Obligate Biotrophic Pathogen Research Articles

Visualization of Host Actin Microfilament Dynamicity upon Obligate Biotrophic Pathogen Infection

Visualization of Host Actin Microfilament Dynamicity upon Obligate Biotrophic Pathogen Infection

Identification and selection of normalization controls for quantitative transcript analysis in Blumeria graminis.

SummaryThe investigation of obligate biotrophic pathogens, for example B lumeria graminis, presents a number of challenges. The sensitivity of many assays is reduced because of the presence of host material. Furthermore, the fungal structures inside and outside of the plant possess very different characteristics. Normalization genes are used in quantitative real‐time polymerase chain reaction (qPCR) to compensate for changes as a result of the quantity and quality of template material. Such genes are used as references against which genes of interest are compared, enabling true quantification. Here, we identified six potential B . graminis and five barley genes for qPCR normalization. The relative changes in abundance of the transcripts were assayed across an infection time course in barley epidermis, in B . graminis epiphytic structures and haustoria. The B . graminis glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), actin (ACT) and histone 3 (H3) genes and the barley GAPDH, ubiquitin (UBI) and α‐tubulin 2B (TUBA2B) genes were optimal normalization controls for qPCR during the infection cycle. These genes were then used for normalization in the quantification of the members of a Candidate Secreted Effector Protein (CSEP) family 21, a conidia‐specific gene and barley genes encoding putative interactors of CSEP0064. The analysis demonstrates the importance of identifying which reference genes are appropriate for each investigation.

Comparative Genomics Including the Early-Diverging Smut Fungus Ceraceosorus bombacis Reveals Signatures of Parallel Evolution within Plant and Animal Pathogens of Fungi and Oomycetes.

Ceraceosorus bombacis is an early-diverging lineage of smut fungi and a pathogen of cotton trees (Bombax ceiba). To study the evolutionary genomics of smut fungi in comparison with other fungal and oomycete pathogens, the genome of C. bombacis was sequenced and comparative genomic analyses were performed. The genome of 26.09 Mb encodes for 8,024 proteins, of which 576 are putative-secreted effector proteins (PSEPs). Orthology analysis revealed 30 ortholog PSEPs among six Ustilaginomycotina genomes, the largest groups of which are lytic enzymes, such as aspartic peptidase and glycoside hydrolase. Positive selection analyses revealed the highest percentage of positively selected PSEPs in C. bombacis compared with other Ustilaginomycotina genomes. Metabolic pathway analyses revealed the absence of genes encoding for nitrite and nitrate reductase in the genome of the human skin pathogen Malassezia globosa, but these enzymes are present in the sequenced plant pathogens in smut fungi. Interestingly, these genes are also absent in cultivable oomycete animal pathogens, while nitrate reductase has been lost in cultivable oomycete plant pathogens. Similar patterns were also observed for obligate biotrophic and hemi-biotrophic fungal and oomycete pathogens. Furthermore, it was found that both fungal and oomycete animal pathogen genomes are lacking cutinases and pectinesterases. Overall, these findings highlight the parallel evolution of certain genomic traits, revealing potential common evolutionary trajectories among fungal and oomycete pathogens, shaping the pathogen genomes according to their lifestyle.

Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.).

The interaction of barley, Hordeum vulgare L., with the powdery mildew fungus Blumeria graminis f. sp. hordei is a well-developed model to investigate resistance and susceptibility to obligate biotrophic pathogens. The 130-Mb Blumeria genome encodes approximately 540 predicted effectors that are hypothesized to suppress or induce host processes to promote colonization. Blumeria effector candidate (BEC)1019, a single-copy gene encoding a putative, secreted metalloprotease, is expressed in haustorial feeding structures, and host-induced gene silencing of BEC1019 restricts haustorial development in compatible interactions. Here, we show that Barley stripe mosaic virus-induced gene silencing of BEC1019 significantly reduces fungal colonization of barley epidermal cells, demonstrating that BEC1019 plays a central role in virulence. In addition, delivery of BEC1019 to the host cytoplasm via Xanthomonas type III secretion suppresses cultivar nonspecific hypersensitive reaction (HR) induced by Xanthomonas oryzae pv. oryzicola, as well as cultivar-specific HR induced by AvrPphB from Pseudomonas syringae pv. phaseolicola. BEC1019 homologs are present in 96 of 241 sequenced fungal genomes, including plant pathogens, human pathogens, and free-living nonpathogens. Comparative analysis revealed variation at several amino acid positions that correlate with fungal lifestyle and several highly conserved, noncorrelated motifs. Site-directed mutagenesis of one of these, ETVIC, compromises the HR-suppressing activity of BEC1019. We postulate that BEC1019 represents an ancient, broadly important fungal protein family, members of which have evolved to function as effectors in plant and animal hosts.

Phenylpropanoid metabolism, hormone biosynthesis and signal transduction-related genes play crucial roles in the resistance of Paulownia fortunei to paulownia witches’ broom phytoplasma infection

Paulownia witches’ broom (PaWB) caused by an obligate biotrophic plant pathogen called phytoplasma, is a devastating disease of paulownia trees over a large part of the world. However, little is known about the molecular mechanisms that underlie phytoplasma pathogenicity in paulownia or about the mode of interactions with host plants. In this study, genome-wide gene expression profiling was used to compare healthy, phytoplasma-infected, and both phytoplasma-infected and 20 mg L−1 methyl methane sulfonate (MMS) treated Paulownia fortunei plants using high-throughput mRNA sequencing analysis. A total of 6571 and 1377 differentially expressed unigenes were identified in the phytoplasma-infected plants versus healthy plants and in 20 mg L−1 MMS-treated plants versus phytoplasma-infected plants, respectively. Expression changes of 16 candidate differentially expressed unigenes were validated by qRT-PCR, indicating significant differences among the three P. fortunei samples. Our analysis showed that dramatic changes occurred in the gene expression profile of P. fortunei after PaWB phytoplasma infection and MMS treatment. The transcription of a large number of genes related to the plant–pathogen interaction, including phenylpropanoid metabolism, hormone biosynthesis and signaling, defense and/or pathogenesis, and signal transduction, were significantly up-regulated in the phytoplasma-infected paulownia and then returned to the levels in the healthy controls after MMS treatment. Our systematic analysis provides comprehensive transcriptomic data about P. fortunei trees infected by PaWB phytoplasma. The findings will help unraveling the molecular mechanisms of plant-phytoplasma interactions and may pave the way for engineering P. fortunei trees with improved properties.

The pepper phosphoenolpyruvate carboxykinase CaPEPCK1 is involved in plant immunity against bacterial and oomycete pathogens.

Phosphoenolpyruvate carboxykinase, a member of the lyase family, is involved in the metabolic pathway of gluconeogenesis in organisms. Although the major function of PEPCK in gluconeogenesis is well established, it is unclear whether this enzyme is involved in plant immunity. Here, we isolated and identified the pepper (Capsicum annuum) PEPCK (CaPEPCK1) gene from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaPEPCK1 was strongly expressed in pepper leaves during the incompatible interaction with avirulent Xcv and in response to environmental stresses, especially salicylic acid (SA) treatment. PEPCK activity was low in healthy leaves but dramatically increased in avirulent Xcv-infected leaves. Knock-down expression of CaPEPCK1 by virus-induced gene silencing resulted in high levels of susceptibility to both virulent and avirulent Xcv infection. CaPEPCK1 silencing in pepper compromised induction of the basal defense-marker genes CaPR1 (pathogenesis-related 1 protein), CaPR10 (pathogenesis-related 10 protein) and CaDEF1 (defensin) during Xcv infection. SA accumulation was also significantly suppressed in the CaPEPCK1-silenced pepper leaves infected with Xcv. CaPEPCK1 in an Arabidopsis overexpression (OX) line inhibited the proliferation of Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis (Hpa). CaPEPCK1-OX plants developed more rapidly, with enlarged leaves, compared to wild-type plants. The T-DNA insertion Arabidopsis orthologous mutants pck1-3 and pck1-4 were more susceptible to the bacterial Pst and oomycete Hpa pathogens than the wild type. Taken together, these results suggest that CaPEPCK positively contributes to plant innate immunity against hemibiotrophic bacterial and obligate biotrophic oomycete pathogens.

Virulence Structure of Blumeria graminis f. sp. tritici and Its Genetic Diversity by ISSR and SRAP Profiling Analyses.

Blumeria graminis f. sp. tritici, which causes wheat powdery mildew, is an obligate biotrophic pathogen that can easily genetically adapt to its host plant. Understanding the virulence structure of and genetic variations in this pathogen is essential for disease control and for breeding resistance to wheat powdery mildew. This study investigated 17 pathogenic populations in Sichuan, China and classified 109 isolates into two distinct groups based on pathogenicity analysis: high virulence (HV, 92 isolates) and low virulence (LV, 17 isolates). Populations from Yibin (Southern region), Xichang (Western region), and Meishan (Middle region) showed lower virulence frequencies than populations from other regions. Many of the previously known resistance genes did not confer resistance in this study. The resistance gene Pm21 displayed an immune response to pathogenic challenge with all populations in Sichuan, and Pm13, Pm5b, Pm2+6, and PmXBD maintained resistance. AMOVA revealed significantly higher levels of variation within populations and lower levels of variation among populations within regions. High levels of gene flow were detected among populations in the four regions. Closely related populations within each region were distinguished by cluster analyses using ISSR and SRAP alleles. Both ISSR and SRAP allele profiling analyses revealed high levels of genetic diversity among pathogenic populations in Sichuan. Although ISSR and SRAP profiling analysis showed similar resolutions, the SRAP alleles appeared to be more informative. We did not detect any significant association between these alleles and the virulence or pathogenicity of the pathogen. Our results suggest that ISSR and SRAP alleles are more efficient for the characterization of small or closely related populations versus distantly related populations.

Candidate Effector Proteins of the Rust Pathogen Melampsora larici-populina Target Diverse Plant Cell Compartments.

Rust fungi are devastating crop pathogens that deliver effector proteins into infected tissues to modulate plant functions and promote parasitic growth. The genome of the poplar leaf rust fungus Melampsora larici-populina revealed a large catalog of secreted proteins, some of which have been considered candidate effectors. Unraveling how these proteins function in host cells is a key to understanding pathogenicity mechanisms and developing resistant plants. In this study, we used an effectoromics pipeline to select, clone, and express 20 candidate effectors in Nicotiana benthamiana leaf cells to determine their subcellular localization and identify the plant proteins they interact with. Confocal microscopy revealed that six candidate effectors target the nucleus, nucleoli, chloroplasts, mitochondria, and discrete cellular bodies. We also used coimmunoprecipitation (coIP) and mass spectrometry to identify 606 N. benthamiana proteins that associate with the candidate effectors. Five candidate effectors specifically associated with a small set of plant proteins that may represent biologically relevant interactors. We confirmed the interaction between the candidate effector MLP124017 and TOPLESS-related protein 4 from poplar by in planta coIP. Altogether, our data enable us to validate effector proteins from M. larici-populina and reveal that these proteins may target multiple compartments and processes in plant cells. It also shows that N. benthamiana can be a powerful heterologous system to study effectors of obligate biotrophic pathogens.

Multi-locus tree and species tree approaches toward resolving a complex clade of downy mildews (Straminipila, Oomycota), including pathogens of beet and spinach

Accurate species determination of plant pathogens is a prerequisite for their control and quarantine, and further for assessing their potential threat to crops. The family Peronosporaceae (Straminipila; Oomycota) consists of obligate biotrophic pathogens that cause downy mildew disease on angiosperms, including a large number of cultivated plants. In the largest downy mildew genus Peronospora, a phylogenetically complex clade includes the economically important downy mildew pathogens of spinach and beet, as well as the type species of the genus Peronospora. To resolve this complex clade at the species level and to infer evolutionary relationships among them, we used multi-locus phylogenetic analysis and species tree estimation. Both approaches discriminated all nine currently accepted species and revealed four previously unrecognized lineages, which are specific to a host genus or species. This is in line with a narrow species concept, i.e. that a downy mildew species is associated with only a particular host plant genus or species. Instead of applying the dubious name Peronospora farinosa, which has been proposed for formal rejection, our results provide strong evidence that Peronospora schachtii is an independent species from lineages on Atriplex and apparently occurs exclusively on Beta vulgaris. The members of the clade investigated, the Peronospora rumicis clade, associate with three different host plant families, Amaranthaceae, Caryophyllaceae, and Polygonaceae, suggesting that they may have speciated following at least two recent inter-family host shifts, rather than contemporary cospeciation with the host plants.

Host-microbe and microbe-microbe interactions in the evolution of obligate plant parasitism.

Research on obligate biotrophic plant parasites, which reproduce only on living hosts, has revealed a broad diversity of filamentous microbes that have independently acquired complex morphological structures, such as haustoria. Genome studies have also demonstrated a concerted loss of genes for metabolism and lytic enzymes, and gain of diversity of genes coding for effectors involved in host defense suppression. So far, these traits converge in all known obligate biotrophic parasites, but unexpected genome plasticity remains. This plasticity is manifested as transposable element (TE)-driven increases in genome size, observed to be associated with the diversification of virulence genes under selection pressure. Genome expansion could result from the governing of the pathogen response to ecological selection pressures, such as host or nutrient availability, or to microbial interactions, such as competition, hyperparasitism and beneficial cooperations. Expansion is balanced by alternating sexual and asexual cycles, as well as selfing and outcrossing, which operate to control transposon activity in populations. In turn, the prevalence of these balancing mechanisms seems to be correlated with external biotic factors, suggesting a complex, interconnected evolutionary network in host-pathogen-microbe interactions. Therefore, the next phase of obligate biotrophic pathogen research will need to uncover how this network, including multitrophic interactions, shapes the evolution and diversity of pathogens.

The sunflower downy mildew pathogen Plasmopara halstedii.

Kingdom Stramenopila; Phylum Oomycota; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Plasmopara; Species Plasmopara halstedii. Sunflower seedling damping off, dwarfing of the plant, bleaching of leaves, starting from veins, and visible white sporulation, initially on the lower side of cotyledons and leaves. Plasmopara halstedii infection may severely impact sunflower seed yield. In spring, germination of overwintered sexual oospores leads to sunflower root infection. Intercellular hyphae are responsible for systemic plant colonization and the induction of disease symptoms. Under humid and fresh conditions, dissemination structures are produced by the pathogen on all plant organs to release asexual zoosporangia. These zoosporangia play an important role in pathogen dissemination, as they release motile zoospores that are responsible for leaf infections on neighbouring plants. Disease control is obtained by both chemical seed treatment (mefenoxam) and the deployment of dominant major resistance genes, denoted Pl. However, the pathogen has developed fungicide resistance and has overcome some plant resistance genes. Research for more sustainable strategies based on the identification of the molecular basis of the interaction are in progress. http://www.heliagene.org/HP, http://lipm-helianthus.toulouse.inra.fr/dokuwiki/doku.php?id=start, https://www.heliagene.org/PlasmoparaSpecies (soon available).

The origin and genetic diversity of the causal agent of Asian soybean rust, Phakopsora pachyrhizi, in South America

A sequence‐based approach was used to investigate molecular genetic variations in Phakopsora pachyrhizi, an obligate biotrophic pathogen that causes Asian soybean rust. In Argentina, the samples came from uredinium‐bearing leaves taken from 11 soybean fields; in Brazil, the samples comprised urediniospores from leaves of 10 soybean genotypes that had been grown in three experimental stations during two growing seasons. PCR‐based cloning techniques were used to generate DNA sequences for two gene regions and alignments were supplemented with data from GenBank. A total of 575 sequences for the internal transcribed spacer region (18 ribotypes) and 160 partial sequences for a housekeeping gene encoding ADP‐ribosylation factor (10 haplotypes) were obtained. Ribotype accumulation curves predicted that about 20 bacterial clones would recover 5–6 ribotypes (c. 70–80% of the total molecular variation) per locality. The samples from the three experimental stations in Brazil displayed most (14 out of 16) ribotypes found worldwide; the lack of genetic structure and differentiation at a diverse geographic scale suggests that both local and distant sources provide airborne inoculum during disease establishment. Soybean genotypes with resistance genes for the Asian soybean rust did not decrease the molecular genetic variation of fungal populations.

A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid.

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.

The Podosphaera xanthii haustorium, the fungal Trojan horse of cucurbit-powdery mildew interactions

The powdery mildew fungi are obligate biotrophic plant pathogens that develop a specialized structure for parasitism termed haustorium, which is responsible for nutrient uptake and factor exchange with the plant. In this work, we present a detailed microscopy analysis of the haustoria of the cucurbit powdery mildew fungus Podosphaera xanthii, a major limiting factor for cucurbit production worldwide. Despite being located inside plant epidermal cells, transmission electron microscopy (TEM) analysis showed the characteristic highly irregular outline of the extrahaustorial membrane that separates the extrahaustorial matrix of haustoria from the cytoplasm of the plant cell. TEM analysis also revealed the presence of some vesicles and electron-dense plaques of material surrounding the haustoria. In confocal microscopy analysis and aniline blue staining we found a positive correlation between haustorial development and deposition of callose, which is distributed as plaques around haustorial complex. In this study, a method for the isolation of P. xanthii haustoria was also adapted, which permitted the analysis of the formation of haustorial lobes and the visualization of vacuoles and the pool of vesicles inside the haustorial complex. Our findings suggested that the haustorial lobes were responsible for vesicular trafficking and most likely act as the main mediators of the fungus-plant dialogue. All of these findings were integrated into a model of the P. xanthii-host cellular interactions.

Interaction of a Blumeria graminis f. sp. hordei effector candidate with a barley ARF‐GAP suggests that host vesicle trafficking is a fungal pathogenicity target

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1-BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana-infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non-adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin-conjugating enzyme, and an ADP ribosylation factor-GTPase-activating protein (ARF-GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF-GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF-GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence-associated host vesicle trafficking.

Messages from Powdery Mildew DNA: How the Interplay with a Host Moulds Pathogen Genomes

The genomes of the barley, Arabidopsis and pea powdery mildew are significantly larger than those of related fungi. This is due to an extraordinary expansion of retro-trasposons that are evident as repetitive elements in the sequence. The protein coding genes are fewer than expected due to an overall reduction in the size of gene families, a reduction in the number of paralogs and because of the loss of certain metabolic pathways. Many of these changes have also been observed in the genomes of other taxonomically unrelated obligate biotrophic pathogens. The only group of genes that bucks the trend of gene loss, are those encoding small secreted proteins that bear the hall marks of effectors.

Members of the NPF3 Transporter Subfamily Encode Pathogen-Inducible Nitrate/Nitrite Transporters in Grapevine and Arabidopsis

Vitis vinifera, the major grapevine species cultivated for wine production, is very susceptible to Erysiphe necator, the causal agent of powdery mildew (PM). This obligate biotrophic fungal pathogen attacks both leaf and berry, greatly affecting yield and quality. To investigate possible mechanisms of nutrient acquisition by successful biotrophs, we characterized a candidate NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY (NPF, formerly NRT1/PTR) member, grapevine NFP3.2, that was up-regulated in E. necator-inoculated susceptible V. vinifera Cabernet Sauvignon leaves, but not in resistant V. aestivalis Norton. Expression in Xenopus laevis oocytes and two-electrode voltage clamp measurements showed that VvNPF3.2 is a low-affinity transporter for both nitrate and nitrite and displays characteristics of NPF members from other plants. We also cloned the Arabidopsis ortholog, AtNPF3.1, and showed that AtNPF3.1 similarly transported nitrate and nitrite with low affinity. With an Arabidopsis triple mutant that is susceptible to E. necator, we found that AtNPF3.1 is up-regulated in the leaves of infected Arabidopsis similarly to VvNPF3.2 in susceptible grapevine leaves. Expression of the reporter β-glucuronidase (GUS) driven by the promoter of VvNPF3.2 or AtNPF3.1 in Arabidopsis indicated that both transporters are expressed in vascular tissue, with expression in major and minor veins, respectively. Interestingly, the promoter of VvNPF3.2 allowed induced expression of GUS in minor veins in PM-infected leaves. Our experiments lay the groundwork for investigating the manipulation of host nutrient distribution by biotrophic pathogens and characterizing physiological variables in the pathogenesis of this difficult to study grapevine disease.

Mla‐ and Rom1‐mediated control of microRNA398 and chloroplast copper/zinc superoxide dismutase regulates cell death in response to the barley powdery mildew fungus

• Barley (Hordeum vulgare L.) Mildew resistance locus a (Mla) confers allele-specific interactions with natural variants of the ascomycete fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease. Significant reprogramming of Mla-mediated gene expression occurs upon infection by this obligate biotrophic pathogen. • We utilized a proteomics-based approach, combined with barley mla, required for Mla12 resistance1 (rar1), and restoration of Mla resistance1 (rom1) mutants, to identify components of Mla-directed signaling. • Loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of chloroplast copper/zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in Rom1 re-established HvSOD1 levels. In addition, both Mla and Rom1 negatively regulated hvu-microRNA398 (hvu-miR398), and up-regulation of miR398 was coupled to reduced HvSOD1 expression. Barley stripe mosaic virus (BSMV)-mediated over-expression of both barley and Arabidopsis miR398 repressed accumulation of HvSOD1, and BSMV-induced gene silencing of HvSod1 impeded Mla-triggered H₂O₂ and hypersensitive reaction (HR) at barley-Bgh interaction sites. • These data indicate that Mla- and Rom1-regulated hvu-miR398 represses HvSOD1 accumulation, influencing effector-induced HR in response to the powdery mildew fungus.

Genetic Relationships in an International Collection of Puccinia horiana Isolates Based on Newly Identified Molecular Markers and Demonstration of Recombination

The obligate biotrophic pathogen Puccinia horiana is the causal agent of chrysanthemum white rust. Although P. horiana is a quarantine organism, it has been able to spread to most chrysanthemum-producing regions in the world since the 1960s; however, the transfer routes are largely obscure. An extremely low level of allelic diversity was observed in a geographically diverse set of eight isolates using complexity reduction of polymorphic sequences (CRoPS) technology. Only 184 of the 16,196 contigs (1.1%) showed one or more single-nucleotide polymorphisms (SNPs). Thirty-two SNPs and one simple-sequence repeat were translated into molecular markers and used to genotype 45 isolates originating from North and South America, Asia, and Europe. In most cases, phylogenetic clustering was related to geographic origin, indicating local establishment. The European isolates mostly grouped in two major populations that may relate to the two historic introductions previously reported. However, evidence of recent geographic transfer was also observed, including transfer events between Europe and South America and between Southeast Asia and Europe. In contrast with the presumed clonal propagation of this microcyclic rust, strong indications of marker recombination were observed, presumably as a result of anastomosis, karyogamy, and somatic meiosis. Recombination and transfer also explain the geographic dispersal of specific markers. A near-to-significant correlation between the genotypic data and previously obtained pathotype data was observed and one marker was associated with the most virulent pathotype group. In combination with a fast SNP detection method, the markers presented here will be helpful tools to further elucidate the transfer pathways and local survival of this pathogen.

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

The Ror1 gene was fine-mapped to the pericentric region of barley chromosome 1HL. 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 0.18cM 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.