Gene Identification in the Obligate Fungal Pathogen Blumeria graminis by Expressed Sequence Tag Analysis

doi:10.1006/fgbi.2001.1281

Fungal Genetics and Biology

Volume 33, Issue 3, August 2001, Pages 195-211

https://doi.org/10.1006/fgbi.2001.1281 Get rights and content

Abstract

Powdery mildew of barley is caused by the obligate fungal pathogen Blumeria graminis f. sp. hordei. Haploid conidia of B. graminis, landing on the barley leaf, germinate to form first a primary germ tube and then an appressorial germ tube. The appressorial germ tube differentiates into a mature appressorium from which direct penetration of host epidermis occurs. Here we present data on 4908 expressed sequence tags obtained from B. graminis conidia. The combined sequences represent 2676 clones describing 1669 individual genes. Comparison with sequences from other pathogenic and nonpathogenic fungi defines hypotheses on the genes required for pathogenicity and growth on the host. The putative roles of some of the identified genes are discussed.

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The infection process can to a large extent be synchronized, which permits identification of genes that are expressed at the different stages of infection. Differential display, SAGE, microarrays, RNA sequencing, and proteome analyses have been used to identify and classify subsets of genes that are required for penetration, haustorial development and reinfection (Carver et al. 1995; Thomas et al. 2001; Grell et al. 2005; Bindschedler et al. 2011; Hacquard et al. 2013). Proteomics and genome sequence analyses of powdery mildew fungi including Bgh have revealed a conservation of the basic ascomycete signal transduction pathways and gene losses in primary and secondary metabolism, carbohydrate-active enzymes, and in uptake of inorganic nitrogen during the evolution towards the obligate biotrophic lifestyle (Spanu et al. 2010; Hacquard et al. 2013; Kusch et al. 2014).
The obligate ascomycete parasitic fungus Blumeria graminis f. sp. hordei (Bgh) has a unique lifestyle as it is completely dependent on living barley leaves as substrate for growth. Genes involved in inorganic nitrogen utilization are notably lacking, and the fungus relies on uptake of host-derived peptides and amino acids. The PTR2 transporter family takes up di- and tri- peptides in a proton coupled process and filamentous fungi typically have two or more di/tri peptide transporters. Here we show that Bgh appear to have one PTR2 that can restore dipeptide uptake in a Saccharomyces cerevisiae PTR2 deletion strain. The Bgh PTR2 gene is expressed in conidia and germinating conidia. During Bgh infection of barley the expression level of the BghPTR2 gene is high in the appressorial germ tube, low in the haustoria and high again during conidiation and secondary infection in the compatible and intermediate resistant interactions. BghPTR2 appears to be important for the initial establishment of fungal infection but not for uptake of di-tri-peptides at the haustorial interface. Based on the expression profile we suggest that BghPTR2 is active in internal transport of nutrient reserves and/or uptake of break down products from the plant surface during the early infection stages.
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Powdery mildews are phytopathogenic ascomycetes that have an obligate biotrophic lifestyle and establish intimate relationships with their plant hosts. A crucial aspect of this plant–fungus interaction is the formation of specialized fungal infection structures termed haustoria. Although located within the cell boundaries of plant epidermal cells, haustoria remain separated from the plant cytoplasm by a host plasma membrane derivative, the extrahaustorial membrane. Haustoria are thought to represent pivotal sites of nutrient uptake and effector protein delivery. We enriched haustorial complexes from Arabidopsis thaliana plants infected with the powdery mildew fungus Golovinomyces orontii and performed in-depth transcriptome analysis by 454-based pyrosequencing of haustorial cDNAs. We assembled 7077 expressed sequence tag (EST) contigs with greater than 5-fold average coverage and analyzed these with regard to the respective predicted protein functions. We found that transcripts coding for gene products with roles in protein turnover, detoxification of reactive oxygen species and fungal pathogenesis are abundant in the haustorial EST contigs, while surprisingly transcripts encoding presumptive nutrient transporters were not highly represented in the haustorial cDNA library. A substantial proportion (∼38%) of transcripts coding for predicted secreted proteins comprises effector candidates. Our data provide valuable insights into the transcriptome of the key infection structure of a model obligate biotrophic phytopathogen.
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Regular ArticleGene Identification in the Obligate Fungal Pathogen Blumeria graminis by Expressed Sequence Tag Analysis

Abstract

J. Mol. Biol.

Physiol. Mol. Plant Pathol.

Mycol. Res.

Fung. Genet. Biol.

Physiol. Mol. Plant Pathol.

Gene

Fung. Genet. Biol.

Fung. Genet. Biol

Physiol. Plant. Pathol.

Physiol. Plant Pathol.

Exp. Mycol.

Curr. Biol.

Fung. Genet. Biol.

Physiol. Mol. Plant Pathol.

Exp. Mycol.

Physiol. Mol. Plant Pathol.

Physiol. Mol. Plant Pathol.

Physiol. Mol. Plant Pathol.

Methods Enzymol.

Gapped BLAST and PSI-BLAST: A new generation of protein database search programs

Nucleic Acids Res.

CEL1: A novel cellulose binding protein secreted by Agaricus bisporus during growth on crystalline cellulose

FEMS Micro. Lett.

Early interactions during powdery mildew infection

Can. J. Bot.

Disparate sequence characteristics of the Erysiphe graminis f. sp. hordei glyceraldehyde-3-phosphate dehydrogenase gene

Curr. Genet.

Biolistic transformation of the obligate pathogenic fungus, Erysiphe graminis f. sp

hordei. Curr. Genet.

PCR cloning and detection of point mutations in the eburicol-1,4-alpha-demethylase (CYP51) gene from Erysiphe graminis f. sp. hordei, a ‘recalcitrant’ fungus

Curr. Genet.

Germination of powdery mildew conidia in vitro on cellulose membranes

Neth. J. Plant Pathol.

Magnaporthe grisea Pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues

Plant Cell

Regular Article
Gene Identification in the Obligate Fungal Pathogen Blumeria graminis by Expressed Sequence Tag Analysis