Identification and Analysis of Powdery Mildew‐Responsive miRNAs in Wheat

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most consistently damaging diseases of common wheat worldwide and greatly affects crop productivity. Recently, several plant microRNAs (miRNAs) have been reported as gene expression regulators related to various adverse environments. However, up to now, less is known on the roles of miRNAs in powdery mildew infection response of wheat. In this study, miRNA expression patterns were investigated for identifying Bgt‐responsive miRNAs in wheat leaves using a plant miRNA microarray platform. A total of 79 miRNAs from 24 families were detected in wheat leaves. Among those, seven miRNAs were further validated to be involved in wheat powdery mildew response and two of them have never been reported. In addition, their target expression profiles showed a negative correlation with that of the seven miRNAs in mock‐ and Bgt‐infected samples furtherly proved, which in turn as the robust evidence, that those seven powdery mildew‐responsive miRNAs are highly reliable. These findings could extend the current view about miRNAs as ubiquitous regulators under stress conditions.     

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

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

Proteomic Analysis of the Defense Response of Wheat to the Powdery Mildew Fungus,Blumeria graminis f. sp. tritici

Powdery mildew of wheat is caused by Blumeria graminis f. sp. tritici (Bgt). Although many wheat cultivars resistant to this disease have been developed, little is known about their resistance mechanisms. The aim of this study was to identify proteins showing changes in abundance during the resistance response of the wheat line N0308 infected by Bgt. In two-dimensional electrophoresis analyses, 45 spots on the gels showed significant changes in abundance at 24, 48, and 72 h after inoculation, as compared to non-inoculated plants. Of these 45 proteins, 44 were identified by mass spectrometry analysis using the NCBInr database of Triticum aestivum (26 spots) and closely related species in the Triticum genus (18 spots). These proteins were associated with the defense response, photosynthesis, metabolism, and other cellular processes in wheat. Most of the up-regulated proteins were identified as stress- and defense-related proteins. In particular, the product of a specific powdery mildew resistance gene (Pm3b and its homolog) and some other defense- and pathogenesis-related proteins were overexpressed. The resistance gene product mediates the immune response and coordinates other cellular processes during the resistance response to Bgt.     

E3 ubiquitin ligase gene CMPG1–V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.)

Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1–V, that encodes a U–box E3 ubiquitin ligase. Expression of the CMPG1–V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1–V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1–V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans‐Golgi network/early endosome vesicles. Transgenic wheat over‐expressing CMPG1–V showed improved broad‐spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid‐responsive genes, H₂O₂ accumulation, and cell‐wall protein cross‐linking at the Bgt infection sites, and the expression of CMPG1–V in H. villosa was increased when treated with salicylic acid, abscisic acid and H₂O₂. These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad‐spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1–V‐mediated powdery mildew resistance.     

Analysis of Virulence in Populations of Wheat Powdery Mildew in Europe

Random samples of spores of wheat powdery mildew were collected from the atmosphere by means of a jet spore sampler while driving through selected wheat growing areas in Europe in 1986. Their single colony progenies were tested in the laboratory forvirulence against wheat differential varieties with powdery mildew resistances Pm2, Pm4b, Pm8, Mli and the combinations Pm2+Pm6, Pm4b+Pm8 and Pm2+Pm4b+Pm8. There were regional differences in virulence frequencies. Virulence on Pm2, Pm2+Pm6 and, less expressed, on Pm4b was most frequent in England and decreased mainly to the east, whereas the reverse was true for Pm8. Virulence to Mli was generally high, and to the combined genotypes generally low. The results suggest that large parts of Europe are an epidemiological unit. The data are discussed with respect to selection caused by the varieties grown, as well as to the influence of wind distribution on the composition of the pathogen population.     

A new powdery mildew resistance allele at the Pm4 wheat locus transferred from einkorn (Triticum monococcum)

Powdery mildew is one of the most destructive foliar diseases of wheat. A set of differential Blumeria graminis f.sp. tritici (Bgt) isolates was used to test the powdery mildew response of a Triticum monococcum-derived resistant hexaploid line, Tm27d2. Segregation analysis of 95 F_2:3 lines from a Chinese Spring/Tm27d2 cross revealed that the resistance of Tm27d2 is controlled by a single dominant gene. Using monosomic analysis and a molecular mapping approach, the resistance gene was localized to the terminal end of chromosome 2AL. The linkage map of chromosome 2AL consisted of nine simple sequence repeat markers and one sequence-tagged site (STS) marker (ResPm4) indicative for the Pm4 locus. According to the differential reactions of 19 wheat cultivars/lines with known powdery mildew resistance genes to 13 Bgt isolates, Tm27d2 carried a new resistance specificity. The complete association of the resistance allele with STS marker ResPm4 indicated that it represented a new allele at the Pm4 locus. This new allele was designated Pm4d. The two flanking markers Xgwm526 and Xbarc122 closely linked to Pm4d at genetic distances of 3.4 and 1.0 cM, respectively, are present in chromosome bin 2AL1-0.85-1.00.     

Non-host resistance of barley is associated with a hydrogen peroxide burst at sites of attempted penetration by wheat powdery mildew fungus

In barley, non-host resistance against the wheat powdery mildew fungus (Blumeria graminis f.sp. tritici, Bgt) is associated with the formation of cell wall appositions and a hypersensitive reaction in which epidermal cells die rapidly in response to fungal attack. In the interaction of barley with the pathogenic barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh), these defence reactions are also associated with accumulation of H₂O₂. To elucidate the mechanism of non-host resistance, the accumulation of H₂O₂ in response to Bgt was studied in situ by histochemical staining with diaminobenzidine. H₂O₂ accumulation was found in cell wall appositions under appressoria from Bgt and in cells undergoing a hypersensitive reaction. A mutation (mlo5) at the barley Mlo locus, that confers broad spectrum resistance to Bgh, did not influence the barley defence phenotype to Bgt. Significantly, Bgt triggered cell death on mlo5-barley while Bgh did not.     

Silencing of copine genes confers common wheat enhanced resistance to powdery mildew

Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a major threat to the production of wheat (Triticum aestivum). It is of great importance to identify new resistance genes for the generation of Bgt‐resistant or Bgt‐tolerant wheat varieties. Here, we show that the wheat copine genes TaBON1 and TaBON3 negatively regulate wheat disease resistance to Bgt. Two copies of TaBON1 and three copies of TaBON3, located on chromosomes 6AS, 6BL, 1AL, 1BL and 1DL, respectively, were identified from the current common wheat genome sequences. The expression of TaBON1 and TaBON3 is responsive to both pathogen infection and temperature changes. Knocking down of TaBON1 or TaBON3 by virus‐induced gene silencing (VIGS) induces the up‐regulation of defence responses in wheat. These TaBON1‐ or TaBON3‐silenced plants exhibit enhanced wheat disease resistance to Bgt, accompanied by greater accumulation of hydrogen peroxide and heightened cell death. In addition, high temperature has little effect on the up‐regulation of defence response genes conferred by the silencing of TaBON1 or TaBON3. Our study shows a conserved function of plant copine genes in plant immunity and provides new genetic resources for the improvement of resistance to powdery mildew in wheat.     

Identification and mapping of two powdery mildew resistance genes in <Emphasis Type="Italic">Triticum boeoticum</Emphasis> L.

Powdery mildew (PM) caused by Blumeria graminis f. sp. tritici (Bgt), is one of the important foliar diseases of wheat that can cause serious yield losses. Breeding for cultivars with diverse resources of resistance is the most promising approach for combating this disease. The diploid A genome progenitor species of wheat are an important resource for new variability for disease resistance genes. An accession of Triticum boeoticum (A^bA^b) showed resistance against a number of Bgt isolates, when tested using detached leaf segments. Inheritance studies in a recombinant inbred line population (RIL), developed from crosses of PM resistant T. boeoticum acc. pau5088 with a PM susceptible T. monococcum acc. pau14087, indicated the presence of two powdery mildew resistance genes in T. boeoticum acc. pau5088. Analysis of powdery mildew infection and molecular marker data of the RIL population revealed that both powdery mildew resistance genes are located on the long arm of chromosome 7A. Mapping was conducted using an integrated linkage map of 7A consisting of SSR, RFLP, STS, and DArT markers. These powdery mildew resistance genes are tentatively designated as PmTb7A.1 and PmTb7A.2. The PmTb7A.2 is closely linked to STS markers MAG2185 and MAG1759 derived from RFLP probes which are linked to powdery mildew resistance gene Pm1. This indicated that PmTb7A.2 might be allelic to Pm1. The PmTb7A.1, flanked by a DArT marker wPt4553 and an SSR marker Xcfa2019 in a 4.3 cM interval, maps proximal to PmT7A.2. PmTb7A.1 is putatively a new powdery mildew resistance gene. The powdery mildew resistance genes from T. boeoticum are currently being transferred to cultivated wheat background through marker-assisted backcrossing, using T. durum as bridging species.     

Characterization of <Emphasis Type="Italic">Pm59</Emphasis>, a novel powdery mildew resistance gene in Afghanistan wheat landrace PI 181356

Key message

A new powdery mildew resistance gene, designated Pm59, was identified in Afghanistan wheat landrace PI 181356, and mapped in the terminal region of the long arm of chromosome 7A.

Abstract

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is an important foliar disease of wheat worldwide. In the Great Plains of the USA, Bgt isolates virulent to widely used powdery mildew resistance genes, such as Pm3a, were previously identified. The objectives of this study were to characterize the powdery mildew resistance gene in Afghanistan landrace PI 181356, which exhibited high resistance to Bgt isolates collected in southern Great Plains, and identify molecular markers for marker-assisted selection. An F₂ population and F_2:3 lines derived from a cross between PI 181356 and OK1059060-126135-3 were used in this study. Genetic analysis indicated that PI 181356 carries a single dominant gene, designated Pm59, in the terminal region of the long arm of chromosome 7A. Pm59 was mapped to an interval between sequence tag site (STS) markers Xmag1759 and Xmag1714 with genetic distances of 0.4 cM distal to Xmag1759 and 5.7 cM proximal to Xmag1714. Physical mapping suggested that Pm59 is in the distal bin 7AL 0.99–1.00. Pm59 is a novel powdery mildew resistance gene, and confers resistance to Bgt isolates collected from the Great Plains and the state of Montana. Therefore, Pm59 can be used to breed powdery mildew-resistant cultivars in these regions. Xmag1759 is ideal for marker-assisted selection of Pm59 in wheat breeding.

     

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

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

Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat

Wheat (Triticum aestivum L.) incurs significant yield losses from powdery mildew, a major fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). enhanced disease resistance1 (EDR1) plays a negative role in the defense response against powdery mildew in Arabidopsis thaliana; however, the edr1 mutant does not show constitutively activated defense responses. This makes EDR1 an ideal target for approaches using new genome‐editing tools to improve resistance to powdery mildew. We cloned TaEDR1 from hexaploid wheat and found high similarity among the three homoeologs of EDR1. Knock‐down of TaEDR1 by virus‐induced gene silencing or RNA interference enhanced resistance to powdery mildew, indicating that TaEDR1 negatively regulates powdery mildew resistance in wheat. We used CRISPR/Cas9 technology to generate Taedr1 wheat plants by simultaneous modification of the three homoeologs of wheat EDR1. No off‐target mutations were detected in the Taedr1 mutant plants. The Taedr1 plants were resistant to powdery mildew and did not show mildew‐induced cell death. Our study represents the successful generation of a potentially valuable trait using genome‐editing technology in wheat and provides germplasm for disease resistance breeding.     

Pathotypes of Blumeria graminis f. sp. tritici,the causal agent of wheat powdery mildew from some regions of Iran

Wheat powdery mildew is controlled mainly by race-specific resistance. To be effective, breeding wheat for resistance to powdery mildew requires knowledge of virulence diversity in local populations of the pathogen. Isolates of Blumeria graminis, collected in 2009 and 2010 from three areas of Iranian production, were analysed for virulence using a host differential series comprised of 16 known genes conferring resistance to powdery mildew. The results showed that high-virulence frequencies to genes Pm1, Pm2, Pm4a, Pm5, Pm6, Pm7, Pm8 and Pm9 were found over both years and across all three areas. Virulence frequencies for Pm3a and Pm3b were intermediate, while virulence frequencies for Pm3a, Pm3c, Pm4a and Pm2, 6 were low. Genes Pm1, 2, 9 and Pm2, 4b, 8 were highly resistant in all regions. Virulence to Pm8 increased to high levels, while virulence to Pm4a decreased across the area surveyed from 2009 to 2010.     

Detection of Latent Infection of Wheat Leaves Caused by Blumeria graminis f.sp. tritici Using Nested PCR

Wheat powdery mildew caused by Blumeria graminis f.sp. tritici (Bgt) is an important and destructive disease worldwide. Detection of latent infection of wheat seedlings is critical to estimate initial inoculum potential of epidemics in the fields. To improve the conventional method, a nested PCR approach had been established in this study to detect latent infections of wheat leaves caused by Bgt. The DNA primer sets including external and internal primer pairs for the nested PCR were designed followed by testing their specificities to Bgt by using Bgt and other fungal species of wheat. Sensitivity test demonstrated that the nested PCR could detect as low as 0.1 fg template DNA and about 10,000 times more sensitive than the standard PCR. Results of artificial inoculation experiments showed that the nested PCR assay can detect a low level of latent infection of wheat seedlings 2 days earlier than did standard PCR. The incidences of latent infection of wheat seedlings determined by the nested PCR linearly correlated with those by the conventional incubation method (r² = 0.66, P = 0.0023). The incidences of latent infection detected with nested PCR were higher than that with the conventional method. This study provides an accurate method to efficiently estimate the initial inoculum potential of wheat powdery mildew epidemics in the fields.     

Molecular mapping of powdery mildew resistance gene <Emphasis Type="Italic">PmHNK</Emphasis> in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivar Zhoumai 22

The Chinese winter wheat cultivar Zhoumai 22 is highly resistant to powdery mildew. The objectives of this study were to map a powdery mildew resistance gene in Zhoumai 22 using molecular markers and investigate its allelism with Pm13. A total of 278 F₂ and 30 BC₁ plants, and 143 F₃ lines derived from the cross between resistant cultivar Zhoumai 22 and susceptible cultivar Chinese Spring were used for resistance gene tagging. The 137 F₂ plants from the cross Zhoumai 22/2761-5 (Pm13) were employed for the allelic test of the resistance genes. Two hundred and ten simple sequence repeat (SSR) markers were used to test the two parents, and resistant and susceptible bulks. Subsequently, seven polymorphic markers were used for genotyping the F₂ and F₃ populations. The results indicated that the powdery mildew resistance in Zhoumai 22 was conferred by a single dominant gene, designated PmHNK tentatively, flanked by seven SSR markers Xgwm299, Xgwm108, Xbarc77, Xbarc84, Xwmc326, Xwmc291 and Xwmc687 on chromosome 3BL. The resistance gene was closely linked to Xwmc291 and Xgwm108, with genetic distances of 3.8 and 10.3 cM, respectively, and located on the chromosome bin 3BL-7-0.63-1.0 in the test with a set of deletion lines. Seedling tests with seven isolates of Blumeria graminis f. sp. tritici (Bgt) and allellic test indicated that PmHNK is different from Pm13, and Pm41 seems also to be different from PmHNK due to its origin from T. dicoccoides and molecular evidence. These results indicate that PmHNK is likely to be a novel powdery mildew resistance gene in wheat.