mlo‐based powdery mildew resistance in hexaploid bread wheat generated by a non‐transgenic TILLING approach

Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non‐transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss‐of‐function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss‐of‐function alleles (mlo) of barley Mlo are known to confer durable broad‐spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo‐A1, TaMlo‐B1 and TaMlo‐D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non‐conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non‐transgenic, powdery mildew‐resistant bread wheat varieties.     

A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs

Background  

To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps.     

Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa

Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.     

Molecular cloning and expression analysis of CmMlo1 in melon

Mlo gene encodes an important transmembrane protein that is involved in biotic/abiotic stresses. Using the method of homologous, we cloned a Mlo gene from melon, named CmMlo1. The gene is 1551 bp in length, encoding 516 amino acids; it has seven-transmembrane domain topology and is a typical transmembrane protein. Localization analysis in onion epidermal cells showed that CmMlo1-GFP is localized to the plasma membrane. RT-PCR results indicated that CmMlo1 is mainly expressed in melon cotyledon and flower, with a tissue-specific distribution manner. CmMlo1 expression is not obvious under powdery mildew stress, but under cadmium stress, its expression was significantly up-regulated, indicating that CmMlo1 is possibly involved in abiotic stress.     

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.     

CAPS and DHPLC Analysis of a Single Nucleotide Polymorphism in the Cytochrome b Gene Conferring Resistance to Strobilurins in Field Isolates of Blumeria graminis f. sp. hordei

A single nucleotide polymorphism in the wheat powdery mildew (Blumeria graminis f. sp. tritici) cytochrome b gene is responsible for resistance to inhibitors of the quinol outer binding site of the cytochrome bc₁ complex (QoI) fungicides. Analysis of a partial sequence of the cytochrome b gene from field isolates resistant and sensitive to QoI fungicides revealed the same point mutation in barley powdery mildew (B. graminis f. sp. hordei). Analysis of 118 and 40 barley powdery mildew isolates using a cleaved amplified polymorphic sequence assay and denaturing high performance liquid chromatography, respectively, confirmed that this single nucleotide polymorphism also confers resistance to QoI fungicides in barley powdery mildew.     

Mapping resistance to powdery mildew in barley reveals a large-effect nonhost resistance QTL

Key message

Resistance factors against non-adapted powdery mildews were mapped in barley. Some QTLs seem effective only to non-adapted mildews, while others also play a role in defense against the adapted form.The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. We investigated which genetic factors determine the nonhost status of barley (Hordeum vulgare L.) to powdery mildews (Blumeria graminis). We set out to verify whether genes involved in nonhost resistance have a wide effectiveness spectrum, and whether nonhost resistance genes confer resistance to the barley adapted powdery mildew. Two barley lines, SusBgt_SC and SusBgt_DC, with some susceptibility to the wheat powdery mildew B. graminis f.sp. tritici (Bgt) were crossed with cv Vada to generate two mapping populations. Each population was assessed for level of infection against four B. graminis ff.spp, and QTL mapping analyses were performed. Our results demonstrate polygenic inheritance for nonhost resistance, with some QTLs effective only to non-adapted mildews, while others play a role against adapted and non-adapted forms. Histology analyses of nonhost interaction show that most penetration attempts are stopped in association with papillae, and also suggest independent layers of defence at haustorium establishment and conidiophore formation. Nonhost resistance of barley to powdery mildew relies mostly on non-hypersensitive mechanisms. A large-effect nonhost resistance QTL mapped to a 1.4 cM interval is suitable for map-based cloning.

     

Pea powdery mildew <Emphasis Type="Italic">er1</Emphasis> resistance is associated to loss-of-function mutations at a <Emphasis Type="Italic">MLO</Emphasis> homologous locus

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F₂ individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.     

The effectivity of Tilletiopsis albescens in biocontrol of powdery mildew

Tilletiopsis albescens grows well on powdery mildew fungi inoculated on barley or cucumber leaves and causes collapse of the colonies. Application of ballistospores or cut mycelium was equally effective for biocontrol, and the effectiveness tended to increase exponentially with the concentration of germinating units (conidia and cut mycelium) applied. Seventy percent relative humidity or more is required for effective biocontrol. Two applications of T. albescens in the period from 3 days before to 3 days after inoculation with powdery mildew were more effective than one. Applications before inoculation or 7 days after inoculation with powdery mildew had little effect. T. albescens followed the powdery mildew as it was disseminated to uninoculated leaves, but this did not result in an effective biocontrol. The potential for using T. albescens for biocontrol of powdery mildews is discussed.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.     

AB-QTL analysis in spring barley. I. Detection of resistance genes against powdery mildew, leaf rust and scald introgressed from wild barley

The objective of this study was to map new resistance genes against powdery mildew (Blumeria graminis f. sp. hordei L.), leaf rust (Puccinia hordei L.) and scald [Rhynchosporium secalis (Oud.) J. Davis] in the advanced backcross doubled haploid (BC₂DH) population S42 derived from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). Using field data of disease severity recorded in eight environments under natural infestation and genotype data of 98 SSR loci, we detected nine QTL for powdery mildew, six QTL for leaf rust resistance and three QTL for scald resistance. The presence of the exotic QTL alleles reduced disease symptoms by a maximum of 51.5, 37.6 and 16.5% for powdery mildew, leaf rust and scald, respectively. Some of the detected QTL may correspond to previously identified qualitative (i.e. Mla) and to quantitative resistance genes. Others may be newly identified resistance genes. For the majority of resistance QTL (61.0%) the wild barley contributed the favourable allele demonstrating the usefulness of wild barley in the quest for resistant cultivars.     

Germination of Erysiphe gratninis f.sp. hordei conidia on barley leaves

Germination of Erysiphe graminis f.sp. hordei conidia on leaves of several barley cultivars was studied in the laboratory. On both detached leaves and intact plants, within 48 h of inoculation a higher proportion of conidia had germinated on the basal and middle portions of the adaxial leaf surface than on the corresponding portions of the abaxial surface. Such differences between surfaces were not observed near the leaf tip. Similar results were obtained with all the cultivars and growth stages tested, and with five isolates of E. graminis, and are consistent with the observation that there is usually less powdery mildew on the abaxial than the adaxial surface of barley leaves. With most of the barley genotype/mildew isolate combinations tested, within 48 h of inoculation higher proportions of conidia germinated on seedlings and juvenile plants than on older plants. Inherited characteristics which affect spore germination on the leaf surface may be important factors in the development of adult-plant resistance of barley to powdery mildew, particularly in certain genotypes.     

Sources of powdery mildew resistance in barley landraces from morocco

A total of forty eight accessions of barley landraces from Morocco were screened for resistance to powdery mildew. Twenty two (46%) of tested landraces showed resistance reactions and thirty four single plant lines were selected. Eleven of these lines were tested in seedling stage with seventeen and another twenty three lines with twenty three isolates of powdery mildew respectively. The isolates were chosen according to the virulence spectra observed on the ‘Pallas’ isolines differential set. Line 229–2–2 was identified with resistance to all prevalent in Europe powdery mildew virulence genes. Lines 230–1–1, 248–1–3 showed susceptible reaction for only one and lines 221–3–2, 227–1–1, 244–3–4 for only two isolates respectively. Three different resistance alleles (Mlat, Mla6, and MLA14) were postulated to be present in tested lines alone or in combination. In thirty (88%) tested lines it was impossible to determine which specific gene or genes for resistance were present. Most probably these lines possessed alleles not represented in the ‘Pallas’ isolines differential set. The distribution of reaction type indicated that about 71% of all reaction types observed were classified as powdery mildew resistance (scores 0, 1 and 2). Majority (79%) of resistance reaction types observed in tested lines was intermediate resistance reaction type two and twenty three lines (68%) showed this reaction for inoculation with more than 50% isolates used. The use of new effective sources of resistance from Moroccan barley landraces for diversification of resistance genes for powdery mildew in barley cultivars was discussed.     

Cultivar-related differences in the distribution of cell-wall-bound thionins in compatible and incompatible interactions between barley and powdery mildew

Leaf-specific thionins of barley (Hordeum vulgare L.) have been identified as a novel class of cell-wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants. The distribution of these polypeptides has been studied in the host-pathogen system of barley and Erisyphe graminis DC.f.sp. hordei Marchal (powdery mildew). Immunogold-labelling of thionins in several barley cultivars indicates that resistance or susceptibility may be attributed to the presence or absence of thionins at the penetration site in walls and papillae of epidermal leaf cells.All of the leaf-specific thionin genes are confined to the distal end of the short arm of chromosome 6 of barley. None of the genes for cultivarspecific resistance to powdery mildew which have previously been mapped on barley chromosomes are found close to this locus.     

Loss of Function in Mlo Orthologs Reduces Susceptibility of Pepper and Tomato to Powdery Mildew Disease Caused by Leveillula taurica

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse tomato and pepper production. In contrast to most powdery mildew species which are epiphytic, L. taurica is an endophytic fungus colonizing the mesophyll tissues of the leaf. In barley, Arabidopsis, tomato and pea, the correct functioning of specific homologues of the plant Mlo gene family has been found to be required for pathogenesis of epiphytic powdery mildew fungi. The aim of this study was to investigate the involvement of the Mlo genes in susceptibility to the endophytic fungus L. taurica. In tomato (Solanum lycopersicum), a loss-of-function mutation in the SlMlo1 gene results in resistance to powdery mildew disease caused by Oidium neolycopersici. When the tomato Slmlo1 mutant was inoculated with L. taurica in this study, it proved to be less susceptible compared to the control, S. lycopersicum cv. Moneymaker. Further, overexpression of SlMlo1 in the tomato Slmlo1 mutant enhanced susceptibility to L. taurica. In pepper, the CaMlo2 gene was isolated by applying a homology-based cloning approach. Compared to the previously identified CaMlo1 gene, the CaMlo2 gene is more similar to SlMlo1 as shown by phylogenetic analysis, and the expression of CaMlo2 is up-regulated at an earlier time point upon L. taurica infection. However, results of virus-induced gene silencing suggest that both CaMlo1 and CaMlo2 may be involved in the susceptibility of pepper to L. taurica. The fact that overexpression of CaMlo2 restored the susceptibility of the tomato Slmlo1 mutant to O. neolycopersici and increased its susceptibility to L. taurica confirmed the role of CaMlo2 acting as a susceptibility factor to different powdery mildews, though the role of CaMlo1 as a co-factor for susceptibility cannot be excluded.     

Fine mapping and chromosome walking towards the Ror1 locus in barley (Hordeum vulgare L.)

Key message

The Ror1 gene was fine-mapped to the pericentric region of barley chromosome 1HL.

Abstract

Recessively inherited loss-of-function alleles of the barley (Hordeum vulgare) Mildew resistance locus o (Mlo) gene confer durable broad-spectrum disease resistance against the obligate biotrophic fungal powdery mildew pathogen Blumeria graminis f.sp. hordei. Previous genetic analyses revealed two barley genes, Ror1 and Ror2, that are Required for mlo-specified resistance and basal defence. While Ror2 was cloned and shown to encode a t-SNARE protein (syntaxin), the molecular nature or Ror1 remained elusive. Ror1 was previously mapped to the centromeric region of the long arm of barley chromosome 1H. Here, we narrowed the barley Ror1 interval to 0.18 cM and initiated a chromosome walk using barley yeast artificial chromosome (YAC) clones, next-generation DNA sequencing and fluorescence in situ hybridization. Two non-overlapping YAC contigs containing Ror1 flanking genes were identified. Despite a high degree of synteny observed between barley and the sequenced genomes of the grasses rice (Oryza sativa), Brachypodium distachyon and Sorghum bicolor across the wider chromosomal area, the genes in the YAC contigs showed extensive interspecific rearrangements in orientation and order. Consequently, the position of a Ror1 homolog in these species could not be precisely predicted, nor was a barley gene co-segregating with Ror1 identified. These factors have prevented the molecular identification of the Ror1 gene for the time being.     

Pyramiding of transgenic <Emphasis Type="Italic">Pm3</Emphasis> alleles in wheat results in improved powdery mildew resistance in the field

Key message

The combined effects of enhanced total transgene expression level and allele-specificity combination in transgenic allele-pyramided Pm3 wheat lines result in improved powdery mildew field resistance without negative pleiotropic effects.

Abstract

Allelic Pm3 resistance genes of wheat confer race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and encode nucleotide-binding domain, leucine-rich repeat (NLR) receptors. Transgenic wheat lines overexpressing alleles Pm3a, b, c, d, f, and g have previously been generated by transformation of cultivar Bobwhite and tested in field trials, revealing varying degrees of powdery mildew resistance conferred by the transgenes. Here, we tested four transgenic lines each carrying two pyramided Pm3 alleles, which were generated by crossbreeding of lines transformed with single Pm3 alleles. All four allele-pyramided lines showed strongly improved powdery mildew resistance in the field compared to their parental lines. The improved resistance results from the two effects of enhanced total transgene expression levels and allele-specificity combinations. In contrast to leaf segment tests on greenhouse-grown seedlings, no allelic suppression was observed in the field. Plant development and yield scores of the pyramided lines were similar to the mean scores of the corresponding parental lines, and thus, the allele pyramiding did not cause any negative effects. On the contrary, in pyramided line, Pm3b × Pm3f normal plant development was restored compared to the delayed development and reduced seed set of parental line Pm3f. Allele-specific RT qPCR revealed additive transgene expression levels of the two Pm3 alleles in the pyramided lines. A positive correlation between total transgene expression level and powdery mildew field resistance was observed. In summary, allele pyramiding of Pm3 transgenes proved to be successful in enhancing powdery mildew field resistance.

     

The phytopathogen powdery mildew affects food-searching behavior and survival of <Emphasis Type="Italic">Coccinella septempunctata</Emphasis>

The diet of entomophagous coccinellids is mainly based on aphids and other food sources such as pollen, nectar, or fungal spores. Knowledge of their foraging behavior on plants infected by powdery mildew and their survival on fungal spores is currently limited. In this study, we investigated the olfactory response of Coccinella septempunctata to odor emission of barley plants infected by powdery mildew and their survival on fungal spores in the presence or absence of aphids. Odors released by powdery-mildew infected plants were more attractive for ladybirds compared to those of uninfected controls. After 3 days, the survival rate of ladybirds feeding only on powdery-mildew spores was less than 50%, while for ladybirds feeding exclusively on Rhopalosiphum padi aphids, the survival rate was close to 90%. After 15 days, the highest survival rate (almost 80%) was observed for ladybirds feeding on plants with both aphids and powdery mildew. Molecular analyses confirmed the presence of fungal spores in ladybird guts when feeding either on powdery mildew or on a mixed diet. Our results provide new insights into foraging behavior of entomophagous coccinellids revealing the potential of powdery mildew to be utilized as important non-essential food in a mixed diet, but also its lethal effect if consumed alone.