The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype

Recessive mlo resistance alleles of the Mlo locus in barley control a non race-specific resistance response to infection by the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. All the mlo alleles analysed stop fungal growth at the same developmental stage within a subcellularly restricted, highly localized cell wall apposition directly beneath the site of abortive fungal penetration. We report that near-isogenic lines carrying the alleles mlo ₁, mlo ₃ or mlo ₅ undergo dramatic spontaneous formation of cell wall appositions, not only in the absence of the fungal pathogen but also in sterile grown plants. A comparative study of spontaneous and infection-triggered cell wall appositions reveals a high degree of similarity with respect to structure, chemical composition and distinct localization within plant tissue. We show that the rate of spontaneous apposition formation is dependent on the genetic background of the plant and that its onset is under developmental control. Furthermore, spontaneous formation of wall appositions is specifically triggered by mlo alleles, since it is unaffected in the presence of the race-specific resistance allele Mlg. We propose a model for the function of the Mlo locus that suggests that both Mlo and mlo alleles control qualitatively the same apposition-based resistance mechanism, which, in the presence of the wild-type Mlo allele, is merely less efficient to provide protection against the currently common races of E. graminis f.sp. hordei.     

Novel induced <Emphasis Type="Italic">mlo</Emphasis> mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

Background  

Recessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility.     

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.     

A high-resolution genetic map and a diagnostic RFLP marker for the Mlg resistance locus to powdery mildew in barley

The semi-dominantly acting Mlg resistance locus in barley confers race-specific resistance to the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. A high-resolution genetic map was constructed at Mlg based on a cross between the near-isogenic barley lines Pallas BC₅ Mlg and Pallas mlg. A total of 2000 F₂ progeny were inspected by cleaved amplified polymorphic sequence (CAPS) analysis, defining a 4.47 cM interval encompassing the resistance locus. Pathogen challenge of the segregants with multiple powdery mildew isolates uncovered a novel resistance specificity in Pallas BC₅ Mlg. Probes from within 4.0 cM of Mlg were mapped in rice, revealing orthologues on five different rice chromosomes and suggesting multiple breaks of chromosomal collinearity in this region between the two grass species. The most tightly Mlg-linked RFLP marker, MWG032, was shown to reliably detect the presence of the resistance allele in a collection of 30 European barley cultivars. Received: 23 March 2000 / Accepted: 20 April 2000     

Differential expression of putative cell death regulator genes in near-isogenic,resistant and susceptible barley lines during interaction with the powdery mildew fungus

We analysed pathogenesis-related expression of genes, that are assumed to be involved in ubiquitous plant defence mechanisms like the oxidative burst, the hypersensitive cell death reaction (HR) and formation of localized cell wall appositions (papillae). We carried out comparative northern blot and RT-PCR studies with near-isogenic barley (Hordeum vulgareL. cv. Pallas) lines (NILs) resistant or susceptible to the powdery mildew fungus race A6 (Blumeria graminis f.sp. hordei, BghA6). The NILs carrying one of the R-genes Mla12, Mlg or the mlo mutant allele mlo5 arrest fungal development by cell wall appositions (mlo5) or a HR (Mla12) or both (Mlg). Expression of an aspartate protease gene, an ascorbate peroxidase gene and a newly identified cysteine protease gene was up-regulated after inoculation with BghA6, whereas the constitutive expression-level of a BAS gene, that encodes an alkyl hydroperoxide reductase, was reduced. Expression of a newly identified barley homologue of a mammalian cell death regulator, Bax inhibitor 1, was enhanced after powdery mildew inoculation. An oxalate oxidase-like protein was stronger expressed in NILS expressing penetration resistance. A so far unknown gene that putatively encodes the large subunit of a superoxide generating NADPH oxidases was constitutively expressed in barley leaves and its expression pattern did not change after inoculation. A newly identified barley Rac1 homologue was expressed constitutively, such as the functionally linked NADPH oxidase gene. Gene expression patterns are discussed with regard to defence mechanisms and signal transduction.     

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.     

Identification of Genes Required for the Function of Non-Race-Specific mlo Resistance to Powdery Mildew in Barley

Recessive alleles (mlo) of the Mlo locus in barley mediate a broad, non-race-specific resistance reaction to the powdery mildew fungus Erysiphe graminis f sp hordei. A mutational approach was used to identify genes that are required for the function of mlo. Six susceptible M2 individuals were isolated after inoculation with the fungal isolate K1 from chemically mutagenized seed carrying the mlo-5 allele. Susceptibility in each of these individuals is due to monogenic, recessively inherited mutations in loci unlinked to mlo. The mutants identify two unlinked complementation groups, designated Ror1 and Ror2 (required for mlo-specified resistance). Both Ror genes are required for the function of different tested mlo alleles and for mlo function after challenge with different isolates of E. g. f sp hordei. A quantitative cytological time course analysis revealed that the host cell penetration efficiency in the mutants is intermediate compared with mlo-resistant and Mlo-susceptible genotypes. Ror1 and Ror2 mutants could be differentiated from each other by the same criterion. The spontaneous formation of cell wall appositions in mlo plants, a subcellular structure believed to represent part of the mlo defense, is suppressed in mlo/ror genotypes. In contrast, accumulation of major structural components in the appositions is seemingly unaltered. We conclude that there is a regulatory function for the Ror genes in mlo-specified resistance and propose a model in which the Mlo wild-type allele functions as a negative regulator and the Ror genes act as positive regulators of a non-race-specific resistance response.     

Elektronenmikroskopische Untersuchungen des Gerstenmehltaus (Erysiphe graminis DC f. sp. hordei Marchal) nach Resistenzinduktion mit mikrobiellen Stoffwechselprodukten

Electron microscopical studies on Mildew of Barley (Erysiphe graminis DC f. sp. hordei Marchal) after induced Resistance with Products of Microbial Metabolism Ultrastructural studies of the infection progress of barley by Erysiphe graminis after induction of resistance with products of microbial metabolism showed that pathogen development was affected through inducer activated defence mechanisms. The formation of papillae-like structures and an accumulation of electron dense material below the host cell wall at the penetration site appeared to be associated with reduced penetration. The haustoria were also altered in the extrahaustorial membrane and electron dense material accumulated at the cell wall of haustorial body and neck, thus apparently impairing efficient functioning and allowing only limited growth of the pathogen.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

The conserved oligomeric Golgi complex is involved in penetration resistance of barley to the barley powdery mildew fungus

Membrane trafficking is vital to plant development and adaptation to the environment. It is suggested that post‐Golgi vesicles and multivesicular bodies are essential for plant defence against directly penetrating fungal parasites at the cell wall. However, the actual plant proteins involved in membrane transport for defence are largely unidentified. We applied a candidate gene approach and single cell transient‐induced gene silencing for the identification of membrane trafficking proteins of barley involved in the response to the fungal pathogen Blumeria graminis f.sp. hordei. This revealed potential components of vesicle tethering complexes [putative exocyst subunit HvEXO70F‐like and subunits of the conserved oligomeric Golgi (COG) complex] and Golgi membrane trafficking (COPIγ coatomer and HvYPT1‐like RAB GTPase) as essential for resistance to fungal penetration into the host cell.     

Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1

Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1_{ΔKL308–309}), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.     

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.     

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.     

Signal Molecules Inducing Systemic Resistance and Susceptibility in Barley Seedlings

Exudate collected from the cut end of barley seedlings exhibited both activities that induced systemic resistance and susceptibility against Erysiphe graminis f. sp. hordei race Hh4 depending on the time after pruning. Exudates collected between 3–6 h after pruning showed maximum activity that induced systemic resistance, whereas those during 9–12 h conversely induced susceptibility in barley seedlings. The accumulation of antifungal substances in barley leaves correlates to the timing, of induced resistance. The antifuntingal substances were watersoluble and severely affected the infection of E. graminis f. sp. hordei.     

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.     

Lauric Acid Exhibits Antifungal Activity Against Plant Pathogenic Fungi

This study aimed at examining the effects of the saturated fatty acid lauric acid on mycelial growth of Rhizoctonia solani and Pythium ultimum and on infection of barley seedlings with Blumeria graminis f. sp. hordei. Mycelial growth of R. solani and P. ultimum in agar culture was significantly reduced by lauric acid at concentrations of 100 μm and above, while no fungal growth occurred in liquid culture at concentrations above 50 μm . Application of lauric acid at concentrations ranging from 250 to 1000 μm to barley leaves before or after inoculation with B. graminis f. sp. hordei led to significant reductions in infection. This study provides the first report of the activity of lauric acid against plant pathogenic fungi and indicates the need for investigation of its mechanism of action.     

Superoxide Generation in Chemically Activated Resistance of Barley in Response to Inoculation with the Powdery Mildew Fungus

In a previous work, a phenotype-specific accumulation of superoxide radical anions (O^??₂) after attack of the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) in near-isogenic barley (Hordeum vulgare L.) lines bearing different Mlx genes for resistance was described (Hückelhoven and Kogel, 1998). We have now a histochemical study of the pathogenesis-related O^??₂ generation in the systemic activated resistance (SAR) response induced in barley cv Pallas by the plant activator 2,6-dichloroisonicotinic acid (DCINA). SAR-specific defence was conducted prevalently characterized by penetration resistance. Fungal arrest was observed before haustorium formation by a highly localized cell wall reinforcement (effective papillae) and, in most cases, by a subsequent hypersensitive cell death (HR). No O^??₂ generation was found in association with these plant defence responses. However, a strong O^??₂ burst in the attacked epidermal cells was detected in the control plants which were not activated by DCINA. This burst coincided with cell wall penetration and subsequent contact of the pathogen with the host plasma membrane. A strong SAR-related O^??₂ burst was induced in the mesophyll tissue beneath the attacked and hypersensitively reacting epidermal cells in plants treated with DCINA. The accumulation of O^??₂ was confined to chloroplasts. The remarkable burst in mesophyll tissue was not followed by mesophyll-HR indicating that chloroplastic O^??₂ generation is not sufficient for the hypersensitive cell death. Since the same pattern of pathogenesis-related O^??₂ accumulation was identified for race-specific response mediated by the Mlg gene for powdery mildew resistance, the present data are consistent with the hypothesis that the SAR phenotype is a phenocopy of the Mlg-type resistance (Kogel et al., 1994).     

Correlation Between Induced Resistance and Host Fluorescence in Barley Inoculated with Erysiphe graminis

Resistance was induced in barley by virulent and avirulent Erysiphe graminis f. sp. hordei and by E. graminis f. sp. tritici. The diameters of fluorescent haloes and papillae at the site of the primary germ tube of the inducers were larger until 12 hours after inoculation with E g. f. sp. tritici than in the corresponding periods after inoculation with E. g. f. sp. hordei. Fluorescence at the site of appressorial lobes of the inducers developed, gradually from 10 and 12 hours after inoculation with E. g. f. sp. hordei and E. g. f. sp. tritici, respectively. Penetration success of single infection units of the challenger was reduced in cells with inducer fluorescence, suggesting that resistance is principally localized to cells previously attacked by the inducer. But penetration success was independent of the average distance to the ten nearest inducer fluorescences, and penetration failures did also occur in cells without inducer fluorescence, suggesting that the induced resistance to some extent is translocated to other epidermal cells. The average diameter of the fluorescent papillae at the site of unsuccessful challenger infection units increased as a result of induced resistance.     

Molecular mapping of a recessive gene for resistance to stripe rust in barley

Barley stripe rust, caused by Puccinia striiformis f. sp. hordei, is one of the most important barley (Hordeum vulgare) diseases in the United States. The disease is best controlled using resistant cultivars. Barley genotype Grannenlose Zweizeilige (GZ) has a recessive gene (rpsGZ) that is effective against all races of P. striiformis f. sp. hordei identified so far in the USA. To develop a molecular map for mapping the gene, F₈ recombinant inbred lines (RILs) were developed from the Steptoe X GZ cross through single-seed descent. Seedlings of the parents and RILs were evaluated for resistance to races PSH-14 and PSH-54 of P. striiformis f. sp. hordei under controlled greenhouse conditions. Genomic DNA was extracted from the parents and 182 F₈ RILs and used for linkage analysis. The resistance gene analog polymorphism (RGAP) technique was used to identify molecular markers for rpsGZ. A linkage group for the gene was constructed with 12 RGAP markers, of which two markers co-segregated with the resistance locus, and two markers were closely linked to the locus with a genetic distance of 0.9 and 2.0 cM, respectively. These four markers were present only in the susceptible parent. The closest marker to the resistance allele was 11.7 cM away. Analyses of two sets of barley chromosome addition lines of wheat with the two RGAP markers that were cosegregating with the susceptibility allele showed that rpsGZ and the markers were located on the long arm of barley chromosome 4H. Further, tests with four simple sequence repeat (SSR) markers confirmed the chromosomal location of the rpsGZ gene and also integrated the RGAP markers into the known SSR-based linkage map of barley. The closest SSR marker EBmac0679 had a genetic distance of 7.5 cM with the gene in the integrated linkage map constructed with the 12 RGAP markers and 4 SSR markers. The information on chromosomal location and molecular markers for rpsGZ should be useful for incorporating this gene into commercial cultivars and combining it with other resistance genes for durable resistance.     

The Relationship between Systemic Resistance Induced by Pruning and Accumulation of Antifungal Substances in Barley Seedlings

Infection by a compatible race of Erysiphe graminis f. sp. hordei on barley secondary leaves was significantly suppressed upon pruning of the primary leaves when E. graminis hordei was inoculated 3–12 h after the pruning, but it, was rather enhanced during 15–21 h. The accumulation of antifungal substances was detected in hot ethanol extracts of barley seedlings from 15–27 h after pruning the primary leaves. Taking the time of the infection process of a challenger (E. graminis, hordei) into consideration, timing of systemic resistance induced upon pruning coincided with the accumulation of antifungal substances.