A putative serine protease, SpSsp1, from Saprolegnia parasitica is recognised by sera of rainbow trout, Oncorhynchus mykiss

Acknowledgements Our work was supported by the BBSRC (BB/C518457/1, BB/G012075/1, BB/J018333/1) (K.L.M., C.J.S., J.S.C., K.S.D., and P.v.W.), the University of Aberdeen (V.L.A., C.J.S., and P.v.W.), MSD Animal Health (J.S.C., K.S.D., and A.H.v.d.B), and The Royal Society (P.v.W.). This work was also supported by a Marie Curie Initial Training Networks with the SAPRO (sustainable approaches to reduce Oomycete (Saprolegnia) infections in aquacultures) grant PITN-GA-2009-238550 (A.H.v.d.B., L.L., C.J.S., P.v.W.). We would like to acknowledge Aberdeen Proteomics for carrying out LC–MS/MS and Laura Grenville-Briggs for valuable discussion and technical help. We are grateful to the Broad Institute (Carsten Russ, Rays Jiang, Brian Haas, and Chad Nusbaum), Brett Tyler (VBI), and P.v.W. for early release of draft supercontigs of the genome sequence of isolate CBS233.65, which helped us identify SpSsp1.Peer reviewedPublisher PD

Cell entry of a host-targeting protein of oomycetes requires gp96

The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants.This work is supported by the [European Community’s] Seventh Framework Programme [FP7/2007–2013] under grant agreement no. [238550] (L.L., J.D.-U., C.J.S., P.v.W.); BBSRC [BBE007120/1, BB/J018333/1 and BB/G012075/1] (F.T., I.d.B., C.J.S., S.W., P.v.W.); Newton Global Partnership Award [BB/N005058/1] (F.T., P.v.W.), the University of Aberdeen (A.D.T., T.R., C.J.S., P.v.W.) and Deutsche Forschungsgemeinschaft [CRC1093] (P.B., T.S.). We would like to acknowledge the Ministry of Higher Education Malaysia for funding INA. We would like to thank Brian Haas for his bioinformatics support. We would like to acknowledge Neil Gow and Johannes van den Boom for critical reading of the manuscript. We would like to acknowledge Svetlana Rezinciuc for technical help with pH-studies

Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

Acknowledgements We thank the Red Cross blood bank in Melbourne for human erythrocytes. We thank Svenja Gunther for expression of GBP130 66–196 proteins; Michelle Gazdik and Chris Burns for help in preparing lipids; Lachlan Whitehead (Centre for Dynamic Imaging, Walter and Eliza Hall Institute) for assistance with quantification of export; and David Bocher for help with generation of STEVOR constructs. This work was supported by the National Health and Medical Research Council of Australia (grants 637406, 1010326, 1049811 and 1057960), a Ramaciotti Foundation Establishment Grant (3197/2010), a Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS, and the Canadian Institutes for Health Research (MOP#130359). J.A.B is an Australian Research Council QEII Fellow, SF was supported by the Research Training Group GRK1459 of the German Research Foundation, and AFC is a Howard Hughes International Scholar.Peer reviewedPublisher PD

The RxLR Motif of the Host Targeting Effector AVR3a of Phytophthora infestans Is Cleaved Before Secretion

Our work is supported by the BBSRC (SW, CJS, PvW), NERC (PvW) and the University of Aberdeen (CJS, PvW, ID). This work was supported by EU East-NMR FP7 Project (Contract 228461) and Polish National Centre for Research and Development under research grant number 178479 (contract number PBS1/A9/ 13/2012) (for IZ). We would like to acknowledge Kevin MacKenzie of the core microscopy facility of the University of Aberdeen for helpful suggestions and Prof. Regine Kahmann for critical reading of the manuscript. On behalf of all authors, the ASPB journals deposit final published articles in PubMed Central for release 12 months after the date of publication (unless a free-access option applies). The final pdf of the article will be open access.Peer reviewedPublisher PD

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Background: Pythium ultimum (P. ultimum) is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions although surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report in a genome outside the metazoans. Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae

Unraveling the sugar code: the role of microbial extracellular glycans in plant-microbe interactions

To defend against microbial invaders but also to establish symbiotic programs, plants need to detect the presence of microbes through the perception of molecular signatures characteristic of a whole class of microbes. Among these molecular signatures, extracellular glycans represent a structurally complex and diverse group of biomolecules that has a pivotal role in the molecular dialog between plants and microbes. Secreted glycans and glycoconjugates such as symbiotic lipochitooligosaccharides or immunosuppressive cyclic beta-glucans act as microbial messengers that prepare the ground for host colonization. On the other hand, microbial cell surface glycans are important indicators of microbial presence. They are conserved structures normally exposed and thus accessible for plant hydrolytic enzymes and cell surface receptor proteins. While the immunogenic potential of bacterial cell surface glycoconjugates such as lipopolysaccharides and peptidoglycan has been intensively studied in the past years, perception of cell surface glycans from filamentous microbes such as fungi or oomycetes is still largely unexplored. To date, only few studies have focused on the role of fungal-derived cell surface glycans other than chitin, highlighting a knowledge gap that needs to be addressed. The objective of this review is to give an overview on the biological functions and perception of microbial extracellular glycans, primarily focusing on their recognition and their contribution to plant-microbe interactions

Auto-aggregation in zoospores of <i>Phytophthora infestans</i>:the cooperative roles of bioconvection and chemotaxis

Phytophthora infestans is a highly destructive plant pathogen. It was the cause of the infamous Irish potato famine in the nineteenth century and remains to this day a significant global problem with associated costs estimated at $3 billion annually. Key to the success of this pathogen is the dispersal of free-swimming cells called zoospores. A poorly understood aspect of zoospore behaviour is auto-aggregation—the spontaneous formation of large-scale patterns in cell density. Current competing hypotheses suggest that these patterns are formed by one of two distinct mechanisms: chemotaxis and bioconvection. In this paper, we present mathematical and experimental results that together provide strong evidence that auto-aggregation can only result from a combination of these mechanisms, each having a distinct, time-separated role. A better understanding of the underlying infection mechanisms of P. infestans and potentially other Phytophthora species will in the longer term lead to advances in preventative treatment and thus potentially significant savings in socio-economic costs

Plant species-specific recognition of long and short beta-1,3-linked glucans is mediated by different receptor systems

Plants survey their environment for the presence of potentially harmful or beneficial microbes. During colonization, cell surface receptors perceive microbe-derived or modified-self ligands and initiate appropriate responses. The recognition of fungal chitin oligomers and the subsequent activation of plant immunity are well described. In contrast, the mechanisms underlying beta-glucan recognition and signaling activation remain largely unexplored. Here, we systematically tested immune responses towards different beta-glucan structures and show that responses vary between plant species. While leaves of the monocots Hordeum vulgare and Brachypodium distachyon can recognize longer (laminarin) and shorter (laminarihexaose) beta-1,3-glucans with responses of varying intensity, duration and timing, leaves of the dicot Nicotiana benthamiana activate immunity in response to long beta-1,3-glucans, whereas Arabidopsis thaliana and Capsella rubella perceive short beta-1,3-glucans. Hydrolysis of the beta-1,6 side-branches of laminarin demonstrated that not the glycosidic decoration but rather the degree of polymerization plays a pivotal role in the recognition of long-chain beta-glucans. Moreover, in contrast to the recognition of short beta-1,3-glucans in A. thaliana, perception of long beta-1,3-glucans in N. benthamiana and rice is independent of CERK1, indicating that beta-glucan recognition may be mediated by multiple beta-glucan receptor systems

The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import

Background The mitochondrial intermembrane space (IMS) is home to proteins fulfilling numerous essential cellular processes, particularly in metabolism and mitochondrial function. All IMS proteins are nuclear encoded and synthesized in the cytosol and must therefore be correctly targeted and transported to the IMS, either through mitochondrial targeting sequences or conserved cysteines and the mitochondrial disulfide relay system. The mitochondrial oxidoreductase MIA40, which catalyzes disulfide formation in the IMS, is imported by the combined action of the protein AIFM1 and MIA40 itself. Here, we characterized the function of the conserved highly negatively charged C-terminal region of human MIA40. Results We demonstrate that the C-terminal region is critical during posttranslational mitochondrial import of MIA40, but is dispensable for MIA40 redox function in vitro and in intact cells. The C-terminal negatively charged region of MIA40 slowed import into mitochondria, which occurred with a half-time as slow as 90 min. During this time, the MIA40 precursor persisted in the cytosol in an unfolded state, and the C-terminal negatively charged region served in protecting MIA40 from proteasomal degradation. This stabilizing property of the MIA40 C-terminal region could also be conferred to a different mitochondrial precursor protein, COX19. Conclusions Our data suggest that the MIA40 precursor contains the stabilizing information to allow for postranslational import of sufficient amounts of MIA40 for full functionality of the essential disulfide relay. We thereby provide for the first time mechanistic insights into the determinants controlling cytosolic surveillance of IMS precursor proteins

Serendipita indica E5 ' NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization

Extracellular adenosine 5'-triphosphate (eATP) is an essential signaling molecule that mediates different cellular processes through its interaction with membrane-associated receptor proteins in animals and plants. eATP regulates plant growth, development, and responses to biotic and abiotic stresses. Its accumulation in the apoplast induces ROS production and cytoplasmic calcium increase mediating a defense response to invading microbes. We show here that perception of extracellular nucleotides, such as eATP, is important in plant-fungus interactions and that during colonization by the beneficial root endophyte Serendipita indica eATP accumulates in the apoplast at early symbiotic stages. Using liquid chromatography-tandem mass spectrometry, and cytological and functional analysis, we show that S. indica secrets SiE5'NT, an enzymatically active ecto-5'-nucleotidase capable of hydrolyzing nucleotides in the apoplast. Arabidopsis thaliana lines producing extracellular SiE5'NT are significantly better colonized, have reduced eATP levels, and altered responses to biotic stresses, indicating that SiE5'NT functions as a compatibility factor. Our data suggest that extracellular bioactive nucleotides and their perception play an important role in fungus-root interactions and that fungal-derived enzymes can modify apoplastic metabolites to promote fungal accommodation