The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat

The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla-RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full-length CC-NBS-LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full-length candidate cDNA, two single-cell expression assays were used to demonstrate complementation of AvrMla6-dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1-independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC-NBS-LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.     

The barley apoptosis suppressor homologue BAX inhibitor-1 compromises nonhost penetration resistance of barley to the inappropriate pathogen Blumeria graminis f. sp. tritici

BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, overexpression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.     

Polyamine metabolism in barley reacting hypersensitively to the powdery mildew fungus Blumeria graminis f. sp. hordei

Polyamine levels and activities of enzymes of polyamine biosynthesis and catabolism were examined in the barley cultivar Delibes (Ml1al + Ml(Ab)) reacting hypersensitively to the powdery mildew fungus, Blumeria graminis f. sp. hordei (race CC220). Levels of free putrescine and spermine and of conjugated forms of putrescine, spermidine and spermine were greatly increased 1–4 d following inoculation of barley with the powdery mildew. These changes in polyamine levels were accompanied by elevated activities of the polyamine biosynthetic enzymes ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and S‐adenosylmethionine decarboxylase (AdoMetDC) and the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO). Activities of two enzymes involved in conjugating polyamines to hydroxycinnamic acids, putrescine hydroxycinnamoyl transferase (PHT) and tyramine feruloyl‐CoA transferase (TFT) were also examined and were found to increase significantly 1–4 d after inoculation. The possibility that the increased levels of free spermine, increased polyamine conjugates, and increased DAO and PAO activities are involved in development of the hypersensitive response of Delibes to powdery mildew infection is discussed.     

Proteogenomics and in silico structural and functional annotation of the barley powdery mildew Blumeria graminis f. sp. hordei

Blumeria graminis is an economically important obligate plant-pathogenic fungus, whose entire genome was recently sequenced and manually annotated using ab initio in silico predictions (Spanu et al. 2010, Science 330, 1543-1546). Employing large scale proteogenomic analysis we are now able to verify independently the existence of proteins predicted by ∼24% of open reading frame models. We compared the haustoria and sporulating hyphae proteomes and identified 71 proteins exclusively in haustoria, the feeding and effector-delivery organs of the pathogen. These proteins are significantly smaller than the rest of the protein pool and predicted to be secreted. Most do not share any similarities with Swiss–Prot or Trembl entries nor possess any identifiable Pfam domains. We used a novel automated prediction pipeline to model the 3D structures of the proteins, identify putative ligand binding sites and predict regions of intrinsic disorder. This revealed that the protein set found exclusively in haustoria is significantly less disordered than the rest of the identified Blumeria proteins or random (and representative) protein sets generated from the yeast proteome. For most of the haustorial proteins with unknown functions no good templates could be found, from which to generate high quality models. Thus, these unknown proteins present potentially new protein folds that can be specific to the interaction of the pathogen with its host.     

Apoplastic pH signaling in barley leaves attacked by the powdery mildew fungus Blumeria graminis f. sp. hordei

To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H(+)- and Ca(2+)-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H(+)- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response.     

Pathogenic divergence of Central European and Australian populations of Blumeria graminis f. sp. hordei

Hosts and pathogens have adapted their response to each other through genetic changes that have arisen during the course of their co‐evolution. In developed countries the longevity of varieties is often short; new varieties frequently possess novel genes with specific resistance to pathogens. The latter must adapt to the resistance genes to maintain pathogenicity. To study this adaptation, 50 Central European and 50 Australian isolates of Blumeria graminis f. sp. hordei (Bgh) were tested on 50 barley differential varieties with different specific resistance genes. All the Central European isolates differed from each other in their virulence combinations and belonged to 50 various pathotypes, whereas Australian isolates comprised 37 pathotypes. None of the pathotypes detected in Central Europe was identical or similar to any of those in Australia. This can be attributed to the much higher number of virulences in Central European isolates that developed over a long period of contact with a range of host varieties containing specific resistance genes. This has led to a gradual divergence of the Australian and the European Bgh populations. In Europe, unlike Australia, new specific resistance genes are still widely used in breeding barley varieties and the divergence of both populations will continue.     

DNA polymorphism among barley NILs of cv. Pallas, carrying genes for resistance to powdery mildew (Blumeria graminis f. sp. hordei)

Barley powdery mildew, caused by the pathogen Blumeria graminis f. sp. hordei is an important disease of barley (Hordeum vulgare L.). The random amplified polymorphic DNA (RAPD) method was used to detect DNA polymorphism among 7 Pallas near-isogenic lines (NILs) carrying Mla3, Mla12, Mlk, Mlp, Mlat, Mlg and MlLa genes for resistance to B. graminis f. sp. hordei. From among 500 random 10-mer primers tested, 3 were specific for NIL P2 (Mla3), 1 for P10 (Mla12), 6 for P17 (Mlk), 46 for P19 (Mlp), 4 for P20 (Mlat), 6 for P21 (Mlg), and 4 for P23 (MlLa). The results of this study demonstrated that the RAPD technique is a useful tool for detecting DNA polymorphism among Pallas NILs.     

Respiratory burst oxidase homologue A of barley contributes to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei

Reactive oxygen intermediates (ROI) are closely related to defence reactions of plants against pathogens. A prominent role in the production of ROI has been attributed to the plant respiratory burst oxidase homologues (RBOH) of the human phagocyte GP91(phox). A barley RBOH, which encodes a putative superoxide (O2*-)) producing NADPH oxidase, is described here. Histochemical analysis of the barley-Blumeria graminis f. sp. hordei (Bgh) interaction showed that O(2*-) is produced locally at the site of penetration. In contrast, hydrogen peroxide (H2O2) is produced in non-penetrated cell wall appositions. A barley RBOHA cDNA was isolated and a minor induction of expression of RBOHA was observed during the interactions of barley with Bgh. Transient RNA interference-mediated gene silencing of HvRBOHA during the penetration process of Bgh led to an increase of basal penetration resistance. The results support a potential role of HvRBOHA in cellular accessibility to Blumeria graminis.     

A proteomic analysis of powdery mildew (Blumeria graminis f.sp. hordei) conidiospores

Conidiospores are the asexual propagation units of many plant-pathogenic fungi. In this article, we report an annotated proteome map of ungerminated conidiospores of the ascomycete barley powdery mildew pathogen, Blumeria graminis f.sp. hordei . Using a combination of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, we have identified the proteins in 180 spots, which probably represent at least 123 distinct fungal gene products. Most of the identified proteins have a predicted function in carbohydrate, lipid or protein metabolism, indicating that the spore is equipped for the catabolism of storage compounds as well as for protein biosynthesis and folding on germination.     

Localisation of genes for resistance against Blumeria graminis f.sp. hordei and Puccinia graminis in a cross between a barley cultivar and a wild barley (Hordeum vulgare ssp. spontaneum) line

The aims of this investigation have been to map new (quantitative) resistance genes against powdery mildew, caused by Blumeria graminis f.sp. hordei L., and leaf rust, caused by Puccinia hordei L., in a cross between the barley ( Hordeum vulgare ssp. vulgare) cultivar "Vada" and the wild barley ( Hordeum vulgare ssp. spontaneum) line "1B-87" originating from Israel. The population consisted of 121 recombinant inbred lines. Resistance against leaf rust and powdery mildew was tested on detached leaves. The leaf rust isolate "I-80" and the powdery mildew isolate "Va-4", respectively, were used for the infection in this experiment. Moreover, powdery mildew disease severity was observed in the field at two different epidemic stages. In addition to other DNA markers, the map included 13 RGA (resistance gene analog) loci. The structure of the data demanded a non-parametric QTL-analysis. For each of the four observations, two QTLs with very high significance were localised. QTLs for resistance against powdery mildew were detected on chromosome 1H, 2H, 3H, 4H and 7H. QTLs for resistance against leaf rust were localised on 2H and 6H. Only one QTL was common for two of the powdery mildew related traits. Three of the seven QTLs were localised at the positions of the RGA-loci. Three of the five powdery mildew related QTLs are sharing their chromosomal position with known qualitative resistance genes. All detected QTLs behaved additively. Possible sources of the distorted segregation observed, the differences between the results for the different powdery mildew related traits and the relation between qualitative and quantitative resistance are discussed.     

Seasonal changes in infection of barley seedlings by Erysiphe graminis f.sp. hordei

Infection of artificially inoculated barley seedlings, exposed out of doors, was variable but there was a seasonal trend. As temperature affects both incubation time and the proportion of successful infections, these two factors will have additive effects on disease development. Infections developed only slowly in winter but, in both 1972 and 1973, conditions became much more favourable to infection in late April or early May.     

RFLP mapping of three new loci for resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) in barley

Three new major, race-specific, resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) were identified in three barley lines, RS42-6*O, RS137-28*E, and HSY-78*A, derived from crosses with wild barley (Hordeum vulgare ssp. spontaneum). The resistance gene origining from wild barley in line RS42-6*O, showed a recessive mode of inheritance, whereas the other wild barley genes were (semi)-dominant. RFLP mapping of these three genes was performed in segregating F₂ populations. The recessive gene in line RS42-6*O, was localized on barley chromosome 1S (7HS), while the (semi)-dominant genes in lines RS137-28*E, and HSY-78*A, were localized on chromosomes 1L (7HL) and 7L (5HL), respectively. Closely linked RFLP clones mapped at distances between 2.6cM and 5.3 cM. Hitherto, specific loci for powdery mildew resistance in barley had not been located on these chromosomes. Furthermore, tests for linkage to the unlocalized resistance gene Mlp revealed free segregation. Therefore, these genes represent new loci and new designations are suggested: mlt (RS42-6*O), Mlf (RS137-28*E), and Mlj (HSY-78*A). Comparisons with mapped QTLs for mildew resistance were made and are discussed in the context of homoeology among the genomes of barley (H-vulgare), wheat (Triticum aestivum), and rye (Secale cereale). Duplications of RFLP bands detected in the neighbourhood of Mlf and mlt might indicate an evolutionary interrelationship to the Mla locus for mildew resistance.