Mildew-Omics: How Global Analyses Aid the Understanding of Life and Evolution of Powdery Mildews

The common powdery mildew plant diseases are caused by ascomycete fungi of the order Erysiphales. Their characteristic life style as obligate biotrophs renders functional analyses in these species challenging, mainly because of experimental constraints to genetic manipulation. Global large-scale (-omics) approaches are thus particularly valuable and insightful for the characterisation of the life and evolution of powdery mildews. Here we review the knowledge obtained so far from genomic, transcriptomic and proteomic studies in these fungi. We consider current limitations and challenges regarding these surveys and provide an outlook on desired future investigations on the basis of the various –omics technologies

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 [7]. 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

Interactions between the powdery mildew effector BEC1054 and barley proteins identify candidate host targets

There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels

Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance

The oxidative burst is an early response to pathogen attack leading to the production of reactive oxygen species (ROS) including hydrogen peroxide. Two major mechanisms involving either NADPH oxidases or peroxidases that may exist singly or in combination in different plant species have been proposed for the generation of ROS. We identified an Arabidopsis thaliana azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H2O2 in response to a Fusarium oxysporum cell-wall preparation. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a type III peroxidase, French bean peroxidase type 1 (FBP1) exhibited an impaired oxidative burst and were more susceptible than wild-type plants to both fungal and bacterial pathogens. Transcriptional profiling and RT-PCR analysis showed that the anti-sense (FBP1) transgenic plants had reduced levels of specific peroxidase-encoding mRNAs, including mRNAs corresponding to Arabidopsis genes At3g49120 (AtPCb) and At3g49110 (AtPCa) that encode two class III peroxidases with a high degree of homology to FBP1. These data indicate that peroxidases play a significant role in generating H2O2 during the Arabidopsis defense response and in conferring resistance to a wide range of pathogens. The Additional file attached below is an Excel spreadsheet of 22,000+ lines containing Supplementary Materials. A PDF version (289 pages) is attached to the main downloadable document

Structure and evolution of barley powdery mildew effector candidates

<p>Abstract</p> <p>Background</p> <p>Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses <it>Blumeria graminis</it> causes one of the most important diseases of cereal crops. <it>B. graminis</it> is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease.</p> <p>Results</p> <p>Here we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite <it>B. graminis</it> f.sp. <it>hordei</it>. The 491 genes encoding these proteins constitute over 7% of this pathogen’s annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100–150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300–400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons.</p> <p>Conclusions</p> <p>We employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in <it>B. graminis</it> f.sp. <it>hordei</it>. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.</p