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1.
Mol Ecol ; 2023 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-36862075

RESUMO

The powdery mildew fungi (Erysiphaceae) are globally distributed plant pathogens with a range of more than 10,000 plant hosts. In this review, we discuss the long- and short-term evolution of these obligate biotrophic fungi and outline their diversity with respect to morphology, lifestyle, and host range. We highlight their remarkable ability to rapidly overcome plant immunity, evolve fungicide resistance, and broaden their host range, for example, through adaptation and hybridization. Recent advances in genomics and proteomics, particularly in cereal powdery mildews (genus Blumeria), provided first insights into mechanisms of genomic adaptation in these fungi. Transposable elements play key roles in shaping their genomes, where even close relatives exhibit diversified patterns of recent and ongoing transposon activity. These transposons are ubiquitously distributed in the powdery mildew genomes, resulting in a highly adaptive genome architecture lacking obvious regions of conserved gene space. Transposons can also be neofunctionalized to encode novel virulence factors, particularly candidate secreted effector proteins, which may undermine the plant immune system. In cereals like barley and wheat, some of these effectors are recognized by plant immune receptors encoded by resistance genes with numerous allelic variants. These effectors determine incompatibility ("avirulence") and evolve rapidly through sequence diversification and copy number variation. Altogether, powdery mildew fungi possess plastic genomes that enable their fast evolutionary adaptation towards overcoming plant immunity, host barriers, and chemical stress such as fungicides, foreshadowing future outbreaks, host range shifts and expansions as well as potential pandemics by these pathogens.

2.
PLoS Pathog ; 15(3): e1007620, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30856238

RESUMO

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.


Assuntos
Ascomicetos/patogenicidade , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Vegetal/imunologia , Plantas/imunologia , RNA de Plantas/metabolismo , RNA Ribossômico/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Plantas/microbiologia , Conformação Proteica , RNA de Plantas/genética , RNA Ribossômico/genética , Homologia de Sequência
3.
Plant Physiol ; 183(1): 385-398, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32123042

RESUMO

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.


Assuntos
Hordeum/metabolismo , Nicotiana/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Ribossomos/metabolismo , Domínio Catalítico
4.
BMC Genomics ; 19(1): 381, 2018 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-29788921

RESUMO

BACKGROUND: Powdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew, Blumeria graminis, has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture of B. graminis forma specialis hordei (Bgh) to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley (Hordeum vulgare). RESULTS: The near-chromosome level assemblies of the Bgh reference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither the SPs nor the TEs reside preferentially in genomic regions with unique features. Extended comparative analysis with different host-specific B. graminis formae speciales revealed the existence of a core suite of SPs, but also isolate-specific SP sets as well as congruence of SP CNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in the Bgh genome. CONCLUSIONS: The characteristics of the Bgh genome (largely retained synteny, CNV of SP genes, recently proliferated TEs and a lack of significant compartmentalization) are consistent with a "one-speed" genome that differs in its architecture and (co-)evolutionary pattern from the "two-speed" genomes reported for several other filamentous phytopathogens.


Assuntos
Ascomicetos/genética , Ascomicetos/fisiologia , Elementos de DNA Transponíveis/genética , Genoma Fúngico/genética , Hordeum/microbiologia , Especificidade de Hospedeiro/genética , Doenças das Plantas/microbiologia , Variações do Número de Cópias de DNA , Duplicação Gênica , Perfilação da Expressão Gênica , Filogenia
5.
New Phytol ; 217(2): 713-725, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29044534

RESUMO

Tritrophic interactions involving a biocontrol agent, a pathogen and a plant have been analyzed predominantly from the perspective of the biocontrol agent. We have conducted the first comprehensive transcriptomic analysis of all three organisms in an effort to understand the elusive properties of Pseudozyma flocculosa in the context of its biocontrol activity against Blumeria graminis f.sp. hordei as it parasitizes Hordeum vulgare. After inoculation of P. flocculosa, the tripartite interaction was monitored over time and samples collected for scanning electron microscopy and RNA sequencing. Based on our observations, P. flocculosa indirectly parasitizes barley, albeit transiently, by diverting nutrients extracted by B. graminis from barley leaves through a process involving unique effectors. This brings novel evidence that such molecules can also influence fungal-fungal interactions. Their release is synchronized with a higher expression of powdery mildew haustorial effectors, a sharp decline in the photosynthetic machinery of barley and a developmental peak in P. flocculosa. The interaction culminates with a collapse of B. graminis haustoria, thereby stopping P. flocculosa growth, as barley plants show higher metabolic activity. To conclude, our study has uncovered a complex and intricate phenomenon, described here as hyperbiotrophy, only achievable through the conjugated action of the three protagonists.


Assuntos
Ascomicetos/fisiologia , Basidiomycota/fisiologia , Hordeum/microbiologia , Controle Biológico de Vetores , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Ascomicetos/genética , Ascomicetos/ultraestrutura , Basidiomycota/ultraestrutura , Transporte Biológico , Celobiose/análogos & derivados , Celobiose/farmacologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glicolipídeos/farmacologia , Hordeum/ultraestrutura , Modelos Biológicos , Fenótipo , Fotossíntese , Transcriptoma/genética
6.
J Proteome Res ; 15(3): 826-39, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26813582

RESUMO

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.


Assuntos
Hordeum/química , Interações Hospedeiro-Patógeno , Proteínas de Plantas/imunologia , Mapeamento de Interação de Proteínas/métodos , Ascomicetos/patogenicidade , Proteínas Fúngicas/toxicidade , Proteínas de Plantas/análise , Ligação Proteica , Espectrometria de Massas em Tandem , Virulência , Leveduras/patogenicidade
7.
Mol Plant Microbe Interact ; 28(9): 968-83, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25938194

RESUMO

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.


Assuntos
Ascomicetos/patogenicidade , Hordeum/microbiologia , Doenças das Plantas/microbiologia , Sequência de Aminoácidos , Ascomicetos/genética , Ascomicetos/metabolismo , Sequência Conservada , Regulação Fúngica da Expressão Gênica/fisiologia , Inativação Gênica , Dados de Sequência Molecular , Filogenia , Folhas de Planta , Vírus de Plantas , Virulência , Xanthomonas/metabolismo
8.
BMC Genomics ; 16: 917, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26556056

RESUMO

BACKGROUND: The Avrk1 and Avra10 avirulence (AVR) genes encode effectors that increase the pathogenicity of the fungus Blumeria graminis f.sp. hordei (Bgh), the powdery mildew pathogen, in susceptible barley plants. In resistant barley, MLK1 and MLA10 resistance proteins recognize the presence of AVRK1 and AVRA10, eliciting the hypersensitive response typical of gene for gene interactions. Avrk1 and Avra10 have more than 1350 homologues in Bgh genome, forming the EKA (Effectors homologous to Avr k 1 and Avr a 10) gene family. RESULTS: We tested the hypothesis that the EKA family originated from degenerate copies of Class I LINE retrotransposons by analysing the EKA family in the genome of Bgh isolate DH14 with bioinformatic tools specially developed for the analysis of Transposable Elements (TE) in genomes. The Class I LINE retrotransposon copies homologous to Avrk1 and Avra10 represent 6.5 % of the Bgh annotated genome and, among them, we identified 293 AVR/effector candidate genes. We also experimentally identified peptides that indicated the translation of several predicted proteins from EKA family members, which had higher relative abundance in haustoria than in hyphae. CONCLUSIONS: Our analyses indicate that Avrk1 and Avra10 have evolved from part of the ORF1 gene of Class I LINE retrotransposons. The co-option of Avra10 and Avrk1 as effectors from truncated copies of retrotransposons explains the huge number of homologues in Bgh genome that could act as dynamic reservoirs from which new effector genes may evolve. These data provide further evidence for recruitment of retrotransposons in the evolution of new biological functions.


Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Hordeum/microbiologia , Elementos Nucleotídeos Longos e Dispersos , Família Multigênica , Doenças das Plantas/microbiologia , Ascomicetos/classificação , Ascomicetos/metabolismo , Biologia Computacional , Sequência Consenso , Genoma Fúngico , Fases de Leitura Aberta , Filogenia , Proteômica
9.
New Phytol ; 207(4): 991-5, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26237564

RESUMO

Plants are the source of most of our food, whether directly or as feed for the animals we eat. Our dinner table is a trophic level we share with the microbes that also feed on the primary photosynthetic producers. Microbes that enter into close interactions with plants need to evade or suppress detection and host immunity to access nutrients. They do this by deploying molecular tools - effectors - which target host processes. The mode of action of effector proteins in these events is varied and complex. Recent data from diverse systems indicate that RNA-interacting proteins and RNA itself are delivered by eukaryotic microbes, such as fungi and oomycetes, to host plants and contribute to the establishment of successful interactions. This is evidence that pathogenic microbes can interfere with the host software. We are beginning to see that pathogenic microbes are capable of hacking into the plants' immunity programs.


Assuntos
Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , RNA de Plantas/metabolismo , Plantas/metabolismo , Plantas/microbiologia , Ligação Proteica
10.
Mol Plant Microbe Interact ; 26(6): 633-42, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23441578

RESUMO

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to ß-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.


Assuntos
Ascomicetos/enzimologia , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Hordeum/microbiologia , Doenças das Plantas/microbiologia , Ribonucleases/genética , Ascomicetos/genética , Ascomicetos/fisiologia , Morte Celular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Hordeum/fisiologia , Interações Hospedeiro-Patógeno , Mutação , Doenças das Plantas/imunologia , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , RNA de Plantas/genética , Ribonucleases/metabolismo , Plântula/microbiologia , Plântula/fisiologia , Especificidade da Espécie
11.
Mol Plant Pathol ; 24(6): 570-587, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36917011

RESUMO

The establishment of host-microbe interactions requires molecular communication between both partners, which may involve the mutual transfer of noncoding small RNAs. Previous evidence suggests that this is also true for powdery mildew disease in barley, which is caused by the fungal pathogen Blumeria hordei. However, previous studies lacked spatial resolution regarding the accumulation of small RNAs upon host infection by B. hordei. Here, we analysed site-specific small RNA repertoires in the context of the barley-B. hordei interaction. To this end, we dissected infected leaves into separate fractions representing different sites that are key to the pathogenic process: epiphytic fungal mycelium, infected plant epidermis, isolated haustoria, a vesicle-enriched fraction from infected epidermis, and extracellular vesicles. Unexpectedly, we discovered enrichment of specific 31-33-base 5'-terminal fragments of barley 5.8S ribosomal RNA in extracellular vesicles and infected epidermis, as well as particular B. hordei transfer RNA fragments in haustoria. We describe canonical small RNAs from both the plant host and the fungal pathogen that may confer cross-kingdom RNA interference activity. Interestingly, we found first evidence of phased small interfering RNAs in B. hordei, a feature usually attributed to plants, which may be associated with the posttranscriptional control of fungal coding genes, pseudogenes, and transposable elements. Our data suggest a key and possibly site-specific role for cross-kingdom RNA interference and noncoding RNA fragments in the host-pathogen communication between B. hordei and its host barley.


Assuntos
Ascomicetos , Hordeum , RNA Fúngico/genética , Ascomicetos/genética , Ascomicetos/metabolismo , Hordeum/microbiologia , RNA de Transferência , Interferência de RNA , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo
12.
BMC Genomics ; 13: 694, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23231440

RESUMO

BACKGROUND: Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis 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. RESULTS: 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 B. graminis f.sp. hordei. 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. CONCLUSIONS: 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 B. graminis f.sp. hordei. 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.


Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Hordeum/microbiologia , Micoses/genética , Micoses/imunologia , Sequência de Aminoácidos , Grão Comestível/microbiologia , Hordeum/metabolismo , Interações Hospedeiro-Patógeno/genética , Dados de Sequência Molecular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteômica , Alinhamento de Sequência
14.
Methods ; 54(4): 432-41, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21453771

RESUMO

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.


Assuntos
Ascomicetos/genética , Proteínas Fúngicas/química , Anotação de Sequência Molecular , Ascomicetos/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genômica , Hordeum/microbiologia , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteoma , Proteômica
15.
Front Microbiol ; 13: 809940, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35283825

RESUMO

Plant microbiomes and immune responses have coevolved through history, and this applies just as much to the phyllosphere microbiome and defense phytohormone signaling. When in homeostasis, the phyllosphere microbiome confers benefits to its host. However, the phyllosphere is also dynamic and subject to stochastic events that can modulate community assembly. Investigations into the impact of defense phytohormone signaling on the microbiome have so far been limited to culture-dependent studies; or focused on the rhizosphere. In this study, the impact of the foliar phytohormone salicylic acid (SA) on the structure and composition of the phyllosphere microbiome was investigated. 16S rRNA amplicons were sequenced from aerial tissues of two Arabidopsis mutants that exhibit elevated SA signaling through different mechanisms. SA signaling was shown to increase community diversity and to result in the colonization of rare, satellite taxa in the phyllosphere. However, a stable core community remained in high abundance. Therefore, we propose that SA signaling acts as a source of intermediate disturbance in the phyllosphere. Predictive metagenomics revealed that the SA-mediated microbiome was enriched for antibiotic biosynthesis and the degradation of a diverse range of xenobiotics. Core taxa were predicted to be more motile, biofilm-forming and were enriched for traits associated with microbe-microbe communication; offering potential mechanistic explanation of their success despite SA-mediated phyllospheric disturbance.

16.
Fungal Genet Biol ; 48(7): 704-13, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21515399

RESUMO

In ascomycetes, mating compatibility is regulated by the mating-type locus, MAT1. The objectives of this study were to identify and sequence genes at the MAT1 locus in the grape powdery mildew fungus, Erysiphe necator, to develop a PCR-based marker for determining mating type in E. necator, and to develop degenerate primers for amplification by PCR of conserved regions of mating-type idiomorphs in other powdery mildew fungi. We identified MAT1-2-1 of the MAT1-2 idiomorph in E. necator based on the homologous sequence in the genome of Blumeria graminis f. sp. hordei and we found MAT1-1-1 and MAT1-1-3 of the MAT1-1 idiomorph from transcriptome sequences of E. necator. We developed and applied a reliable PCR-based multiplex marker to confirm that genotype correlated with mating phenotype, which was determined by pairing with mating-type tester isolates. Additionally, we used the marker to genotype populations of E. necator from different Vitis spp. from throughout the USA. We found both mating types were present in all populations and mating-type ratios did not deviate from 1:1. The mating-type genes in E. necator are similar to those of other Leotiomycetes; however, the structure of the MAT1 locus in E. necator, like the MAT1-2 idiomorph of B. graminis, is markedly different from other ascomycetes in that it is greatly expanded and may contain a large amount of repetitive DNA. As a result, we were unable to amplify and sequence either idiomorph in its entirety. We designed degenerate primers that amplify conserved regions of MAT1-1 and MAT1-2 in E. necator, Podosphaera xanthii, Microsphaera syringae, and B. graminis, representing the major clades of the Erysiphales. These degenerate primers or sequences obtained in this study from these species can be used to identify and sequence MAT1 genes or design mating-type markers in other powdery mildew fungi as well.


Assuntos
Ascomicetos/classificação , Ascomicetos/genética , Ordem dos Genes , Genes Fúngicos Tipo Acasalamento , Técnicas de Tipagem Micológica , Reação em Cadeia da Polimerase/métodos , Primers do DNA/genética , DNA Fúngico/química , DNA Fúngico/genética , Dados de Sequência Molecular , Micologia/métodos , Análise de Sequência de DNA , Estados Unidos , Vitis/microbiologia
17.
Fungal Genet Biol ; 48(3): 327-34, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20955813

RESUMO

The two fungal pathogens Blumeria graminis f. sp. tritici (B.g. tritici) and hordei (B.g. hordei) cause powdery mildew specifically in wheat or barley. They have the same life cycle, but their growth is restricted to the respective host. Here, we compared the sequences of two loci in both cereal mildews to determine their divergence time and their relationship with the evolution of their hosts. We sequenced a total of 273.3kb derived from B.g. tritici BAC sequences and compared them with the orthologous regions in the B.g. hordei genome. Protein-coding genes were colinear and well conserved. In contrast, the intergenic regions showed very low conservation mostly due to different integration patterns of transposable elements. To estimate the divergence time of B.g. tritici and B.g. hordei, we used conserved intergenic sequences including orthologous transposable elements. This revealed that B.g. tritici and B.g. hordei have diverged about 10 million years ago (MYA), two million years after wheat and barley (12 MYA). These data suggest that B.g. tritici and B.g. hordei have co-evolved with their hosts during most of their evolutionary history after host divergence, possibly after a short phase of host expansion when the same pathogen could still grow on the two diverged hosts.


Assuntos
Ascomicetos/genética , Evolução Molecular , Hordeum/microbiologia , Doenças das Plantas/microbiologia , Polimorfismo Genético , Triticum/microbiologia , Elementos de DNA Transponíveis , DNA Fúngico/química , DNA Fúngico/genética , DNA Intergênico , Especiação Genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência , Sintenia
18.
Mol Cell Proteomics ; 8(10): 2368-81, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19602707

RESUMO

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity.


Assuntos
Ascomicetos/química , Ascomicetos/genética , Proteínas Fúngicas , Genoma Fúngico , Hordeum/microbiologia , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Ascomicetos/citologia , Ascomicetos/patogenicidade , Biologia Computacional , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Doenças das Plantas/microbiologia
19.
Appl Environ Microbiol ; 75(2): 542-6, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19011063

RESUMO

In this study, we have constructed and expressed inverted repeat chimeras from the first exons of the six known hydrophobins of the fungus Cladosporium fulvum, the causal agent of tomato leaf mold. We used quantitative PCR to measure specifically the expression levels of the hydrophobins. The targeted genes are silenced to different degrees, but we also detected clear changes in the expression levels of nontargeted genes. This work highlights the difficulties that are likely to be encountered when attempting to silence more than one gene in a multigene family.


Assuntos
Cladosporium/genética , Cladosporium/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Inativação Gênica , Marcação de Genes , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Expressão Gênica , Solanum lycopersicum , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
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