuclear Technology Research, Sichuan Academy of Agricultural Sciences) for irradiation of wheat seeds with 60Co-γ-ray. The authors thank Prof. Yong Q. Gu (USDA-ARS Western Regional Research Center) for constructive comments on this manuscript. We are also grateful to Prof. Emerita Paula Jameson (University of Canterbury, New Zealand), financially supported by the “Double Hundred” Plan for Foreign Experts in Shandong Province, China, for critical review and editing of this manuscript. All authors declare no conflict of interest.

Published: November 9, 2022

Footnotes

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information can be found online at https://doi.org/10.1016/j.xplc.2022.100472.

Contributor Information

Anli Gao, Email: algao@henu.edu.cn.

Pengtao Ma, Email: ptma@ytu.edu.cn.

Huagang He, Email: hghe@ujs.edu.cn.

Accession numbers

The genomic sequence of Pm12 has been deposited in GenBank under accession number MN334940.

Supplemental information

Document S1. Supplemental Figures 1–4 and Supplemental Tables 1–4
mmc1.pdf (474.3KB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (2.9MB, pdf)

References

  1. An D., Ma P., Zheng Q., Fu S., Li L., Han F., Han G., Wang J., Xu Y., Jin Y., et al. Development and molecular cytogenetic identification of a new wheat-rye 4R chromosome disomic addition line with resistances to powdery mildew, stripe rust and sharp eyespot. Theor. Appl. Genet. 2019;132:257–272. doi: 10.1007/s00122-018-3214-3. [DOI] [PubMed] [Google Scholar]
  2. An D., Zheng Q., Zhou Y., Ma P., Lv Z., Li L., Li B., Luo Q., Xu H., Xu Y. Molecular cytogenetic characterization of a new wheat-rye 4R chromosome translocation line resistant to powdery mildew. Chromosome Res. 2013;21:419–432. doi: 10.1007/s10577-013-9366-8. [DOI] [PubMed] [Google Scholar]
  3. Avni R., Lux T., Minz-Dub A., Millet E., Sela H., Distelfeld A., Deek J., Yu G., Steuernagel B., Pozniak C., et al. Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement. Plant J. 2022;110:179–192. doi: 10.1111/tpj.15664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Avni R., Nave M., Barad O., Baruch K., Twardziok S.O., Gundlach H., Hale I., Mascher M., Spannagl M., Wiebe K., et al. Wild emmer genome architecture and diversity elucidate wheat evolution and domestication. Science. 2017;357:93–97. doi: 10.1126/science.aan0032. [DOI] [PubMed] [Google Scholar]
  5. Bahrini I., Ogawa T., Kobayashi F., Kawahigashi H., Handa H. Overexpression of the pathogen-inducible wheat TaWRKY45 gene confers disease resistance to multiple fungi in transgenic wheat plants. Breed Sci. 2011;61:319–326. doi: 10.1270/jsbbs.61.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bai S., Liu J., Chang C., Zhang L., Maekawa T., Wang Q., Xiao W., Liu Y., Chai J., Takken F.L.W., et al. Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance. PLoS Pathog. 2012;8:e1002752. doi: 10.1371/journal.ppat.1002752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baudin M., Hassan J.A., Schreiber K.J., Lewis J.D. Analysis of the ZAR1 immune complex reveals determinants for immunity and molecular interactions. Plant Physiol. 2017;174:2038–2053. doi: 10.1104/pp.17.00441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bauer S., Yu D., Lawson A.W., Saur I.M.L., Frantzeskakis L., Kracher B., Logemann E., Chai J., Maekawa T., Schulze-Lefert P. The leucine-rich repeats in allelic barley MLA immune receptors define specificity towards sequenceunrelated powdery mildew avirulence effectors with a predicted common RNase-like fold. PLoS Pathog. 2021;17:e1009223. doi: 10.1371/journal.ppat.1009223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bendahmane A., Farnham G., Moffett P., Baulcombe D.C. Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato. Plant J. 2002;32:195–204. doi: 10.1046/j.1365-313x.2002.01413.x. [DOI] [PubMed] [Google Scholar]
  10. Bernoux M., Burdett H., Williams S.J., Zhang X., Chen C., Newell K., Lawrence G.J., Kobe B., Ellis J.G., Anderson P.A., Dodds P.N. Comparative analysis of the flax immune receptors L6 and L7 suggests an equilibrium-based switch activation model. Plant Cell. 2016;28:146–159. doi: 10.1105/tpc.15.00303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bie T., Zhao R., Jiang Z., Gao D., Zhang B., He H. Efficient marker-assisted screening of structural changes involving Haynaldia villosa chromosome 6V using a double-distal-marker strategy. Mol. Breeding. 2015;35:34. [Google Scholar]
  12. Bie T., Zhao R., Zhu S., Chen S., Cen B., Zhang B., Gao D., Jiang Z., Chen T., Wang L., et al. Development and characterization of marker MBH1 simultaneously tagging genes Pm21 and PmV conferring resistance to powdery mildew in wheat. Mol. Breeding. 2015;35:189. [Google Scholar]
  13. Bourras S., Kunz L., Xue M., Praz C.R., Müller M.C., Kälin C., Schläfli M., Ackermann P., Flückiger S., Parlange F., et al. The AvrPm3-Pm3 effector-NLR interactions control both race-specific resistance and host-specificity of cereal mildews on wheat. Nat. Commun. 2019;10:2292. doi: 10.1038/s41467-019-10274-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Bourras S., McNally K.E., Ben-David R., Parlange F., Roffler S., Praz C.R., Oberhaensli S., Menardo F., Stirnweis D., Frenkel Z., et al. Multiple avirulence loci and allele-specific effector recognition control the Pm3 race-specific resistance of wheat to powdery mildew. Plant Cell. 2015;27:2991–3012. doi: 10.1105/tpc.15.00171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Burland T.G. DNASTAR’s Lasergene sequence analysis software. Methods Mol. Biol. 2000;132:71–91. doi: 10.1385/1-59259-192-2:71. [DOI] [PubMed] [Google Scholar]
  16. Cao A., Xing L., Wang X., et al. Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat. Proc. Natl. Acad. Sci. USA. 2011;108:7727–7732. doi: 10.1073/pnas.1016981108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Césari S., Kanzaki H., Fujiwara T., Bernoux M., Chalvon V., Kawano Y., Shimamoto K., Dodds P., Terauchi R., Kroj T. The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance. EMBO J. 2014;33:1941–1959. doi: 10.15252/embj.201487923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Cesari S., Moore J., Chen C., Webb D., Periyannan S., Mago R., Bernoux M., Lagudah E.S., Dodds P.N. Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins. Proc. Natl. Acad. Sci. USA. 2016;113:10204–10209. doi: 10.1073/pnas.1605483113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Chen P., You C., Hu Y., Chen S., Zhou B., Cao A., Wang X. Radiation-induced translocations with reduced Haynaldia villosa chromatin at the Pm21 locus for powdery mildew resistance in wheat. Mol. Breeding. 2013;31:477–484. [Google Scholar]
  20. Dong H., Bai L., Zhang Y., Zhang G., Mao Y., Min L., Xiang F., Qian D., Zhu X., Song C.P. Modulation of guard cell turgor and drought tolerance by a peroxisomal acetate-malate shunt. Mol. Plant. 2018;11:1278–1291. doi: 10.1016/j.molp.2018.07.008. [DOI] [PubMed] [Google Scholar]
  21. El Kasmi F., Chung E.H., Anderson R.G., Li J., Wan L., Eitas T.K., Gao Z., Dangl J.L. Signaling from the plasma-membrane localized plant immune receptor RPM1 requires self-association of the full-length protein. Proc. Natl. Acad. Sci. USA. 2017;114:E7385–E7394. doi: 10.1073/pnas.1708288114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gao A., Hu M., Gong Y., Dong R., Jiang Y., Zhu S., Ji J., Zhang D., Li S., He H. Pm21 CC domain activity modulated by intramolecular interactions is implicated in cell death and disease resistance. Mol. Plant Pathol. 2020;21:975–984. doi: 10.1111/mpp.12943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gaurav K., Arora S., Silva P., Sánchez-Martín J., Horsnell R., Gao L., Brar G.S., Widrig V., John Raupp W., Singh N., et al. Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement. Nat. Biotechnol. 2022;40:422–431. doi: 10.1038/s41587-021-01058-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Guo W., Xin M., Wang Z., Yao Y., Hu Z., Song W., Yu K., Chen Y., Wang X., Guan P., et al. Origin and adaptation to high altitude of Tibetan semi-wild wheat. Nat. Commun. 2020;11:5085. doi: 10.1038/s41467-020-18738-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. He H., Du H., Liu R., Liu T., Yang L., Gong S., Tang Z., Du H., Liu C., Han R., et al. Characterization of a new gene for resistance to wheat powdery mildew on chromosome 1RL of wild rye Secale sylvestre. Theor. Appl. Genet. 2021;134:887–896. doi: 10.1007/s00122-020-03739-1. [DOI] [PubMed] [Google Scholar]
  26. He H., Ji J., Li H., Tong J., Feng Y., Wang X., Han R., Bie T., Liu C., Zhu S. Genetic diversity and evolutionary analyses reveal the powdery mildew resistance gene Pm21 undergoing diversifying selection. Front. Genet. 2020;11:489. doi: 10.3389/fgene.2020.00489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. He H., Ji Y., Zhu S., Li B., Zhao R., Jiang Z., Bie T. Genetic, physical and comparative mapping of the powdery mildew resistance gene Pm21 originating from Dasypyrum villosum. Front. Plant Sci. 2017;8:1914. doi: 10.3389/fpls.2017.01914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. He H., Liu R., Ma P., Du H., Zhang H., Wu Q., Yang L., Gong S., Liu T., Huo N., et al. Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI 1796. Theor. Appl. Genet. 2021;134:53–62. doi: 10.1007/s00122-020-03681-2. [DOI] [PubMed] [Google Scholar]
  29. He H., Zhu S., Jiang Z., Ji Y., Wang F., Zhao R., Bie T. Comparative mapping of powdery mildew resistance gene Pm21 and functional characterization of resistance-related genes in wheat. Theor. Appl. Genet. 2016;129:819–829. doi: 10.1007/s00122-016-2668-4. [DOI] [PubMed] [Google Scholar]
  30. He H., Zhu S., Sun W., Gao D., Bie T. Efficient development of Haynaldia villosa chromosome 6VS-specific DNA markers using a CISP-IS strategy. Plant Breed. 2013;132:290–294. [Google Scholar]
  31. He H., Zhu S., Zhao R., Jiang Z., Ji Y., Ji J., Qiu D., Li H., Bie T. Pm21, encoding a typical CC-NBS-LRR protein, confers broad-spectrum resistance to wheat powdery mildew disease. Mol. Plant. 2018;11:879–882. doi: 10.1016/j.molp.2018.03.004. [DOI] [PubMed] [Google Scholar]
  32. Hewitt T., Müller M.C., Molnár I., Mascher M., Holušová K., Šimková H., Kunz L., Zhang J., Li J., Bhatt D., et al. A highly differentiated region of wheat chromosome 7AL encodes a Pm1a immune receptor that recognises its corresponding AvrPm1a effector from Blumeria graminis. New Phytol. 2021;229:2812–2826. doi: 10.1111/nph.17075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hurni S., Brunner S., Buchmann G., Herren G., Jordan T., Krukowski P., Wicker T., Yahiaoui N., Mago R., Keller B. Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew. Plant J. 2013;76:957–969. doi: 10.1111/tpj.12345. [DOI] [PubMed] [Google Scholar]
  34. International Wheat Genome Sequencing Consortium (IWGSC), IWGSC RefSeq Principal Investigators, Appels R., Eversole K., Feuillet C., Keller B., Rogers J., Stein N., IWGSC Whole-Genome Assembly Principal Investigators, Pozniak C.J., et al. Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science. 2018;361:eaar7191. doi: 10.1126/science.aar7191. [DOI] [PubMed] [Google Scholar]
  35. Jia J., Devos K.M., Chao S., Miller T.E., Reader S.M., Gale M.D. RFLP-based maps of the homoeologous group-6 chromosomes of wheat and their application in that tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat. Theor. Appl. Genet. 1996;92:559–565. doi: 10.1007/BF00224558. [DOI] [PubMed] [Google Scholar]
  36. Krattinger S.G., Lagudah E.S., Spielmeyer W., Singh R.P., Huerta-Espino J., McFadden H., Bossolini E., Selter L.L., Keller B. A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat. Science. 2009;323:1360–1363. doi: 10.1126/science.1166453. [DOI] [PubMed] [Google Scholar]
  37. Kumar S., Stecher G., Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016;33:1870–1874. doi: 10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Letunic I., Doerks T., Bork P. SMART: recent updates, new developments and status in 2015. Nucleic Acids Res. 2015;43:D257–D260. doi: 10.1093/nar/gku949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Li L.F., Zhang Z.B., Wang Z.H., Li N., Sha Y., Wang X.F., Ding N., Li Y., Zhao J., Wu Y., et al. Genome sequences of the five Sitopsis species of Aegilops and the origin of polyploid wheat B-subgenome. Mol. Plant. 2022;15:488–503. doi: 10.1016/j.molp.2021.12.019. [DOI] [PubMed] [Google Scholar]
  40. Li M., Dong L., Li B., Wang Z., Xie J., Qiu D., Li Y., Shi W., Yang L., Wu Q., et al. A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytol. 2020;228:1027–1037. doi: 10.1111/nph.16761. [DOI] [PubMed] [Google Scholar]
  41. Ling H.Q., Ma B., Shi X., Liu H., Dong L., Sun H., Cao Y., Gao Q., Zheng S., Li Y., et al. Genome sequence of the progenitor of wheat A subgenome Triticum urartu. Nature. 2018;557:424–428. doi: 10.1038/s41586-018-0108-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Liu L., Zhang Y., Tang S., Zhao Q., Zhang Z., Zhang H., Dong L., Guo H., Xie Q. An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana. Plant J. 2010;61:893–903. doi: 10.1111/j.1365-313X.2009.04109.x. [DOI] [PubMed] [Google Scholar]
  43. Liu Y.G., Huang N. Efficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR. Plant Mol. Biol. Rep. 1998;16:175–181. [Google Scholar]
  44. Lu P., Guo L., Wang Z., Li B., Li J., Li Y., Qiu D., Shi W., Yang L., Wang N., et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat. Commun. 2020;11:680. doi: 10.1038/s41467-020-14294-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Luo M.C., Gu Y.Q., Puiu D., Wang H., Twardziok S.O., Deal K.R., Huo N., Zhu T., Wang L., Wang Y., et al. Genome sequence of the progenitor of the wheat D genome Aegilops tauschii. Nature. 2017;551:498–502. doi: 10.1038/nature24486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Maccaferri M., Harris N.S., Twardziok S.O., Pasam R.K., Gundlach H., Spannagl M., Ormanbekova D., Lux T., Prade V.M., Milner S.G., et al. Durum wheat genome highlights past domestication signatures and future improvement targets. Nat. Genet. 2019;51:885–895. doi: 10.1038/s41588-019-0381-3. [DOI] [PubMed] [Google Scholar]
  47. McIntosh R.A., Dubcovsky J., Rogers W.J., Morris C., Xia X.C. KOMUGI Wheat Genetic Resource Database; 2017. Catalogue of Gene Symbols for Wheat: 2017 Supplement.https://shigen.nig.ac. jp/wheat/komugi/genes/symbolClassList.jsp [Google Scholar]
  48. Miller T.E., Reader S.M., Ainsworth C.C., Summer R.W. In: Cereal Breeding Related to Integrated Cereal Production: Proceedings of the EUCARPIA Conference. Jorna M., Shootmaker L., editors. Pudoc; Wageningen, Netherlands: 1988. The introduction of a major gene for resistance to powdery mildew of wheat, Erysiphe graminis f. sp. tritici, from Aegilops speltoides into wheat, Triticum aestivum; pp. 179–183. [Google Scholar]
  49. Moffett P., Farnham G., Peart J., Baulcombe D.C. Interaction between domains of a plant NBS–LRR protein in disease resistance-related cell death. EMBO J. 2002;21:4511–4519. doi: 10.1093/emboj/cdf453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Moore J.W., Herrera-Foessel S., Lan C., Schnippenkoetter W., Ayliffe M., Huerta-Espino J., Lillemo M., Viccars L., Milne R., Periyannan S., et al. A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat. Genet. 2015;47:1494–1498. doi: 10.1038/ng.3439. [DOI] [PubMed] [Google Scholar]
  51. Müller M.C., Kunz L., Schudel S., Lawson A.W., Kammerecker S., Isaksson J., Wyler M., Graf J., Sotiropoulos A.G., Praz C.R., et al. Ancient variation of the AvrPm17 gene in powdery mildew limits the effectiveness of the introgressed rye Pm17 resistance gene in wheat. Proc. Natl. Acad. Sci. USA. 2022;119 doi: 10.1073/pnas.2108808119. e2108808119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Niewoehner A.S., Leath S. Virulence of Blumeria graminis f. sp. tritici on winter wheat in the eastern United States. Plant Dis. 1998;82:64–68. doi: 10.1094/PDIS.1998.82.1.64. [DOI] [PubMed] [Google Scholar]
  53. Praz C.R., Bourras S., Zeng F., Sánchez-Martín J., Menardo F., Xue M., Yang L., Roffler S., Böni R., Herren G., et al. AvrPm2 encodes an RNase-like avirulence effector which is conserved in the two different specialized forms of wheat and rye powdery mildew fungus. New Phytol. 2016;213:1301–1314. doi: 10.1111/nph.14372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Qi L.L., Wang S.L., Chen P.D., Liu D.J., Gill B.S. Identification and physical mapping of three Haynaldia villosa chromosome-6V deletion lines. Theor. Appl. Genet. 1998;97:1042–1046. [Google Scholar]
  55. Qi X., Cui F., Yu L., Ding A., Li J., Chen G., Wang H. Molecular tagging wheat powdery mildew resistance gene Pm21 by EST-SSR and STS markers. Mol. Plant Breed. 2010;8:1–6. [Google Scholar]
  56. Rairdan G.J., Collier S.M., Sacco M.A., Baldwin T.T., Boettrich T., Moffett P. The coiled-coil and nucleotide binding domains of the potato Rx disease resistance protein function in pathogen recognition and signaling. Plant Cell. 2008;20:739–751. doi: 10.1105/tpc.107.056036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sánchez-Martín J., Keller B. Contribution of recent technological advances to future resistance breeding. Theor. Appl. Genet. 2019;132:713–732. doi: 10.1007/s00122-019-03297-1. [DOI] [PubMed] [Google Scholar]
  58. Sánchez-Martín J., Steuernagel B., Ghosh S., Herren G., Hurni S., Adamski N., Vrána J., Kubaláková M., Krattinger S.G., Wicker T., et al. Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biol. 2016;17:221. doi: 10.1186/s13059-016-1082-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sánchez-Martín J., Widrig V., Herren G., Wicker T., Zbinden H., Gronnier J., Spörri L., Praz C.R., Heuberger M., Kolodziej M.C., et al. Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins. Nat. Plants. 2021;7:327–341. doi: 10.1038/s41477-021-00869-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Sato K., Abe F., Mascher M., Haberer G., Gundlach H., Spannagl M., Shirasawa K., Isobe S. Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar ‘Fielder. DNA Res. 2021;28:dsab008. doi: 10.1093/dnares/dsab008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Shen Q.H., Saijo Y., Mauch S., Biskup C., Bieri S., Keller B., Seki H., Ulker B., Somssich I.E., Schulze-Lefert P. Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science. 2007;315:1098–1103. doi: 10.1126/science.1136372. [DOI] [PubMed] [Google Scholar]
  62. Singh S.P., Hurni S., Ruinelli M., Brunner S., Sanchez-Martin J., Krukowski P., Peditto D., Buchmann G., Zbinden H., Keller B. Evolutionary divergence of the rye Pm17 and Pm8 resistance genes reveals ancient diversity. Plant Mol. Biol. 2018;98:249–260. doi: 10.1007/s11103-018-0780-3. [DOI] [PubMed] [Google Scholar]
  63. Song W., Xie C., Du J., Xie H., Liu Q., Ni Z., Yang T., Sun Q.,, Liu Z. A “one-marker-for-two-genes” approach for efficient molecular discrimination of Pm12 and Pm21 conferring resistance to powdery mildew in wheat. Mol. Breeding. 2009;23:357–363. [Google Scholar]
  64. Song W., Xie H., Liu Q., Xie C., Ni Z., Yang T., Sun Q., Liu Z. Molecular identification of Pm12-carrying introgression lines in wheat using genomic and EST-SRR markers. Euphytica. 2007;158:95–102. [Google Scholar]
  65. Thompson J.D., Higgins D.G., Gibson T.J. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Walkowiak S., Gao L., Monat C., Haberer G., Kassa M.T., Brinton J., Ramirez-Gonzalez R.H., Kolodziej M.C., Delorean E., Thambugala D., et al. Multiple wheat genomes reveal global variation in modern breeding. Nature. 2020;588:277–283. doi: 10.1038/s41586-020-2961-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wan Q., Wang T., Ma Y., Liu W., Cao Z., Qi Y., Zhang C., Ding K., Fan J., Zhou Y., et al. Monitoring and analysis of virulence of Blumeria graminis f.sp. tritici population in Anhui Province. Acta Phytopathol. Sin. 2021;51:741–747. (In Chinese) [Google Scholar]
  68. Wang G.F., Ji J., El-Kasmi F., Dangl J.L., Johal G., Balint-Kurti P.J. Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity. PLoS Pathog. 2015;11:e1004674. doi: 10.1371/journal.ppat.1004674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wang J., Hu M., Wang J., Qi J., Han Z., Wang G., Qi Y., Wang H.W., Zhou J.M., Chai J. Reconstitution and structure of a plant NLR resistosome conferring immunity. Science. 2019;364:eaav5870. doi: 10.1126/science.aav5870. [DOI] [PubMed] [Google Scholar]
  70. Wang J., Wang J., Hu M., Wu S., Qi J., Wang G., Han Z., Qi Y., Gao N., Wang H.W., et al. Ligand-triggered allosteric ADP release primes a plant NLR complex. Science. 2019;364:eaav5868. doi: 10.1126/science.aav5868. [DOI] [PubMed] [Google Scholar]
  71. Wulff B.B.H., Moscou M.J. Strategies for transferring resistance into wheat: from wide crosses to GM cassettes. Front. Plant Sci. 2014;5:692. doi: 10.3389/fpls.2014.00692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Wulff B.B.H., Heese A., Tomlinson-Buhot L., Jones D.A., de la Peña M., Jones J.D.G. The major specificity-determining amino acids of the tomato Cf-9 disease resistance protein are at hypervariable solvent-exposed positions in the central leucine-rich repeats. Mol. Plant Microbe Interact. 2009;22:1203–1213. doi: 10.1094/MPMI-22-10-1203. [DOI] [PubMed] [Google Scholar]
  73. Xie J., Guo G., Wang Y., Hu T., Wang L., Li J., Qiu D., Li Y., Wu Q., Lu P., et al. A rare single nucleotide variant in Pm5e confers powdery mildew resistance in common wheat. New Phytol. 2020;228:1011–1026. doi: 10.1111/nph.16762. [DOI] [PubMed] [Google Scholar]
  74. Xing L., Hu P., Liu J., Witek K., Zhou S., Xu J., Zhou W., Gao L., Huang Z., Zhang R., et al. Pm21 from Haynaldia villosa encodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat. Mol. Plant. 2018;11:874–878. doi: 10.1016/j.molp.2018.02.013. [DOI] [PubMed] [Google Scholar]
  75. Yahiaoui N., Srichumpa P., Dudler R., Keller B. Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat. Plant J. 2004;37:528–538. doi: 10.1046/j.1365-313x.2003.01977.x. [DOI] [PubMed] [Google Scholar]
  76. Zhang D., Zhu K., Dong L., Liang Y., Li G., Fang T., Guo G., Wu Q., Xie J., Chen Y., et al. Wheat powdery mildew resistance gene Pm64 derived from wild emmer (Triticum turgidum var. dicoccoides) is tightly linked in repulsion with stripe rust resistance gene Yr5. Crop J. 2019;7:761–770. [Google Scholar]
  77. Zhang J., Jiang Y., Guo Y., Li G., Yang Z., Xu D., Xuan P. Identification of novel chromosomal aberrations induced by 60Co-γ-irradiation in wheat-Dasypyrum villosum lines. Int. J. Mol. Sci. 2015;16:29787–29796. doi: 10.3390/ijms161226134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Zhang S., Zhang R., Song G., Gao J., Li W., Han X., Chen M., Li Y., Li G. Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat. BMC Plant Biol. 2018;18:302. doi: 10.1186/s12870-018-1496-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Zhao G., Zou C., Li K., Wang K., Li T., Gao L., Zhang X., Wang H., Yang Z., Liu X., et al. The Aegilops tauschii genome reveals multiple impacts of transposons. Nat. Plants. 2017;3:946–955. doi: 10.1038/s41477-017-0067-8. [DOI] [PubMed] [Google Scholar]
  80. Zhao Y.B., Liu M.X., Chen T.T., Ma X., Li Z.K., Zheng Z., Zheng S.R., Chen L., Li Y.Z., Tang L.R., et al. Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism. Sci. Adv. 2022;8:eabq5108. doi: 10.1126/sciadv.abq5108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Zhu S., Ji Y., Ji J., Bie T., Gao A., He H. Fine physical bin mapping of the powdery mildew resistance gene Pm21 based on chromosomal structural variations in wheat. Int. J. Mol. Sci. 2018;19:643. doi: 10.3390/ijms19020643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Zou S., Wang H., Li Y., Kong Z., Tang D. The NB-LRR gene Pm60 confers powdery mildew resistance in wheat. New Phytol. 2018;218:298–309. doi: 10.1111/nph.14964. [DOI] [PubMed] [Google Scholar]

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