TaCRT3 Is a Positive Regulator of Resistance to Blumeria graminis f. sp. tritici in Wheat.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most prevalent diseases of wheat worldwide and can lead to severe yield reductions. Identifying genes involved in powdery mildew resistance will be useful for disease resistance breeding and control. Calreticulin (CRT) is a member of multigene family widely found in higher plants and is associated with a variety of plant physiological functions and defense responses. However, the role of CRT in wheat resistance to powdery mildew remains unclear. TaCRT3 was identified from the proteomic sequence of an incompatible interaction between the wheat (Triticum aestivum) cultivar Xingmin 318 and the Bgt isolate E09. Following analysis of transient expression of the GFP-TaCRT3 fusion protein in Nicotiana benthamiana leaves, TaCRT3 was localized in the nucleus, cytoplasm, and cell membrane. Transcript expression levels of TaCRT3 were significantly upregulated in the wheat-Bgt incompatible interaction. More critically, knockdown of TaCRT3 using virus-induced gene silencing resulted in attenuated resistance to Bgt in wheat. Histological analysis showed a significant increase in Bgt development in TaCRT3-silenced plants, whereas the pathogen-related gene was significantly downregulated in TaCRT3-silenced leaves. In addition, overexpression of TaCRT3 in wheat enhanced the resistance to powdery mildew, the growth of Bgt was significantly inhibited, and the area of H2O2 near the infection site and the expression of defense-related genes of the salicylic acid pathway significantly increased. These findings imply that TaCRT3 may act as a disease resistance factor that positively regulates resistance to powdery mildew, during which SA signaling is probably activated.

Diversity and similarity of wheat powdery mildew resistance among three allelic functional genes at the Pm60 locus.

Cultivated wheat is continually exposed to various pathogens. Blumeria graminis f. sp. tritici (Bgt) causes powdery mildew disease and significant yield loss. Pm60 was cloned from Triticum urartu and confers race-specific powdery mildew resistance in wheat. Pm60a and Pm60b are allelic variants of Pm60 and have two leucine-rich repeat motifs deletions and insertions, respectively, which were detected in other T. urartu accessions. Through map-based cloning, virus-induced gene silencing, and stable transformation assays, we demonstrated that Pm60a and Pm60b conferred Bgt E09 resistance resembling that provided by Pm60. However, the homozygous Pm60a (but not Pm60 or Pm60b) transformants driven by the native promoters lacked race-specific resistance when they were inoculated with Bgt E18. As all three T. urartu accessions contained the three foregoing alleles, they had high resistance to Bgt E18. Pyramiding Pm60a with either of the allelic genes in F1 plants did not cause mutual allele suppression or interference with Bgt E18 resistance. Deletion (but not insertion) of the two leucine-rich repeat motifs in Pm60a substantially narrowed the resistance spectrum. In T. urartu accession PI428210, we identified another locus adjacent to Pm60a and resistant to Bgt E18. Characterization of the alleles at the Pm60 locus revealed their diversity and similarity and may facilitate wheat breeding for resistance to powdery mildew disease caused by B. graminis f. sp. tritici.

Regulation of hormone pathways in wheat infested by Blumeria graminis f. sp. tritici.

BACKGROUND: Wheat powdery mildew is an obligate biotrophic pathogen infecting wheat, which can pose a serious threat to wheat production. In this study, transcriptome sequencing was carried out on wheat leaves infected by Blumeria graminis f. sp. tritici from 0 h to 7 d. RESULTS: KEGG and GO enrichment analysis revealed that the upstream biosynthetic pathways and downstream signal transduction pathways of salicylic acid, jasmonic acid, and ethylene were highly enriched at all infection periods. Trend analysis showed that the expressions of hormone-related genes were significantly expressed from 1 to 4 d, suggesting that 1 d-4 d is the main period in which hormones play a defensive role. During this period of time, the salicylic acid pathway was up-regulated, while the jasmonic acid and ethylene pathways were suppressed. Meanwhile, four key modules and 11 hub genes were identified, most of which were hormone related. CONCLUSION: This study improves the understanding of the dynamical responses of wheat to Blumeria graminis f. sp. tritici infestation at the transcriptional level and provides a reference for screening core genes regulated by hormones.

Wheat Class III Peroxidase TaPOD70 Is a Potential Susceptibility Factor Negatively Regulating Wheat Resistance to Blumeria graminis f. sp. tritici.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important diseases on wheat worldwide and can lead to a large reduction in wheat production. Class III peroxidases (PODs), a kind of secretory enzyme and members of a multigene family in higher plants, have been linked to various plant physiological functions and defensive responses. However, the role of PODs in wheat resistance to Bgt remains unclear. TaPOD70, a class III POD gene, was identified from the proteomics sequencing of the incompatible interaction between wheat (Triticum aestivum) cultivar Xingmin 318 and Bgt isolate E09. After transient expression of the TaPOD70-GFP fusion protein in Nicotiana benthamiana leaves, TaPOD70 was located in the membrane region. Yeast secretion assay showed that TaPOD70 was a secretory protein. Furthermore, Bax-induced programmed cell death was inhibited by transient expression of TaPOD70 in N. benthamiana. The transcript expression level of TaPOD70 was significantly upregulated in the wheat-Bgt compatible interaction. More crucially, knocking down TaPOD70 using virus-induced gene silencing increased wheat resistance to Bgt compared with the control plants. In response to Bgt, histological analyses indicated that hyphal development of Bgt was significantly reduced, whereas H2O2 production was enhanced in TaPOD70-silenced leaves. These findings imply that TaPOD70 may act as a susceptibility factor, adversely regulating wheat resistance to Bgt.

Resistance to Powdery Mildew Is Conferred by Different Genetic Loci at the Adult-Plant and Seedling Stages in Winter Wheat Line Tianmin 668.

Winter wheat line Tianmin 668 was crossed with susceptible cultivar Jingshuang 16 to develop 216 recombinant inbred lines (RILs) for dissecting its adult-plant resistance (APR) and all-stage resistance (ASR) against powdery mildew. The RIL population was genotyped on a 16K genotyping by target sequencing single-nucleotide polymorphism array and phenotyped in six field trials and in the greenhouse. Three loci-QPmtj.caas-2BL, QPmtj.caas-2AS, and QPmtj.caas-5AL-conferring APR to powdery mildew were detected on chromosomes 2BL, 2AS, and 5AL, respectively, of Tianmin 668. The effect of resistance to powdery mildew for QPmtj.caas-2BL was greater than that of the other two loci. A Kompetitive allele-specific PCR marker specific for QPmtj.caas-2BL was developed and verified on 402 wheat cultivars or breeding lines. Results of virulence and avirulence patterns to 17 Blumeria graminis f. sp. tritici isolates, bulked segregant analysis-RNA-sequencing, and a genetic linkage mapping identified a resistance allele at locus Pm4 in Tianmin 668 based on the seedling phenotypes of the RIL population. The PCR-based DNA sequence alignment and cosegregation of the functional marker with the phenotypes of the RIL population demonstrated that Pm4d was responsible for the ASR to isolate Bgt1 in Tianmin 668. The dissection of genetic loci for APR and ASR may facilitate the application of Tianmin 668 in developing powdery mildew-resistant wheat cultivars.

QTL Mapping Reveals Both All-Stage and Adult-Plant Resistance to Powdery Mildew in Chinese Elite Wheat Cultivars.

Powdery mildew caused by Blumeria graminis f. sp. tritici is a threat to wheat production in China. Mapping quantitative trait loci (QTL) for resistance to powdery mildew and developing breeder-friendly markers are important initial steps in breeding resistant cultivars. An all-stage resistance gene and several QTL were identified using a population of 254 recombinant inbred lines developed from a Jingdong 8/Aikang 58 cross. The population was evaluated for powdery mildew resistance across six field environments over three consecutive growing seasons utilizing two different mixtures of B. graminis f. sp. tritici isolates, named #Bgt-HB and #Bgt-BJ. Using genotypic data obtained from the Wheat TraitBreed 50K single-nucleotide polymorphism array, seven stable QTL were identified on chromosome arms 1DL, 2AL, 2DS, 4DL, 5AL, 6BL.1, and 6BL.2. The QTL on 2AL conferred all-stage resistance to B. graminis f. sp. tritici race E20 in greenhouse tests and explained up to 52% of the phenotypic variance in field trials but was resistant only against #Bgt-HB. The gene involved in this QTL was predicted to be Pm4a based on genome location and gene sequence. QPmja.caas-1DL, QPmja.caas-4DL, and QPmja.caas-6BL.1 were identified as potentially new QTL for powdery mildew resistance. QPmja.caas-2DS and QPmja.caas-6BL.1 were effective against both B. graminis f. sp. tritici mixtures, indicating their probable broad-spectrum resistance. A Kompetitive allele-specific PCR marker closely linked to QPmja.caas-2DS was developed and validated in a panel of 286 wheat cultivars. Because both Jingdong 8 and Aikang 58 have been leading cultivars and breeding parents, the QTL and marker reported represent valuable resources for wheat researchers and breeders.

Genetic Analysis and Molecular Identification of the Powdery Mildew Resistance in 116 Elite Wheat Cultivars/Lines.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive disease worldwide. Host resistance is the preferred method for limiting the disease epidemic, protecting the environment, and minimizing economic losses. In the present study, the reactions to powdery mildew for a collection of 600 wheat cultivars and breeding lines from different wheat-growing regions were tested using the Bgt isolate E09. Next, 116 resistant genotypes were identified and then crossed with susceptible wheat cultivars/lines to produce segregating populations for genetic analysis. Among them, 87, 19, and 10 genotypes displayed single, dual, and multiple genic inheritance, respectively. To identify the Pm gene(s) in those resistant genotypes, 16 molecular markers for 13 documented Pm genes were used to test the resistant and susceptible parents and their segregating populations. Of the 87 wheat genotypes that fitted the monogenic inheritance, 75 carried the Pm2a allele. Three, two, one, and two genotypes carried Pm21, Pm6, Pm4, and the recessive genes pm6 and pm42, respectively. Four genotypes did not carry any of the tested genes, suggesting that they might have other uncharacterized or new genes. The other 29 wheat cultivars/lines carried two or more of the tested Pm genes and/or other untested genes, including Pm2, Pm5, Pm6, and/or pm42. It was obvious that Pm2 was widely used in wheat production, whereas Pm1, Pm24, Pm33, Pm34, Pm35, Pm45, and Pm47 were not detected in any of these resistant wheat genotypes. This study clarified the genetic basis of the powdery mildew resistance of these wheat cultivars/lines to provide information for their rational utilization in different wheat-growing regions. Moreover, some wheat genotypes which may have novel Pm gene(s) were mined to enrich the diversity of resistance source.

Identification of a Pm4 Allele Conferring Powdery Mildew Resistance in Wheat Breeding Line GR18-1.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a serious fungal wheat disease of wheat worldwide. Host resistance is considered to be the most environmentally friendly and efficient approach against this disease. Wheat breeding line GR18-1 showed resistance to powdery mildew at both seedling and adult stages for several years. Genetic analysis indicated that a single dominant gene, tentatively designated as PmGR-18, conferred powdery mildew resistance in GR18-1. Bulked segregant analysis and marker analysis showed that PmGR-18 was located in the Pm4 interval on chromosome arm 2AL and was flanked by the markers Xwgrc763 and Xwgrc872, respectively, with genetic distances of 0.5 and 1.0 cM corresponding to a physical interval of 1.13 Mb based on the Chinese Spring reference genome sequence v2.1. Using homology-based cloning and Sanger sequencing, we found that the sequence of PmGR-18 was totally consistent with that of Pm4d. qRT-PCR analysis showed that the expression levels of two splicing variants Pm4d_V1 and Pm4d_V2 in GR18-1 were significantly upregulated after inoculating with Bgt isolate E09, and the level of Pm4d_V2 was significantly lower than that of Pm4d_V1 at most of the time points, suggesting a different resistance pattern may be involved in the genotype. To facilitate the transfer of PmGR-18 in marker-assisted selection (MAS) breeding, the flanked markers Xwgrc763 and Xwgrc872 and the functional marker JS717/JS718 were tested and confirmed to enable the tracking of PmGR-18 when it transferred into those susceptible cultivars.

Identification of specificity-defining amino acids of the wheat immune receptor Pm2 and powdery mildew effector AvrPm2.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) act as intracellular sensors for pathogen-derived effector proteins and trigger an immune response, frequently resulting in the hypersensitive cell death response (HR) of the infected host cell. The wheat (Triticum aestivum) NLR Pm2 confers resistance against the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) if the isolate contains the specific RNase-like effector AvrPm2. We identified and isolated seven new Pm2 alleles (Pm2e-i) in the wheat D-genome ancestor Aegilops tauschii and two new natural AvrPm2 haplotypes from Bgt. Upon transient co-expression in Nicotiana benthamiana, we observed a variant-specific HR of the Pm2 variants Pm2a and Pm2i towards AvrPm2 or its homolog from the AvrPm2 effector family, BgtE-5843, respectively. Through the introduction of naturally occurring non-synonymous single nucleotide polymorphisms and structure-guided mutations, we identified single amino acids in both the wheat NLR Pm2 and the fungal effector proteins AvrPm2 and BgtE-5843 responsible for the variant-specific HR of the Pm2 variants. Exchanging these amino acids led to a modified HR of the Pm2-AvrPm2 interaction and allowed the identification of the effector head epitope, a 20-amino-acid long unit of AvrPm2 involved in the HR. Swapping of the AvrPm2 head epitope to the non-HR-triggering AvrPm2 family member BgtE-5846 led to gain of a HR by Pm2a. Our study presents a molecular approach to identify crucial effector surface structures involved in the HR and demonstrates that natural and induced diversity in an immune receptor and its corresponding effectors can provide the basis for understanding and modifying NLR-effector specificity.

Thinopyrum intermedium TiAP1 interacts with a chitin deacetylase from Blumeria graminis f. sp. tritici and increases the resistance to Bgt in wheat.

The biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt) is a crucial factor causing reduction in global wheat production. Wild wheat relatives, for example Thinopyrum intermedium, is one of the wild-used parents in wheat disease-resistant breeding. From T. intermedium line, we identified the aspartic protease gene, TiAP1, which is involved in resistance against Bgt. TiAP1 is a secreted protein that accumulates in large amounts at the infection sites of Bgt and extends to the intercellular space. Yeast two-hybrid, luciferase complementation imaging and bimolecular florescent complimentary analysis showed that TiAP1 interacted with the chitin deacetylase (BgtCDA1) of Bgt. The yeast expression, purification and in vitro test confirmed the chitin deacetylase activity of BgtCDA1. The bombardment and VIGS-mediated host-induced gene silencing showed that BgtCDA1 promotes the invasion of Bgt. Transcriptome analysis showed the cell wall xylan metabolism, lignin biosynthesis-related and defence genes involved in the signal transduction were up-regulated in the transgenic TiAP1 wheat induced by Bgt. The TiAP1 in wheat may inactivate the deacetylation function of BgtCDA1, cause chitin oligomers expose to wheat chitin receptor, then trigger the wheat immune response to inhibit the growth and penetration of Bgt, and thereby enhance the resistance of wheat to pathogens.

Genome-Wide Association Mapping of Resistance to Powdery Mildew in Regional Trials of Wheat Mainly from China.

Wheat powdery mildew (Blumeria graminis f. sp. tritici [Bgt]) is a widespread disease that causes significant economic losses to common wheat (Triticum aestivum L.) crops worldwide. To identify effective resistance genes, we evaluated 120 common wheat accessions mainly from different wheat producing regions in China for responses to different Bgt isolates in the seedling stage and to natural infection in three field trials and genotyped them with a wheat 55K iSelect single-nucleotide polymorphism array for a genome-wide association study. A total of 26 loci were identified, which explained 6.6 to 26.2% of the phenotypic variation depending on individual locus. Of the 26 loci, 10 were detected in the A genomes, 10 in the B genomes, and only 6 in the D genome. Sixteen loci overlapped with known powdery mildew resistance genes or quantitative trait loci, and the remaining 10 loci were potentially novel. This study improves the understanding of the genetic structure of wheat powdery mildew resistance and provides germplasms and information on genes and markers for breeding new wheat cultivars with effective resistance to powdery mildew.

Bio-Inspired Rhamnolipids, Cyclic Lipopeptides and a Chito-Oligosaccharide Confer Protection against Wheat Powdery Mildew and Inhibit Conidia Germination.

Plant protection is mainly based on the application of synthetic pesticides to limit yield losses resulting from diseases. However, the use of more eco-friendly strategies for sustainable plant protection has become a necessity that could contribute to controlling pathogens through a direct antimicrobial effect and/or an induction of plant resistance. Three different families of natural or bioinspired compounds originated from bacterial or fungal strains have been evaluated to protect wheat against powdery mildew, caused by the biotrophic Blumeria graminis f.sp. tritici (Bgt). Thus, three bio-inspired mono-rhamnolipids (smRLs), three cyclic lipopeptides (CLPs, mycosubtilin (M), fengycin (F), surfactin (S)) applied individually and in mixtures (M + F and M + F + S), as well as a chitosan oligosaccharide (COS) BioA187 were tested against Bgt, in planta and in vitro. Only the three smRLs (Rh-Eth-C12, Rh-Est-C12 and Rh-Succ-C12), the two CLP mixtures and the BioA187 led to a partial protection of wheat against Bgt. The higher inhibitor effects on the germination of Bgt spores in vitro were observed from smRLs Rh-Eth-C12 and Rh-Succ-C12, mycosubtilin and the two CLP mixtures. Taking together, these results revealed that such molecules could constitute promising tools for a more eco-friendly agriculture.

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

The development of improved plant nucleotide-binding, leucine-rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3A2/F2 . PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race-specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.

A highly differentiated region of wheat chromosome 7AL encodes a Pm1a immune receptor that recognizes its corresponding AvrPm1a effector from Blumeria graminis.

Pm1a, the first powdery mildew resistance gene described in wheat, is part of a complex resistance (R) gene cluster located in a distal region of chromosome 7AL that has suppressed genetic recombination. A nucleotide-binding, leucine-rich repeat (NLR) immune receptor gene was isolated using mutagenesis and R gene enrichment sequencing (MutRenSeq). Stable transformation confirmed Pm1a identity which induced a strong resistance phenotype in transgenic plants upon challenge with avirulent Blumeria graminis (wheat powdery mildew) pathogens. A high-density genetic map of a B. graminis family segregating for Pm1a avirulence combined with pathogen genome resequencing and RNA sequencing (RNAseq) identified AvrPm1a effector gene candidates. In planta expression identified an effector, with an N terminal Y/FxC motif, that induced a strong hypersensitive response when co-expressed with Pm1a in Nicotiana benthamiana. Single chromosome enrichment sequencing (ChromSeq) and assembly of chromosome 7A suggested that suppressed recombination around the Pm1a region was due to a rearrangement involving chromosomes 7A, 7B and 7D. The cloning of Pm1a and its identification in a highly rearranged region of chromosome 7A provides insight into the role of chromosomal rearrangements in the evolution of this complex resistance cluster.

Molecular Characterization of All-Stage and Adult-Plant Resistance Loci Against Powdery Mildew in Winter Wheat Cultivar Liangxing 99 Using BSR-Seq Technology.

Winter wheat cultivar Liangxing 99, which carries gene Pm52, is resistant to powdery mildew at both seedling and adult-plant stages. An F2:6 recombinant inbred line population from cross Liangxing 99 × Zhongzuo 9504 was phenotyped with Blumeria graminis f. sp. tritici isolate Bgt27 at the adult-plant stage in four field tests and the seedling stage in a greenhouse test. The analysis of bulk segregant RNA sequencing (BSR-Seq) identified a single-nucleotide polymorphism-enriched locus, Qaprpm.caas.2B, on chromosome 2BL in the same genomic interval of Pm52 associated with the all-stage resistance (ASR) and Qaprpm.caas.7A on chromosome 7AL associated with the adult-plant resistance (APR) against the disease. Qaprpm.caas.2B was detected in a 1.3 cM genetic interval between markers Xicscl726 and XicsK128 in which Pm52 was placed with a range of logarithm of odds (LOD) values from 28.1 to 34.6, and the phenotype variations explained in terms of maximum disease severity (MDS) ranged from 45 to 52%. The LOD peak of Qaprpm.caas.7A was localized in a 4.6 cM interval between markers XicsK7A8 and XicsK7A26 and explained the phenotypic variation of MDS ranging from 13 to 16%. The results of this study confirmed Pm52 for ASR and identified Qaprpm.caas.7A for APR to powdery mildew in Liangxing 99.

A rare single nucleotide variant in Pm5e confers powdery mildew resistance in common wheat.

Powdery mildew poses severe threats to wheat production. The most sustainable way to control this disease is through planting resistant cultivars. We report the map-based cloning of the powdery mildew resistance allele Pm5e from a Chinese wheat landrace. We applied a two-step bulked segregant RNA sequencing (BSR-Seq) approach in developing tightly linked or co-segregating markers to Pm5e. The first BSR-Seq used phenotypically contrasting bulks of recombinant inbred lines (RILs) to identify Pm5e-linked markers. The second BSR-Seq utilized bulks of genetic recombinants screened from a fine-mapping population to precisely quantify the associated genomic variation in the mapping interval, and identified the Pm5e candidate genes. The function of Pm5e was validated by transgenic assay, loss-of-function mutants and haplotype association analysis. Pm5e encodes a nucleotide-binding domain leucine-rich-repeat-containing (NLR) protein. A rare nonsynonymous single nucleotide variant (SNV) within the C-terminal leucine rich repeat (LRR) domain is responsible for the gain of powdery mildew resistance function of Pm5e, an allele endemic to wheat landraces of Shaanxi province of China. Results from this study demonstrate the value of landraces in discovering useful genes for modern wheat breeding. The key SNV associated with powdery mildew resistance will be useful for marker-assisted selection of Pm5e in wheat breeding programs.

A CNL protein in wild emmer wheat confers powdery mildew resistance.

Powdery mildew, a fungal disease caused by Blumeria graminis f. sp. tritici (Bgt), has a serious impact on wheat production. Loss of resistance in cultivars prompts a continuing search for new sources of resistance. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, WEW), the progenitor of both modern tetraploid and hexaploid wheats, harbors many powdery mildew resistance genes. We report here the positional cloning and functional characterization of Pm41, a powdery mildew resistance gene derived from WEW, which encodes a coiled-coil, nucleotide-binding site and leucine-rich repeat protein (CNL). Mutagenesis and stable genetic transformation confirmed the function of Pm41 against Bgt infection in wheat. We demonstrated that Pm41 was present at a very low frequency (1.81%) only in southern WEW populations. It was absent in other WEW populations, domesticated emmer, durum, and common wheat, suggesting that the ancestral Pm41 was restricted to its place of origin and was not incorporated into domesticated wheat. Our findings emphasize the importance of conservation and exploitation of the primary WEW gene pool, as a valuable resource for discovery of resistance genes for improvement of modern wheat cultivars.

Identification of a Recessive Gene PmQ Conferring Resistance to Powdery Mildew in Wheat Landrace Qingxinmai Using BSR-Seq Analysis.

Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt), a biotrophic fungal species. It is very important to mine new powdery mildew (Pm) resistance genes for developing resistant wheat cultivars to reduce the deleterious effects of the disease. This study was carried out to characterize the Pm gene in Qingxinmai, a winter wheat landrace from Xinjiang, China. Qingxinmai is resistant to many Bgt isolates collected from different wheat fields in China. F1, F2, and F2:3 generations of the cross between Qingxinmai and powdery mildew susceptible line 041133 were developed. It was confirmed that a single recessive gene, PmQ, conferred the seedling resistance to a Bgt isolate in Qingxinmai. Bulked segregant analysis-RNA-Seq (BSR-Seq) was performed on the bulked homozygous resistant and susceptible F2:3 families, which detected 57 single nucleotide polymorphism (SNP) variants that were enriched in a 40 Mb genomic interval on chromosome arm 2BL. Based on the flanking sequences of the candidate SNPs extracted from the Chinese Spring reference genome, 485 simple sequence repeat (SSR) markers were designed. Six polymorphic SSR markers, together with nine markers that were anchored on chromosome arm 2BL, were used to construct a genetic linkage map for PmQ. This gene was placed in a 1.4 cM genetic interval between markers Xicsq405 and WGGBH913 corresponding to 4.9 Mb physical region in the Chinese Spring reference genome. PmQ differed from most of the other Pm genes identified on chromosome arm 2BL based on its position and/or origin. However, this gene and Pm63 from an Iranian common wheat landrace were located in a similar genomic region, so they may be allelic.

Fine Mapping a Broad-Spectrum Powdery Mildew Resistance Gene in Chinese Landrace Datoumai, PmDTM, and Its Relationship with Pm24.

Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a globally important wheat disease causing severe yield losses, and deployment of resistant varieties is the preferred choice for managing this disease. Chinese wheat landrace Datoumai was resistant to 22 of 23 Bgt isolates at the seedling stage. Genetic analysis based on the inoculation of Bgt isolate E09 on the F1, F2, and F2:3 populations derived from the cross Datoumai × Huixianhong revealed that the powdery mildew resistance of Datoumai is controlled by a single dominant gene, temporarily designated as PmDTM. Bulked segregant analysis and simple sequence repeat mapping with 200 F2 plants showed that PmDTM was located in the same genetic region as Pm24 on chromosome 1DS. To fine map PmDTM, 12 critical recombinants were identified from 1,192 F2 plants and delimited PmDTM to a 0.5-cM Xhnu58800 to Xhnu59000 interval covering 180.5 Kb (38,728,125 to 38,908,656 bp) on chromosome 1DS, and only one highly confident gene, TraesCS1D02G058900, was annotated within this region. TraesCS1D02G058900 encodes a receptor-like serine/threonine-protein kinase (STK), and a 6-bp deletion in exon 5 may confer the resistance to powdery mildew. Allele frequency analysis indicated that the STK allele with 6-bp deletion was only present in three landraces (Datoumai, Chiyacao [Pm24], and Hulutou) and was absent in all of the 353 Chinese modern cultivars and 147 foreign cultivars. These results demonstrate that PmDTM is mapped to the same locus as Pm24 and can be widely used to enhance powdery mildew resistance in wheat growing regions worldwide.

Histone Deacetylase TaHDT701 Functions in TaHDA6-TaHOS15 Complex to Regulate Wheat Defense Responses to Blumeria graminis f.sp. tritici.

Powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt) leads to severe economic losses in bread wheat (Triticum aestivum L.). To date, only a few epigenetic modulators have been revealed to regulate wheat powdery mildew resistance. In this study, the histone deacetylase 2 (HD2) type histone deacetylase TaHDT701 was identified as a negative regulator of wheat defense responses to Bgt. Using multiple approaches, we demonstrated that TaHDT701 associates with the RPD3 type histone deacetylase TaHDA6 and the WD40-repeat protein TaHOS15 to constitute a histone deacetylase complex, in which TaHDT701 could stabilize the TaHDA6-TaHOS15 association. Furthermore, knockdown of TaHDT701, TaHDA6, and TaHOS15 resulted in enhanced wheat powdery mildew resistance, suggesting that the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex negatively regulates wheat defense responses to Bgt. Moreover, chromatin immunoprecipitation assays revealed that TaHDT701 could function in concert with TaHOS15 to recruit TaHDA6 to the promoters of defense-related genes such as TaPR1, TaPR2, TaPR5, and TaWRKY45. In addition, silencing of TaHDT701, TaHDA6, and TaHOS15 resulted in the up-regulation of TaPR1, TaPR2, TaPR5, and TaWRKY45 accompanied with increased histone acetylation and methylation, as well as reduced nucleosome occupancy, at their promoters, suggesting that the TaHDT701-TaHDA6-TaHOS15 histone deacetylase complex suppresses wheat powdery mildew resistance by modulating chromatin state at defense-related genes.