Dataset Information

The Ustilago hordei-Barley Interaction Is a Versatile System for Characterization of Fungal Effectors.

ABSTRACT: Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

SUBMITTER: Okmen B

PROVIDER: S-EPMC7912019 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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The Ustilago hordei-Barley Interaction Is a Versatile System for Characterization of Fungal Effectors.

Ökmen Bilal B Schwammbach Daniela D Bakkeren Guus G Neumann Ulla U Doehlemann Gunther G

Journal of fungi (Basel, Switzerland) 20210127 2

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei-barley pathosystem as a model to characterize effectors from ...[more]

PMID: 33513785

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Project description:Barley covered smut (CS) pathogen Ustilago hordei genome was mined for microsatellite distribution and their application in defining population structure and genetic variation. To dissect the molecular variation and genetic structure of U. hordei, 59 fungal isolates representing two distinct agro-ecological zones of India were analyzed by employing simple sequence repeats (SSRs). Using bioinformatic approaches, a total of 100,239 and 137,442 microsatellites were identified from 20.13 and 26.94 Mb of assembled genomic sequences of Uh364 and Uh4857-4 isolates of U. hordei, respectively. Penta-nucleotides (31.29 and 29.75%) followed by tri-nucleotide (28.27 and 29.88%) were most prevalent in both the genomes. Out of them, 15 polymorphic microsatellites showing conservancies in both the genomes were selected for exploring population genetic structure of U. hordei. An average of two alleles per microsatellite marker was generated with band size ranging from 180 to 850 bp. Polymorphic information content (PIC) varied between 0.095 and 0.37. Fifty-nine isolates were distributed in two distinct groups with about 65% genetic similarity according to UPGMA clustering and population structure analysis (K = 2). Gene flow analysis (Nm = 1.009) reflected moderate gene flow among the analyzed population. An analysis of molecular variance (AMOVA) displayed high level of genetic variation within population (87%) and low variation among populations (13%). Linkage disequilibrium (LD) analysis indicated positively significant but relatively low standardized index of association (SIA) value in both the population sets (SIA = 0.181), advocating a state of LD with epidemic population structure. In conclusion, the newly developed neutral SSR markers are highly polymorphic within U. hordei and will be useful for revealing evolutionary history and providing deep insight into the population dynamics of U. hordei in India as well as facilitating developing management strategies for CS of barley.

Project description:The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.

Project description:The success of plant pathogenic fungi mostly relies on their arsenal of virulence factors that are expressed and delivered into the host tissue during colonization. The biotrophic fungal pathogen Ustilago hordei causes covered smut disease on both barley and oat. The biotrophic interaction of the fungus with its host plant starts with formation of appressorium and subsequent penetration of invasive hyphae into the plant cell, in which the plasma membrane of the infected cell gets invaginated and encases the penetrating hypha. After penetration, the fungus grows towards and in later stages along the vascular bundles, which contain assimilates and metabolites, representing a source of nutrients for the fungus. In this study, we combined cytological, genomics and molecular biological methods to achieve a better understanding of the molecular interactions in the U. hordei-barley pathosystem. This study focuses on the genome-wide Agilent Microarray-based transcriptome analysis of U. hordei during early stages (20 hpi, 40 hpi, 3 dpi, 6 dpi) of the biotrophic interaction in comparison to axenic culture growth. This should give insights into the molecular processes during these stages by the identification of regulated genes. Furthermore, the regulation of putative secreted enzymes and effector proteins can be studied. Transcriptome analysis of U. hordei at different stages of host infection revealed 273 effector gene candidates with differentially expressed transcript levels. Furthermore, U. hordei transcriptionally activates core-effector genes that may suppress even non-host early defense responses. It could be shown that about one half of the U. hordei open reading frames are differentially regulated during at least one of the analyzed stages. Based on the transcriptome data, some of them were identified to be involved in stage-specific processes, e.g. in the nutrient acquisition by the induction of carbohydrate transporters during the later stages of infection. Furthermore, a set of strongly induced genes was identified, which encode for secreted proteins. The acquired datasets show a strong induction of stage-specific secreted effector proteins, whose functions remain unclear. Based on the expression values, a group of 16 promising candidates was chosen for functional characterization. The success of plant pathogenic fungi mostly relies on their arsenal of virulence factors that are expressed and delivered into the host tissue during colonization. The biotrophic fungal pathogen Ustilago hordei causes covered smut disease on both barley and oat. The biotrophic interaction of the fungus with its host plant starts with formation of appressorium and subsequent penetration of invasive hyphae into the plant cell, in which the plasma membrane of the infected cell gets invaginated and encases the penetrating hypha. After penetration, the fungus grows towards and in later stages along the vascular bundles, which contain assimilates and metabolites, representing a source of nutrients for the fungus. In this study, we combined cytological, genomics and molecular biological methods to achieve a better understanding of the molecular interactions in the U. hordei-barley pathosystem. This study focuses on the genome-wide Agilent Microarray-based transcriptome analysis of U. hordei during early stages (20 hpi, 40 hpi, 3 dpi, 6 dpi) of the biotrophic interaction in comparison to axenic culture growth. This should give insights into the molecular processes during these stages by the identification of regulated genes. Furthermore, the regulation of putative secreted enzymes and effector proteins can be studied. Transcriptome analysis of U. hordei at different stages of host infection revealed 273 effector gene candidates with differentially expressed transcript levels. Furthermore, U. hordei transcriptionally activates core-effector genes that may suppress even non-host early defense responses. It could be shown that about one half of the U. hordei open reading frames are differentially regulated during at least one of the analyzed stages. Based on the transcriptome data, some of them were identified to be involved in stage-specific processes, e.g. in the nutrient acquisition by the induction of carbohydrate transporters during the later stages of infection. Furthermore, a set of strongly induced genes was identified, which encode for secreted proteins.The acquired datasets show a strong induction of stage-specific secreted effector proteins, whose functions remain unclear. Based on the expression values, a group of 16 promising candidates was chosen for functional characterization.

Dataset Information

The Ustilago hordei-Barley Interaction Is a Versatile System for Characterization of Fungal Effectors.

Publications

The <i>Ustilago hordei</i>-Barley Interaction Is a Versatile System for Characterization of Fungal Effectors.

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