Pm21, Encoding a Typical CC-NBS-LRR Protein, Confers Broad-Spectrum Resistance to Wheat Powdery Mildew Disease

Common wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) is one of the most widely cultivated cereal crops, providing ∼20% of the calories consumed by humans (Fu et al., 2009Fu D. Uauy C. Distelfeld A. Blechl A. Epstein L. Chen X. Sela H. Fahima T. Dubcovsky J. A kinase-START gene confers temperature-dependent resistance to wheat stripe rust.Science. 2009; 323: 1357-1360Crossref PubMed Scopus (505) Google Scholar). However, wheat production is constantly challenged by powdery mildew disease, which occurs globally and is caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt). Utilization of powdery mildew resistance (Pm) genes is the most effective and economical way to control powdery mildew disease. To date, more than 100 Pm genes or alleles have been documented (McIntosh et al., 2017McIntosh R.A. Dubcovsky J. Rogers W.J. Morris C. Xia X.C. Catalogue of gene symbols for wheat: 2017 supplement. KOMUGI Wheat Genetic Resource Database, 2017https://shigen.nig.ac.jp/wheat/komugi/genes/symbolClassList.jspGoogle Scholar). Some of them can provide highly effective resistance against powdery mildew, for examples, Pm12, Pm21, and Pm37. Among them, Pm21 originating from the Triticeae grass Dasypyrum villosum (2n = 2x = 14, VV) confers high resistance or immunity to all tested Bgt races. The wheat-D. villosum translocation line T6AL.6VS harboring Pm21 has been widely used in wheat breeding in China since 1995 (Chen et al., 2013Chen 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. Breed. 2013; 31: 477-484Crossref Scopus (62) Google Scholar). Nevertheless, the genetic basis of the broad-spectrum resistance conferred by Pm21 remains unclear. Due to the lack of recombination between 6VS and the wheat homoeologous counterpart (Qi et al., 1998Qi 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: 1042-1046Crossref Scopus (34) Google Scholar), the conventional map-based cloning strategy in wheat background is not effective for isolating Pm21. Recently, we discovered Bgt-susceptible D. villosum resources and constructed a genetic population using resistant and susceptible D. villosum lines, which allows the fine mapping of Pm21. Specifically, we mapped Pm21 to a 0.01 cM interval, which corresponds to a 112.5 kb genomic region in the model plant Brachypodium distachyon. Moreover, Pm21 was found to co-segregate with a resistance gene analog (RGA) marker (He et al., 2017He 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: 1914Crossref PubMed Scopus (24) Google Scholar). Here, we report the isolation of Pm21 and its functional validation using multiple means. The RGA marker and its surrounding genomic sequences were amplified from the resistant D. villosum line DvRes-1 by PCR and sequenced (Supplemental Figure 1). Then, a 17 732-bp genomic sequence harboring three genes (DvPP2C, DvRGA2, and DvRGA1) and a separated fragment containing DvRGA3 were assembled (Figure 1A). Genetic analysis showed that the three RGA genes all co-segregated with Pm21 in the F2 population developed by crossing Bgt-resistant (DvRes-1) and -susceptible (DvSus-1) D. villosum lines (Supplemental Figure 2). The genomic sequence of DvRGA1 was 2986 bp with two exons and one intron, whereas that of DvRGA2 was 3699 bp with three exons and two introns (Figure 1B). Both genes were intact and capable of yielding typical CC-NBC-LRR proteins upon conceptual translation. The exon-intron pattern of DvRGA3 was similar to that of DvRGA2, but its coding sequence for the LRR domain was disrupted by several premature stop codons, indicating that it is a pseudogene. Both DvRGA1 and DvRGA2 were constitutively expressed in the leaves of DvRes-1 without Bgt challenge, whereas the transcripts of DvRGA3 was undetectable (Supplemental Figure 3), consistent with the foregoing suggestion of DvRGA3 being a pseudogene. Quantitative RT–PCR (qPCR) showed that DvRGA1 and DvRGA2 had similar expression patterns in the Pm21-carrying cultivar Yangmai 18 inoculated with the Bgt isolate ZY01, whereas the transcript level of DvRGA2 was higher than that of DvRGA1 (Figure 1C). To investigate the involvement of DvRGA1 and DvRGA2 in Pm21-mediated resistance, we silenced these two genes in Yangmai 18 individually using virus-induced gene silencing (VIGS) mediated by Barley stripe mosaic virus (BSMV). We found that silencing of DvRGA2, but not that of DvRGA1, allowed abundant development of Bgt colonies with disease symptoms and fungal sporulation on the leaves (Figure 1D and Supplemental Figure 4), indicating that DvRGA2 is associated with Pm21 resistance. Subsequently, ethyl methanesulfonate (EMS)-induced mutagenesis was performed on Yangmai 18, and a total of 58 independent susceptible mutants were identified from 6408 M2 families (Figure 1E). Except Y18-S6, which had a deletion spanning the whole Pm21 locus (He et al., 2017He 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: 1914Crossref PubMed Scopus (24) Google Scholar), the remaining 57 susceptible mutants each harbored a mutated DvRGA2 sequence. Among them, 55 had single-base mutations in DvRGA2, and the other two, Y18-S35 and Y18-S43, had two-base changes. Of the 59 mutation sites, 43 rendered amino acid changes and 16 caused premature stop codons (Figure 1F and Supplemental Table 1). Concomitantly, we checked the flanking genes DvPP2C and DvRGA1 in 11 of the 58 susceptible mutants randomly selected and found no mutations in DvPP2C and DvRGA1. Southern blotting analysis also indicated that DvRGA2 is a single copy gene in D. villosum (Supplemental Figure 5). These results suggest that DvRGA2 is necessary for Pm21-mediated Bgt resistance in wheat. To validate if DvRGA2 is sufficient for conferring broad-spectrum Bgt resistance, DvRGA2 with its native promoter was transformed into a susceptible wheat cultivar Kenong 199 by particle bombardment. Of the 16 T1 lines obtained, six were validated as transgenic lines containing DvRGA2 (Supplemental Figure 6). Quantitative RT–PCR analysis showed that DvRGA2 was expressed in each of these six transgenic lines (Supplemental Figure 7). The T2 plants generated from these six lines showed immunity to the Bgt isolate YZ01 throughout the whole growth period (Figure 1G and 1H). Their T2 seedlings also conferred immunity to the other 17 purified Bgt isolates and 15 mixed Bgt cultures collected from different regions of China (Supplemental Table 2). Thus, we conclude that DvRGA2 is Pm21 and can confer broad-spectrum resistance to powdery mildew disease in transgenic wheat. Among the CC-NBS-LRR proteins reported in wheat, PM21 shared the highest identity (35.0%) with the stem rust resistance protein SR22, but poor identities (<20.0%) with the powdery mildew resistance proteins PM2, PM3b, and PM8 (Supplemental Figure 8). We searched for Pm21 orthologs in the common wheat genome and found that they were present but all disrupted by retrotransposon-like elements in the second intron. Disruptions in Pm21 orthologs were also observed in the species closely related to common wheat, such as T. dicoccoides, T. urartu, Aegilops speltoides, and Ae. tauschii (Supplemental Figure 9). It appears that the structural variations occur after the divergence between D. villosum and the above species. Hence, Pm21 is likely a relatively ancient Pm gene in the tribe Triticeae, but its orthologs in common wheat and closely related species have become pseudogenes. To investigate the diversity of Pm21 alleles, a total of 72 sequences (including Pm21) were obtained from 63 D. villosum accessions resistant to powdery mildew. Among them, 38 non-redundant alleles were used for further study. The encoding sequences of these alleles had 91.7%–100% identities, and their putative amino acid sequences shared 86.5%–100% identities (Supplemental Figure 10 and Supplemental Table 3). Nonsynonymous (Ka) and synonymous (Ks) nucleotide substitution rates of full-length proteins, different domains, and non-domain regions were determined. A relatively high Ka/Ks ratio (4.02) was observed only on the predicted solvent-exposed LRR residues (Meyers et al., 1998Meyers B.C. Shen K.A. Rohani P. Gaut B.S. Michelmore R.W. Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.Plant Cell. 1998; 11: 1833-1846Crossref Scopus (229) Google Scholar), indicating that diversifying selection occurs in these residues (Supplemental Table 4). In the past, it was notoriously difficult to isolate Pm21 using the map-based cloning strategy because of no recombination between the alien chromosome 6VS and the wheat homoeologous counterpart, and even between two different 6V chromosomes, in wheat background (Qi et al., 1998Qi 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: 1042-1046Crossref Scopus (34) Google Scholar). Central to the success of our studies is the discovery and utilization of Bgt-susceptible accessions, which enabled the fine mapping of Pm21 to a small genomic region (He et al., 2017He 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: 1914Crossref PubMed Scopus (24) Google Scholar). In this report, the BSMV-VIGS, EMS mutant analysis and transgenic validation data corroborate with each other, and support that DvRGA2 is Pm21. More importantly, we confirm that the isolated DvRGA2 confers broad-spectrum Bgt resistance via inoculating DvRGA2 expressing transgenic plants with a diverse panel of Bgt isolates, which is consistent with past findings of Pm21 as a broad-spectrum Bgt resistance gene in wheat breeding studies. While we were preparing this paper, another group of scientists reported that NLR1-V was a candidate of Pm21 using RGA capture and PacBio sequencing (Xing et al., 2017Xing L. Hu P. Liu J. Cui C. Wang H. Di Z. Zhou S. Xu J. Gao L. Huang Z. et al.NLR1-V, a CC-NBS-LRR encoding gene, is a potential candidate gene of the wheat powdery mildew resistance gene Pm21.bioRxiv. 2017; https://doi.org/10.1101/114058Crossref Google Scholar). We noticed that DvRGA2 and NLR1-V encode identical proteins but have minor indel polymorphisms in the second intron. Thus, DvRGA2 and NLR1-V may be allelic. Broad-spectrum resistance genes are highly desirable for developing elite crop cultivars, but only a few such genes have been isolated to date, e.g., Yr36 (Fu et al., 2009Fu D. Uauy C. Distelfeld A. Blechl A. Epstein L. Chen X. Sela H. Fahima T. Dubcovsky J. A kinase-START gene confers temperature-dependent resistance to wheat stripe rust.Science. 2009; 323: 1357-1360Crossref PubMed Scopus (505) Google Scholar), Lr67 (Moore et al., 2015Moore 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: 1494-1498Crossref PubMed Scopus (365) Google Scholar), PigmR (Deng et al., 2017Deng Y. Zhai K. Xie Z. Yang D. Zhu X. Liu J. Wang X. Qin P. Yang Y. Zhang G. et al.Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance.Science. 2017; 355: 962-965Crossref PubMed Scopus (311) Google Scholar), and Sw-5b (Zhu et al., 2017Zhu M. Jiang L. Bai B. Zhao W. Chen X. Li J. Liu Y. Chen Z. Wang B. Wang C. et al.The intracellular immune receptor Sw-5b confers broad-spectrum resistance to Tospoviruses through recognition of a conserved 21-amino acid viral effector epitope.Plant Cell. 2017; 29: 2214-2232Crossref PubMed Scopus (51) Google Scholar). Pm21 (DvRGA2) represents a valuable new addition to this type of genes. It has been demonstrated that the broad-spectrum resistance conferred by Sw-5b, a CC-NBS-LRR protein, stems from the recognition of a conserved pathogen effector. Our evolutionary analysis suggests that Pm21 is a relatively ancient powdery mildew resistance gene. Therefore, we hypothesize that the CC-NBS-LRR protein specified by Pm21 might perceive a conserved effector element from different Bgt isolates in conferring broad-spectrum powdery mildew disease resistance. This hypothesis is supported by the high conservation of Pm21 in Bgt-resistant D. villosum accessions. Although some solvent-exposed residues in the LRR domain of PM21 appear to undergo diversifying selection, this does not lead to the loss of Bgt resistance. Thus, comparison of variant resistance alleles may facilitate identification of the amino acid residues critical to the function of the LRR domain of PM21 in the future. In conclusion, we have cloned Pm21 and validated its function as a broad-spectrum Bgt resistance gene through transgenic expression. The cloned Pm21 is valuable for efficient development of Bgt-resistant wheat crops through molecular breeding. Since Pm21 and its wheat orthologs have different evolutionary fates, it may also aid further analysis of the different mechanisms underlying the evolution of important disease resistance genes in Triticeae plants. This study was supported by grants from the National Natural Science Foundation of China (31471497), the Innovation Foundation of Jiangsu Academy of Agricultural Sciences (ZX(17)2011), the Natural Science Foundation of Jiangsu Province (BK20130503 and BK20151319), and the Foundation of Jiangsu University (13JDG103).