Map-based cloning of the gene Pm21 that confers broad spectrum resistance to wheat powdery mildew

Common wheat (Triticum aestivum L.) is one of the most important cereal crops. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a continuing threat to wheat production. The Pm21 gene, originating from Dasypyrum villosum, confers high resistance to all known Bgt races and has been widely applied in wheat breeding in China. In this research, we identify Pm21 as a typical coiled-coil, nucleotide-binding site, leucine-rich repeat gene by an integrated strategy of resistance gene analog (RGA)-based cloning via comparative genomics, physical and genetic mapping, BSMV-induced gene silencing (BSMV-VIGS), large-scale mutagenesis and genetic transformation. Common wheat (Triticum aestivum L.) is the most widely grown cereal crop occupying ~17% of all cultivated land worldwide and providing ~20% of the calories consumed by humankind (Fu et al. 2009). However, wheat production is challenged constantly by powdery mildew, which is caused by Blumeria graminis f. sp. tritici (Bgt). Utilization of powdery mildew resistance (Pm) genes is an effective and economical way to reduce yield losses caused by Bgt. Up to now, more than 100 Pm genes in wheat and its relatives have been documented (McIntosh et al. 2017). Among them, Pm21 that originates from Dasypyrum villosum confers a high level of resistance to all known Bgt races (Chen et al. 1995; Cao et al. 2011). It is important to clarify the genetic basis and functional mechanism of Pm21. Previously, several candidate genes, including Stpk-V and DvUPK located in chromosome 6VS bin FL0.45-0.58 carrying Pm21, were reported to be required by Pm21 resistance (Cao et al. 2011; He et al. 2016); however, due to lack of a fine map, the relationships of these candidate genes and Pm21 are unclear. In the present study, four D. villosum lines (DvSus-1 ~ DvSus-4) susceptible to Bgt isolate YZ01 at the seedling stage were identified from a total of 110 accessions (Fig. 1E and Table S1). Fine genetic mapping of Pm21 was conducted on an F2 population derived from a cross between resistant line DvRes-1 carrying Pm21 and susceptible line DvSus-1. Among the total 10,536 F2 plants, 64 recombinants between markers 6VS-00.1 and Xcfe164 (Qi et al. 2010) on 6VS were identified. Pm21 was then mapped to a 0.01-cM interval flanked by the markers 6VS-08.4b and 6VS-10b (Fig. 1A and Fig. S1), in which, genes DvEXO70 (6VS-08.8b; encoding a putative exocyst complex component EXO70A1-like protein) and DvPP2C (6VS-09b) co-segregated with Pm21 (Fig. S2), whereas candidate genes reported previously, such as Stpk-V (Cao et al. 2011) , were not. A conserved coiled-coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR)-encoding resistance gene analog (RGA) locus was found between wheat and Brachypodium by comparative mapping (Fig. 1B, 1C and 1D). Subsequently, a 17,732-bp genomic sequence harboring three complete genes, viz., DvPP2C, encoding a protein phosphatase (He et al. 2016), DvRGA2 and DvRGA1, were obtained together with a separated gene DvRGA3 from the resistant D. villosum line DvRes-1 by PCR (Fig. S3). Genetic analysis demonstrated that all the above RGAs, DvRGA1 (6VS-09.6b), DvRGA2 (6VS-09.4b) and DvRGA3 (6VS-09.8b) co-segregated with Pm21 (Fig. 1A, Fig. S1 and Fig. S2). Further physical mapping, by using the susceptible deletion line Y18-S16 identified from an EMS-induced Yangmai 18 population, showed that the entire genetic interval carrying Pm21 was missing in Y18-S16 (Fig. S1). The genomic sequence of DvRGA1 is 2,986 bp in length with 2 exons and 1 intron, and the corresponding open reading frame (ORF) is 2,736 bp. The nucleotide sequence of DvRGA2 spans 3,699 bp, harboring 3 exons and 2 introns, with a 2,730-bp ORF. The exon-intron structure of DvRGA3 was similar to that of DvRGA2 but there was an additional repeat sequence in the second intron and several premature stop codons in the third exon (Fig. S4), suggesting that DvRGA3 is a pseudogene. Transcriptional analysis demonstrated that DvRGA1 and DvRGA2 were transcribed in D. villosum seedlings whereas DvRGA3 was not. Quantitative real-time RT-PCR (qPCR) showed that DvRGA1 and DvRGA2 were both enhanced at the transcriptional level following Bgt infection, and they shared similar transcription patterns during infection of Yangmai 18 (Fig. S5). To confirm which RGA(s) corresponded to Pm21, the DvRGA1 and DvRGA2 alleles were cloned from susceptible D. villosum lines DvSus-1 ~ DvSus-4 as well as susceptible wheat addition line DA6V#1 (Sears 1953; Qi et al, 1998) and then sequenced. All the five susceptible lines had variations in the DvRGA2 gene. Interestingly, DvSus-2 and DvSus-3 had a common mutation and DvSus-4 and DA6V#1 shared another mutation in DvRGA2. DvSus-1 ~ DvSus-3 had abnormal DvRGA1 alleles whereas DvSus-4 and DA6V#1 did not (Table S2). The silencing effects of DvRGA1 and DvRGA2 in Yangmai 18 were analyzed using BSMV-VIGS technology. Silencing of DvRGA2 allowed normal development of powdery mildew with macroscopic disease symptoms and sporulation on leaves at 10 days post-inoculation (Fig. 2A and 2B). However, no obvious effects were observed after silencing of DvRGA1 and DvEXO70 (Fig. S6). Silencing of DvPP2C led to sporulation but without significant macroscopic disease development (He et al. 2016) . These results indicated that DvRGA2 is required for Pm21 resistance. To detect if DvRGA2 is sufficient for the resistance, DvRGA2 with its native promoter was transformed into susceptible cv Kenong 199 by particle bombardment. Six of the 16 T1 families identified with markers MBH1 and 6VS-09.4 were positively transgenic and showed immunity to Bgt isolate YZ01 and to Bgt isolate mixtures collected from different regions (Fig. 2C). We concluded that DvRGA2 expressed by its native promoter confers resistance to powdery mildew in wheat. To verify whether DvRGA2 is Pm21, Yangmai 18 carrying Pm21 was mutagenized with EMS. Fifty eight independent susceptible mutants were identified among 6,408 M2 families (Fig. 2D). Except for Y18-S16, which had a deletion spanning the Pm21 locus, each of the other 57 susceptible mutants harbored a mutated DvRGA2 sequence. Among them, 55 each had a single-base mutation in DvRGA2, whereas the other two, Y18-S35 and Y18-S43, each had two-base changes. Sixteen of the 59 mutation sites caused premature stop codons and 43 caused changes in amino acids (Fig. 2E, Table S3). We also checked the flanking genes DvPP2C (He et al. 2016) and DvRGA1 in 11 randomly selected mutants, and found no differences from that in untreated Yangmai 18. We concluded that DvRGA2 alone is Pm21. Amino acid sequence analysis showed that DvRGA2 protein had relatively high identities with BRADI3G03874, BRADI3G03878, BRADI3G03882 and BRADI3G03935 (51.2 ~ 60.5%). Among the CC-NBS-LRR proteins reported in wheat, DvRGA2 shared highest identity (35.0%) with stem rust resistance proteins SR22 but lower identities with powdery mildew resistance proteins PM2 (15.2%), PM3b (18.7%) and PM8 (19.8%) (Fig. S7). We searched for Pm21 orthologs in the wheat genome and found that they are present but disrupted by a transposon-like element (6AS) or not completely assembled (6BS and 6DS) in the second intron. Pm21 orthologs in other related species, such as T. urartu, Aegilops speltoides and Ae. tauschii, the donors of wheat subgenomes, are all disrupted by transposon-like elements (Fig. S8). It appears that the events causing structural abnormalities of Pm21 orthologs occurred after divergence of D. villosum and the other species. In the past, it was extremely difficult to clone Pm21 in wheat background by map-based strategy due to lack of recombination between alien chromosome 6VS and wheat homoeologous chromosomes. The present break-through came with the discovery of several powdery mildew susceptible D. villosum lines, allowing construction of a high density genetic map of chromosome 6VS within that species. Although several genes had been reported to be required for Pm21 resistance (Cao et al. 2011; He et al. 2016), and even overexpression of Stpk-V conferred high resistance to powdery mildew in transgenic wheat, none apart from DvPP2C was located in the genetic interval carrying Pm21. Although fine genetic mapping allows gene isolation by using the map-based cloning, the method is usually dependent on a bacterial artificial chromosome (BAC) library that is time consuming, labour intensive and expensive to develop. Given that most of the identified disease resistance (R) genes in wheat, such as Pm2 (Sánchez-Martín et al. 2016) , Pm3 (Yahiaoui et al. 2004) , Lr10 (Feuillet et al. 2003) , Sr33 (Periyannan et al. 2013) and Sr35 (Saintenac et al. 2013), encode CC-NBS-LRR proteins, we preferentially isolated CC-NBS-LRR-encoding resistance gene analogs (RGAs) by PCR according to a conserved RGA locus common to wheat and Brachypodium. Among the candidates isolated, DvRGA2 was shown to be identical to Pm21 via BSMV-VIGS, genetic transformation and large-scale mutagenesis. Broad spectrum resistances are commonly controlled by non-NBS-LRR genes, such as Yr36 (Fu et al. 2009), Lr34 (Krattinger et al. 2009) and Lr67 (Moore et al. 2015), rather than by NBS-LRR-encoding genes that confer race-specific resistance, such as Pm2 (Sánchez-Martín et al. 2016) and Pm3 (Yahiaoui et al. 2004). Nevertheless, researches show that several NBS-LRR-encoding genes can confer broad spectrum resistances, such as potato late blight resistance gene RB (Song et al. 2003) and rice blast resistance gene Pi9 (Qu et al. 2006). Here, we demonstrated that the broad spectrum resistance of Pm21 is also conferred by a single CC-NBS-LRR-encoding gene. It was proposed that Pm21 is a relatively ancient Pm gene, whose product may perceive a conserved effector(s) from different Bgt races. Since Pm21 is a single CC-NBS-LRR-encoding gene, the question arises as to whether it will continue to confer durable resistance to powdery mildew. Analysis of nine independent mutations in Pm21 revealed single amino acid changes in the LRR domain that were correlated with loss of resistance to Bgt isolate YZ01. Among them, the mutations in Y18-S9 and Y18-S20 (Table S3) involved changes in solvent-exposed LRR residues that are considered to control specific recognition of the pathogen (Meyers et al. 1998; Wulff et al. 2009). In the recent years, wheat varieties carrying Pm21 are increasingly being planted in China (Bie et al. 2015), which would accelerate Bgt evolution, and the risk of losing Pm21 resistance would arise. So, it will be a great challenge to maintain the resistance of Pm21 in the future. One practical way may be pyramiding other Pm gene(s) into wheat varieties carrying Pm21. Materials and Methods Plant materials and pathogen inoculation A total of 110 accessions of Dasypyrum villosum were collected from the Germplasm Resources Information Network (GRIN) (51), GRIN Czech (16), Genebank Information System of IPK Gatersleben (GBIS-IPK) (35), Nordic Genetic Resource Center (NordGen) (7) and Cytogenetics Institute, Nanjing Agricultural University (CI-NAU) (1). The susceptible addition line DA6V#1 (Sears 1953; Qi et al. 1998) was provided by GRIN (Table S1). Powdery mildew resistant wheat cultivar (cv) Yangmai 18 carrying a pair of translocated T6AL.6VS chromosomes (Pm21) and susceptible cv Yangmai 9 were developed at Yangzhou Academy of Agricultural Sciences (YAAS). All plants were inoculated with Bgt isolate YZ01, a predominant isolate collected from Yangzhou (He et al. 2016), by dusting from sporulating susceptible plants and powdery mildew responses were assessed at 8 days post-inoculation. Bgt isolate YZ01 was maintained on cv Yangmai 9 seedlings. DNA isolation and development of molecular markers Genomic DNA was extracted from fresh leaves of seedlings by the CTAB method (Murray and Thompson 1980). DNA markers were reported previously (He et al. 2016; Qi et al. 2010; Cao et al. 2006) or newly developed using CISP and CISP-IS strategies based on collinearity among Brachypodium, rice and Triticeae species, as described by He et al. (2013) . All primers used in this study are listed in Table S4. Genetic mapping An F2 population was derived from the cross between the resistant D. villosum line DvRes-1 carrying the Pm21 gene and seedling-susceptible line DvSus-1 (Table S1) newly found in this study. Powdery mildew responses of F2 plants were determined at the one-leaf stage. For molecular analysis, PCR amplifications were performed in a Peltier Thermal Cycler (Bio-Rad, USA) in 25 μl volumes containing 1×PCR buffer, 0.2 mM of each dNTP, 2 μM of each primer, 1 unit of Taq DNA polymerase, and 1 μl of DNA template. PCR was carried out with an initial denaturation at 94°C for 3 min, 35 cycles of 20 s at 94°C, 30 s at 60°C, 1 min at 72°C, and a final extension for 5 min at 72°C. PCR products were separated in 6 ~ 12% non-denaturing polyacrylamide gels, silver stained, and photographed. Chi-squared (χ) tests were used to determine the goodness-of-fit of the observed segregation ratios to theoretical Mendelian ratios. PCR amplification of candidate RGAs Degenerative primers used for cloning of candidate genes were designed according to the conserved sequences of predicted RGAs in wheat and Brachypodium in the orthologous regions of the Pm21 locus. Fragments of candidate RGAs were PCR-amplified from genomic DNA of D. villosum line DvRes-1 carrying Pm21. Thermal-asymmetric-interlaced (TAIL) PCR (Liu and Huang 1998) and Long-range (LR) PCR (Song et al. 2003) with LA Taq DNA polymerase (TaKaRa, Japan) were further used to clone unknown DNA fragments close to the candidate genes. Quantitative real-time RT-PCR (qPCR) Total RNA was isolated from wheat and D. villosum leaves inoculated or non-inoculated with Bgt isolate ZY01, using TRIzol reagent (Life Technologies, USA). First-strand cDNA was then synthesized from 2 μg of total RNA using a PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Quantitative real time RT-PCR (qPCR) was performed in an ABI 7300 Real Time PCR System (Life Technologies, USA) as described by He et al. (2016) . The wheat actin gene (TaACT) was used as reference gene as reported (Bahrini et al. 2011). All reactions were run in three technical replicates for each cDNA sample. Sequence analysis The genome sequences of Brachypodium, rice and wheat were obtained from Brachypodium distachyon genome assemblies v2.0 (http://www.brachypodium.org), rice genome pseudomolecule release 7 (http://rice.plantbiology.msu.edu), and the IWGSC Sequence Repository (http://wheat-urgi. versailles.inra.fr), respectively. Genes were predicted using the FGENESH tool (Solovyev et al. 2006), and then re-annotated by using the BLAST program (Johnson et al. 2008) in combination with the SMART program (Letunic et al. 2015). Protein domain prediction and multiple sequence alignment analysis were performed by the SMART and CLUSTAL W (Thompson et al. 1994) tools, respectively. Phylogenetic tree was constructed by the Neighbor-Joining method in the MEGA4 software (Tamura et al. 2007). Functional analysis of candidate genes by BSMV-VIGS BSMV-VIGS (Hein et al. 2005; Scofield et al. 2005) was utilized to investigate the potential involvement of the candidate genes in wheat cv Yangmai 18. Gene fragments were amplified from the first-strand cDNA of D. villosum, digested with EcoRI/SalI, and then inserted in reverse orientation into a modified BSMV: γ vector. The details of silencing of target genes were described in our previous work (He et al. 2016). Wheat transformation The vectors pAHC25 (Christensen and Quail 1996) was digested with HindIII, and then the large fragment containing the bar gene was ligated with the multiple cloning sites (SmaISnaBIEcoRVStuINotI), generating pAHC25-MCS2. The 5,890-bp genomic DNA of DvRGA2, containing a 1,779-bp native promoter sequence and a 412-bp downstream sequence, was PCR-amplified using PrimeSTAR Max Premix (TaKaRa, Japan) according to the manufacturer’s guideline. After digestion with SmaI and NotI, DvRGA2 was inserted into pAHC25-MCS2. After confirmed by sequencing, the construct was transformed into susceptible wheat cv Kenong 199 using the PDS-1000/He biolistic particle delivery system (Bio-Rad). T1 plants were tested for presence of the transgene by PCR-amplification using markers MBH1 (Bie et al. 2015) and 6VS-09.4, located in the promoter region and coding region of DvRGA2, respectively. Marker 6VS-09.6 derived from DvRGA1 was also used as a negative control. The positively identified T1 plants were inoculated with Bgt isolate YZ01 and mixed isolates collected from different regions of China. Mutation analysis About 10,000 seeds of Yangmai 18 were treated with 0.8% ethyl methanesulfonate (EMS), and 6,408 M2 families were obtained. About 100 seeds of each M2 family were screened for mutants that were susceptible to powdery mildew. Susceptibility of mutants was confirmed in adult plant tests. The DvRGA2 gene in each mutant line was obtained by RT-PCR, inserted into pAHC25-MCS1 after digestion with SmaI/SpeI, and sequenced by the Sanger method. pAHC25-MCS1 was derived from the vector pAHC25, in which, the gus gene was replaced by multiple cloning sites (SmaINotIMluISpeISacI). Each mutation was verified by sequencing the PCR product harboring the candidate mutation site. As controls, the DvRGA1 and DvPP2C genes in 11 randomly selected mutants were also obtained and sequenced by the same method. The distribution of all DvRGA2 mutations in sequences from susceptible wheat lines was analyzed at the protein domain and motif levels according to previous descriptions (Meyers et al. 1999; Dilbirligi and Gill 2003). The DvRGA1 and DvRGA2 genes in the susceptible and resistant D. villosum lines were amplified by LR PCR from genomic templates, cloned into the pMD18-T vector, and sequenced. Acknowledgments: This research was supported by Grants from the National Natural Science Foundation of China (31471497), the Natural Science Foundation of Jiangsu Province (BK20130503) and the Innovation Foundation of Jiangsu Academy of Agricultural Sciences [ZX(17)2011]. The authors are grateful to the Germplasm Resources Information Network (GRIN), GRIN Czech, Genebank Information System (GBIS) of the IPK Gatersleben, Nordic Genetic Resource Center (NordGen), and Cytogenetics Institute, Nanjing Agricultural University (CI-NAU) for providing D. villosum accessions. 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