80 results found
Spanu P, Przydacz M, Jones R, et al., 2020, Mode of action of the catalytic site in the N-terminal ribosome-inactivating domain of barley JIP60, Plant Physiology, ISSN: 0032-0889
Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein from barley (Hordeum vulgare) and is involved in the plant immune response dependent on the jasmonate hormones. Here, we demonstrate that transient expression in Nicotiana benthamiana of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163 to 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 Tyrosine 96, Glutamate 202, Arginine 205, and Tryptophan 235 in the presumptive active site of JIP60 are conserved in 815 plant ribosome inactivating proteins (RIP) in the Pfam database identified using HMMER as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose a role for these amino acids in JIP60 activity (depurination of adenosine in ribosomes). This study provides insight into the catalytic mechanism of this protein.
Thieron HS, Panstruga R, Spanu PD, 2019, Establishing a system to study cross-kingdom communication via extracellular vesicles between the crop plant Hordeum vulgare and the biotrophic fungus Blumeria graminis f. sp. hordei, 18th Congress of International-Society-for-Molecular-Plant-Microbe-Interactions (IS-MPMI), Publisher: AMER PHYTOPATHOLOGICAL SOC, Pages: 30-31, ISSN: 0894-0282
Przydacz M, Bruderer M, Belmans G, et al., 2019, Structure/function determination of JIP60 mode of action: The ribosome inactivating protein targeted by RNase-like powdery mildew effectors, 18th Congress of International-Society-for-Molecular-Plant-Microbe-Interactions (IS-MPMI), Publisher: AMER PHYTOPATHOLOGICAL SOC, Pages: 165-165, ISSN: 0894-0282
Zhang R, Spanu PD, Davies L, et al., 2019, Grapevine (Vitis vinifera) receptor-like cytoplasmic kinases VvPBL27 and VvPBS1 connect chitin perception to MAPK activation, 18th Congress of International-Society-for-Molecular-Plant-Microbe-Interactions (IS-MPMI), Publisher: AMER PHYTOPATHOLOGICAL SOC, Pages: 80-80, ISSN: 0894-0282
Barsoum M, Sabelleck B, Spanu PD, et al., 2019, Rumble in the Effector Jungle: Candidate Effector Proteins in Interactions of Plants with Powdery Mildew and Rust Fungi, CRITICAL REVIEWS IN PLANT SCIENCES, Vol: 38, Pages: 255-279, ISSN: 0735-2689
Li L, Collier B, Spanu PD, 2019, Isolation of Powdery Mildew Haustoria from Infected Barley, BIO-PROTOCOL, Vol: 9
Yu S-C, Kuemmel F, Skoufou-Papoutsaki M-N, et al., 2019, Yeast transformation efficiency is enhanced by TORC1- and eisosome-dependent signalling, Microbiology Open, Vol: 8, ISSN: 2045-8827
Transformation of baker's yeast (Saccharomyces cerevisiae) plays a key role in several experimental techniques, yet the molecular mechanisms underpinning transformation are still unclear. The addition of amino acids to the growth and transformation medium increases transformation efficiency. Here, we show that target of rapamycin complex 1 (TORC1) activated by amino acids enhances transformation via ubiquitin‐mediated endocytosis. We created mutants of the TORC1 pathway, alpha‐arrestins, and eisosome‐related genes. Our results demonstrate that the TORC1‐Npr1‐Art1/Rsp5 pathway regulates yeast transformation. Based on our previous study, activation of this pathway results in up to a 200‐fold increase in transformation efficiency, or greater. Additionally, we suggest DNA may be taken up by domains at the membrane compartment of Can1 (MCC) in the plasma membrane formed by eisosomes. Yeast studies on transformation could be used as a platform to understand the mechanism of DNA uptake in mammalian systems, which is clinically relevant to optimize gene therapy.
Pennington HG, Rhian J, Kwon S, et al., 2019, The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA, PLoS Pathogens, Vol: 15, ISSN: 1553-7366
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.
Frantzeskakis L, Kracher B, Kusch S, et al., 2018, Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen, BMC GENOMICS, Vol: 19, ISSN: 1471-2164
BackgroundPowdery 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).ResultsThe 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.ConclusionsThe 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.
Thordal-Christensen H, Birch PRJ, Spanu PD, et al., 2018, Why did filamentous plant pathogens evolve the potential to secrete hundreds of effectors to enable disease?, Molecular Plant Pathology, Vol: 19, Pages: 781-785, ISSN: 1364-3703
Bourras S, Praz CR, Spanu PD, et al., 2018, Cereal powdery mildew effectors: a complex toolbox for an obligate pathogen., Current Opinion in Microbiology, Vol: 46, Pages: 26-33, ISSN: 1369-5274
Cereal powdery mildews are major pathogens of cultivated monocot crops, and all are obligate biotrophic fungi that can only grow and reproduce on living hosts. This lifestyle is combined with extreme host specialization where every mildew subspecies (referred to as forma specialis) can only infect one plant species. Recently there has been much progress in our understanding of the possible roles effectors play in this complex host-pathogen interaction. Here, we review current knowledge on the origin, evolution, and mode of action of cereal mildew effectors, with a particular focus on recent advances in the identification of bona fide effectors and avirulence effector proteins from wheat and barley powdery mildews.
Belanger RR, Laur J, Labbe C, et al., 2017, An unusual menage a trois in the phyllosphere, Annual Meeting the American-Phytopathological-Society (APS), Publisher: AMER PHYTOPATHOLOGICAL SOC, Pages: 152-152, ISSN: 0031-949X
Laur J, Ramakrishnan GB, Labbe C, et al., 2017, Pseudozyma flocculosa, a new paradigm in the mode of action of biological control agents, Annual Meeting of the Northeastern Division of the American Phytopathological Society, Publisher: AMER PHYTOPATHOLOGICAL SOC, Pages: 202-202, ISSN: 0031-949X
Laur J, Ramakrishnan GB, Labbé C, et al., 2017, Effectors involved in fungal-fungal interaction lead to a rare phenomenon of hyperbiotrophy in the tritrophic system biocontrol agent-powdery mildew-plant., New Phytologist, Vol: 217, Pages: 713-725, ISSN: 0028-646X
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.
Spanu PD, Panstruga R, 2017, Editorial: Biotrophic Plant-Microbe Interactions, Frontiers in Plant Science, Vol: 8, ISSN: 1664-462X
Laur JR, Ramakrishnan G, Labbe C, et al., 2017, A transcriptomic approach to elucidate the mode of action of the biocontrol agent Pseudozyma flocculosa toward powdery mildews, Joint Meeting of the APS-Caribbean-Division and the Latin-American-Phytopathological-Society and the Mexican-Society-of-Phytopathology, Publisher: American Phytopathological Society, Pages: 9-9, ISSN: 0031-949X
Spanu PD, 2017, Cereal immunity against powdery mildews targets RNase-Like Proteins associated with Haustoria (RALPH) effectors evolved from a common ancestral gene, New Phytologist, Vol: 213, Pages: 969-971, ISSN: 0028-646X
This article is a Commentary on Praz et al., 213: 1301–1314
Spanu PD, Yu SC, Dawson A, et al., 2016, Nutrient supplements boost yeast transformation efficiency, Scientific Reports, Vol: 6, ISSN: 2045-2322
Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per μg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening.
Fuggetta MP, De Mico A, Cottarelli A, et al., 2016, Synthesis and Enantiomeric Separation of a Novel Spiroketal Derivative: A Potent Human Telomerase Inhibitor with High in Vitro Anticancer Activity, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 59, Pages: 9140-9149, ISSN: 0022-2623
Vela-Corcía D, Bautista R, de Vicente A, et al., 2016, De novo Analysis of the Epiphytic Transcriptome of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii and Identification of Candidate Secreted Effector Proteins., PLOS One, Vol: 11, ISSN: 1932-6203
The cucurbit powdery mildew fungus Podosphaera xanthii is a major limiting factor for cucurbit production worldwide. Despite the fungus's agronomic and economic importance, very little is known about fundamental aspects of P. xanthii biology, such as obligate biotrophy or pathogenesis. To design more durable control strategies, genomic information about P. xanthii is needed. Powdery mildews are fungal pathogens with large genomes compared with those of other fungi, which contain vast amounts of repetitive DNA sequences, much of which is composed of retrotransposons. To reduce genome complexity, in this work we aimed to obtain and analyse the epiphytic transcriptome of P. xanthii as a starting point for genomic research. Total RNA was isolated from epiphytic fungal material, and the corresponding cDNA library was sequenced using a 454 GS FLX platform. Over 676,562 reads were obtained and assembled into 37,241 contigs. Annotation data identified 8,798 putative genes with different orthologues. As described for other powdery mildew fungi, a similar set of missing core ascomycete genes was found, which may explain obligate biotrophy. To gain insight into the plant-pathogen relationships, special attention was focused on the analysis of the secretome. After this analysis, 137 putative secreted proteins were identified, including 53 candidate secreted effector proteins (CSEPs). Consistent with a putative role in pathogenesis, the expression profile observed for some of these CSEPs showed expression maxima at the beginning of the infection process at 24 h after inoculation, when the primary appressoria are mostly formed. Our data mark the onset of genomics research into this very important pathogen of cucurbits and shed some light on the intimate relationship between this pathogen and its host plant.
Bindschedler LV, Panstruga R, Spanu PD, 2016, Mildew-omics: how global analyses aid the understanding of life and evolution of powdery mildews, Frontiers in Plant Science, Vol: 7, ISSN: 1664-462X
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.
Albaser A, Kazana E, Bennett MH, et al., 2016, Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) β-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil, Journal of Agricultural and Food Chemistry, Vol: 64, Pages: 1520-1527, ISSN: 1520-5118
A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm–3 min–1 mg–1, respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.
Pennington HG, Gheorghe DM, Damerum A, et al., 2016, Interactions between the Powdery Mildew Effector BEC1054 and Barley Proteins Identify Candidate Host Targets, Journal of Proteome Research, Vol: 15, Pages: 826-839, ISSN: 1535-3907
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.
Amselem J, Vigouroux M, Oberhaensli S, et al., 2015, Evolution of the EKA family of powdery mildew avirulence-effector genes from the ORF 1 of a LINE retrotransposon, BMC Genomics, Vol: 16, ISSN: 1471-2164
BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.
Pennington HG, Li L, Spanu PD, 2015, Identification and selection of normalization controls for quantitative transcript analysis in Blumeria graminis, Molecular Plant Pathology, Vol: 17, Pages: 625-633, ISSN: 1464-6722
The investigation of obligate biotrophic pathogens, for example Blumeria 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.
Whigham E, Qi S, Mistry D, et al., 2015, Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.), Molecular Plant-Microbe Interactions, Vol: 28, Pages: 968-983, ISSN: 1943-7706
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.
Spanu PD, 2015, RNA-protein interactions in plant disease: hackers at the dinner table, NEW PHYTOLOGIST, Vol: 207, Pages: 991-995, ISSN: 0028-646X
Kanhayuwa L, Coutts R, Spanu P, 2014, A novel mycovirus in the human pathogenic fungus Aspergillus fumigatus and its application as a virus-induced gene silencing (VIGS) vector, FEBS EMBO 2014 Conference, Publisher: WILEY-BLACKWELL, Pages: 649-649, ISSN: 1742-464X
Panstruga R, Spanu PD, 2014, Powdery mildew genomes reloaded., New Phytol, Vol: 202, Pages: 13-14
Spanu PD, 2014, Messages from Powdery Mildew DNA: How the Interplay with a Host Moulds Pathogen Genomes, JOURNAL OF INTEGRATIVE AGRICULTURE, Vol: 13, Pages: 233-236, ISSN: 2095-3119
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