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To cause rice blast disease, the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on the selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signaling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls the expression of at least 60 temporally coregulated effector genes, preventing their transcription during the prepenetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for the orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.

To cause rice blast disease the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signalling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls expression of at least 60 temporally co-regulated effector genes, preventing their transcription during the pre-penetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.

Wheat, the most important food crop, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America, and to guide pre-emptive wheat breeding for blast resistance.

Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development—including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis—as well as controlling a additional set of virulence-associated transcription factor–encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention. Comparative RNA-seq, ChIP–seq and quantitative phosphoproteomics reveal how the blast fungus uses the Pmk1 MAP kinase to regulate a network of transcription factors that orchestrate the complex transcriptome changes necessary for infecting rice plants.

Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy, photosynthesis and osmotrophy. Traits associated with the latter two functions arose independently multiple times in the eukaryotes. The Fungi successfully coupled osmotrophy with filamentous growth, and similar traits are also manifested in the Pseudofungi (oomycetes and hyphochytriomycetes). Both the Fungi and the Pseudofungi encompass a diversity of plant and animal parasites. Genome-sequencing efforts have focused on host-associated microbes (mutualistic symbionts or parasites), providing limited comparisons with free-living relatives. Here we report the first draft genome sequence of a hyphochytriomycete ‘pseudofungus’;Hyphochytrium catenoides. Using phylogenomic approaches, we identify genes of recent viral ancestry, with related viral derived genes also present on the genomes of oomycetes, suggesting a complex history of viral coevolution and integration across the Pseudofungi.H. catenoideshas a complex life cycle involving diverse filamentous structures and a flagellated zoospore with a single anterior tinselate flagellum. We use genome comparisons, drug sensitivity analysis and high-throughput culture arrays to investigate the ancestry of oomycete/pseudofungal characteristics, demonstrating that many of the genetic features associated with parasitic traits evolved specifically within the oomycete radiation. Comparative genomics also identified differences in the repertoire of genes associated with filamentous growth between the Fungi and the Pseudofungi, including differences in vesicle trafficking systems, cell-wall synthesis pathways and motor protein repertoire, demonstrating that unique cellular systems underpinned the convergent evolution of filamentous osmotrophic growth in these two eukaryotic groups.

Rice blast is a pervasive and devastating disease that threatens rice production across the world. In spite of its importance to global food security, however, the underlying biology of plant infection by the blast fungus Magnaporthe oryzae remains poorly understood. In particular, it is unclear how the fungus elaborates a specialised infection cell, the appressorium, in response to surface signals from the rice leaf. Here, we report the identification of a network of temporally co-regulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We have functionally characterised this network of transcription factors and demonstrated the operation of a hierarchical transcriptional control system. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which represses hyphal-associated gene expression and simultaneously induces major physiological changes required for appressorium development, including cell cycle arrest, autophagic cell death, turgor generation and melanin biosynthesis. Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organisation, polarised exocytosis and effector gene expression necessary for plant tissue invasion.

Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development-including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis-as well as controlling a additional set of virulence-associated transcription factor-encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention.

The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morphogenetic changes, essential for its ability to cause disease. The smo mutation was identified > 25 years ago, and affects the shape and development of diverse cell types in M. oryzae, including conidia, appressoria, and asci. All attempts to clone the SMO1 gene by map-based cloning or complementation have failed over many years. Here, we report the identification of SMO1 by a combination of bulk segregant analysis and comparative genome analysis. SMO1 encodes a GTPase-activating protein, which regulates Ras signaling during infection-related development. Targeted deletion of SMO1 results in abnormal, nonadherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants. SMO1 is necessary for the organization of microtubules and for septin-dependent remodeling of the F-actin cytoskeleton at the appressorium pore. Smo1 physically interacts with components of the Ras2 signaling complex, and a range of other signaling and cytoskeletal components, including the four core septins. SMO1 is therefore necessary for the regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection.

Background: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. Results: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. Conclusions: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins. Fil: Armaleo, Daniele. University of Duke; Estados Unidos Fil: Müller, Olaf. University of Duke; Estados Unidos Fil: Lutzoni, François. University of Duke; Estados Unidos Fil: Andrésson, Ólafur S.. University of Iceland; Islandia Fil: Blanc, Guillaume. Université de Toulon; Francia Fil: Bode, Helge B.. Goethe Universitat Frankfurt; Alemania Fil: Collart, Frank R.. University Of Illinois At Chicago; ; Estados Unidos Fil: Dal Grande, Francesco. Senckenberg Biodiversity and Climate Research Center; Alemania Fil: Dietrich, Fred. University of Duke; Estados Unidos Fil: Grigoriev, Igor V.. University of California; Estados Unidos. US Department of Energy Joint Genome Institute; Estados Unidos Fil: Joneson, Suzanne. University of Duke; Estados Unidos Fil: Kuo, Alan. No especifíca; Fil: Larsen, Peter E.. University of Illinois; Estados Unidos Fil: Logsdon, John M.. University of Iowa; Estados Unidos Fil: Lopez, David. Gilead Sciences Incorporated; Estados Unidos Fil: Martin, Francis. Université de Lorraine; Francia Fil: May, Susan P.. University of Duke; Estados Unidos Fil: McDonald, Tami R.. University of Duke; Estados Unidos Fil: Merchant, Sabeeha S.. University of California at Berkeley; Estados Unidos Fil: Miao, Vivian. University of British Columbia; Canadá Fil: Morin, Emmanuelle. Université de Lorraine; Francia Fil: Oono, Ryoko. University of California; Estados Unidos Fil: Pellegrini, Matteo. University of California at Los Angeles; Estados Unidos Fil: Rubinstein, Nimrod. No especifíca; Fil: Sánchez Puerta, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Savelkoul, Elizabeth. University of Iowa; Estados Unidos Fil: Schmitt, Imke. Goethe Universitat Frankfurt; Alemania Fil: Slot, Jason C.. Ohio State University; Estados Unidos Fil: Soanes, Darren. University of Exeter; Reino Unido Fil: Szövényi, Péter. Universitat Zurich; Suiza Fil: Talbot, Nicholas J.. The Sainsbury Laboratory; Reino Unido Fil: Veneault-Fourrey, Claire. Université de Lorraine; Francia Fil: Xavier, Basil B.. Universiteit Antwerp; Bélgica

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat, and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the United Kingdom. Specifically, all eight rice blast isolates tested in this study produced the WB12 amplicon. In addition, comparative genomics of the WB12 nucleotide sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast and encourage further development of the assay to ensure its value in diagnosis.

The fungal phytopathogen Magnaporthe oryzae causes blast disease in cereals such as rice and finger millet worldwide. In this study, we assessed genetic diversity of 160 isolates from nine sub-Saharan Africa (SSA) and other principal rice producing countries and conducted a genome-wide association study (GWAS) to identify the genomic regions associated with virulence of M. oryzae. GBS of isolates provided a large and high-quality 617K single nucleotide polymorphism (SNP) dataset. Disease ratings for each isolate was obtained by inoculating them onto differential lines and locally-adapted rice cultivars. Genome-wide association studies were conducted using the GBS dataset and sixteen disease rating datasets. Principal Component Analysis (PCA) was used an alternative to population structure analysis for studying population stratification from genotypic data. A significant association between disease phenotype and 528 SNPs was observed in six GWA analyses. Homology of sequences encompassing the significant SNPs was determined to predict gene identities and functions. Seventeen genes recurred in six GWA analyses, suggesting a strong association with virulence. Here, the putative genes/genomic regions associated with the significant SNPs are presented.

The pathogenic life cycle of the rice blast fungusMagnaporthe oryzaeinvolves a series of morphogenetic changes, essential for its ability to cause disease. Thesmomutation was identified more than twenty-five years ago and affects the shape and development of diverse cell types inM. oryzae,including conidia, appressoria and asci. All attempts to clone theSMO1gene by map-based cloning and/or complementation, have failed over many years. Here, we report the identification ofSMO1by a combination of bulk segregant analysis and comparative genome analysis.SMO1encodes a GTPase-activating protein (GAP), which regulates Ras signalling during infection-related development. Targeted deletion ofSMO1results in abnormal, non-adherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants.SMO1is necessary for organisation of microtubules and for septin-dependent remodelling of the F-actin cytoskeleton at the appressorium pore. Smo1 physically interacts with components of the Ras2 signaling complex, and a range of other signalling and cytoskeletal components, including the four core septins.SMO1is therefore necessary for regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection.

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific gene editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based gene editing method. It has previously been reported in M. oryzae that transformation with plasmids expressing Cas9 can generate specific mutations using sgRNAs, directing the endonuclease to specific genes. We show, however, that expression of Cas9 is highly toxic to M. oryzae, rendering this approach impractical. We demonstrate that using purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs), provides an alternative and very effective gene editing procedure. When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the UK. In addition, comparative genomics of the WB12 sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast.

Summary Protein kinase C constitutes a family of serine–threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of M agnaporthe oryzae. First, all attempts to generate a target deletion of PKC 1, the single copy protein kinase C‐encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC 1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2‐encoding gene, MDL 2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacement with an analogue‐sensitive PKC 1AS allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expression associated with cell wall re‐modelling, autophagy, signal transduction and secondary metabolism. When considered together, these results suggest protein kinase C is essential for growth and development of M . oryzae with extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease.

Highlights • We generated a Z. tritici codon-optimized gene for green fluorescent protein (ZtGFP). • In epi-fluorescence and confocal microscopy, ZtGFP is brighter and more stable than eGFP. • We provide 3 vectors that carry AcGFP, eGFP and ZtGFP for yeast recombination-based cloning. • The vectors carry carboxin resistance for targeted integration. • The carboxin resistance conveying vectors integrate as single copies into the sdi1 locus., Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20–30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.

Made available in DSpace on 2018-12-11T17:06:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-10-03 Background: In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16% of the cultivated wheat area in Bangladesh, with yield losses reaching up to 100%. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields. Results: Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae. Conclusion: Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent. Bangabandhu Sheikh Mujibur Rahman Agricultural University Department of Biotechnology Institute of Integrative Biology ETH Zurich Plant Pathology INRA UMR 385 Biologie et génétique des interactions plantes-pathogènes BGPI University of Exeter College of Life and Environmental Sciences Norwich Research Park Earlham Institute Bangladesh Agricultural Research Institute Argo-Environmental Remote Sensing and Modeling Lab, Joydebpur 1701 Directorate of Grains Industry Department of Agriculture and Food Western Australia (DAFWA), 3 Baron-Hay Court Brazilian Agricultural Research Enterprise - EMBRAPA Wheat/Trigo University of São Paulo State - UNESP Department of Crop Protection Rural Engineering and Soil Science, IlhaSolteira Campus CIRAD UMR 385 Biologie et génétique des interactions plantes-pathogènes BGPI Norwich Research Park The Sainsbury Laboratory Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10 INRA UMR 1290 Biologie et Gestion des Risques en agriculture BIOGER Norwich Research Park John Innes Centre University of São Paulo State - UNESP Department of Crop Protection Rural Engineering and Soil Science, IlhaSolteira Campus

The blast fungus Magnaporthe oryzae gains entry to its host plant by means of a specialized pressure-generating infection cell called an appressorium, which physically ruptures the leaf cuticle

Gene transfer has been identified as a prevalent and pervasive phenomenon and an important source of genomic innovation in bacteria. The role of gene transfer in microbial eukaryotes seems to be of a reduced magnitude but in some cases can drive important evolutionary innovations, such as new functions that underpin the colonization of different niches. The aim of this review is to summarize published cases that support the hypothesis that horizontal gene transfer (HGT) has played a role in the evolution of phytopathogenic traits in fungi and oomycetes. Our survey of the literature identifies 46 proposed cases of transfer of genes that have a putative or experimentally demonstrable phytopathogenic function. When considering the life-cycle steps through which a pathogen must progress, the majority of the HGTs identified are associated with invading, degrading, and manipulating the host. Taken together, these data suggest HGT has played a role in shaping how fungi and oomycetes colonize plant hosts.

This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/1462-2920.12470

The Ustilago maydis Hook protein Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional early endosome trafficking by controlling attachment of both kinesin-3 and dynein., Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3– and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein.

The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative β‐1,3‐glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+, which carry a putative carbohydrate‐binding module, and the GH72− Gels, without this motif. We reveal that M. oryzae GH72 + GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3‐glucans have a higher degree of polymerization and are less branched than the wild‐type strain. The mutant showed significant differences in global patterns of gene expression, a hyper‐branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium‐mediated plant infection.

Trichoderma species are ubiquitous soil fungi that hold enormous potential for the development of credible alternatives to agrochemicals and synthetic fertilizers in sustainable crop production. In this paper, we show that substantial improvements in plant productivity can be met by genetic modification of a plant-growth-promoting and biocontrol strain of Trichoderma hamatum, but that these improvements are obtained in the absence of disease pressure only. Using a quantitative monoclonal antibody-based ELISA, we show that an N-acetyl-β-d-glucosaminidase-deficient mutant of T. hamatum, generated by insertional mutagenesis of the corresponding gene, has impaired saprotrophic competitiveness during antagonistic interactions with Rhizoctonia solani in soil. Furthermore, its fitness as a biocontrol agent of the pre-emergence damping-off pathogen Sclerotinia sclerotiorum is significantly reduced, and its ability to promote plant growth is constrained by the presence of both pathogens. This work shows that while gains in T. hamatum-mediated plant-growth-promotion can be met through genetic manipulation of a single beneficial trait, such a modification has negative impacts on other aspects of its biology and ecology that contribute to its success as a saprotrophic competitor and antagonist of soil-borne pathogens. The work has important implications for fungal morphogenesis, demonstrating a clear link between hyphal architecture and secretory potential. Furthermore, it highlights the need for a holistic approach to the development of genetically modified Trichoderma strains for use as crop stimulants and biocontrol agents in plant agriculture.

Horizontal gene transfer (HGT) can radically alter the genomes of microorganisms, providing the capacity to adapt to new lifestyles, environments, and hosts. However, the extent of HGT between eukaryotes is unclear. Using whole-genome, gene-by-gene phylogenetic analysis we demonstrate an extensive pattern of cross-kingdom HGT between fungi and oomycetes. Comparative genomics, including the de novo genome sequence of Hyphochytrium catenoides , a free-living sister of the oomycetes, shows that these transfers largely converge within the radiation of oomycetes that colonize plant tissues. The repertoire of HGTs includes a large number of putatively secreted proteins; for example, 7.6% of the secreted proteome of the sudden oak death parasite Phytophthora ramorum has been acquired from fungi by HGT. Transfers include gene products with the capacity to break down plant cell walls and acquire sugars, nucleic acids, nitrogen, and phosphate sources from the environment. Predicted HGTs also include proteins implicated in resisting plant defense mechanisms and effector proteins for attacking plant cells. These data are consistent with the hypothesis that some oomycetes became successful plant parasites by multiple acquisitions of genes from fungi.

Since the first attempts to classify the evolutionary history of trypanosomes, there have been conflicting reports regarding their true phylogenetic relationships and, in particular, their relationships with other vertebrate trypanosomatids, e.g. Leishmania sp., as well as with the many insect parasitising trypanosomatids. Perhaps the issue that has provided most debate is that concerning the monophyly (or otherwise) of genus Trypanosoma and, even with the advent of molecular methods, the findings of numerous studies have varied significantly depending on the gene sequences analysed, number of taxa included, choice of outgroup and phylogenetic methodology. While of arguably limited applied importance, resolution of the question as to whether or not trypanosomes are monophyletic is critical to accurate evaluation of competing, mutually exclusive evolutionary scenarios for these parasites, namely the 'vertebrate-first' or 'insect-first' hypotheses. Therefore, a new approach, which could overcome previous limitations was needed. At its most simple, the problem can be defined within the framework of a trifurcated tree with three hypothetical positions at which the root can be placed. Using BLASTp and whole-genome gene-by-gene phylogenetic analyses of Trypanosoma brucei, Trypanosoma cruzi, Leishmania major and Naegleria gruberi, we have identified 599 gene markers - putative homologues - that were shared between the genomes of these four taxa. Of these, 75 homologous gene families that demonstrate monophyly of the kinetoplastids were identified. We then used these data sets in combination with an additional outgroup, Euglena gracilis, coupled with large-scale gene concatenation and diverse phylogenetic techniques to investigate the relative branching order of T. brucei, T. cruzi and L. major. Our findings confirm the monophyly of genus Trypanosoma and demonstrate that

A growing body of data suggests that fungi have gained genes by horizontal gene transfer (HGT). This is an exciting result because fungi at first glance represent the most recalcitrant of all organisms to gene transfer, possessing robust cell walls and having lost phagotrophic capacities because they feed exclusively by osmotrophy. Nonetheless, a number of mechanisms have been implicated in gene transfer including: anastomosis of cellular structures, conjugation-like transfer between bacteria and yeasts, and exchange of supernumerary chromosomes. Despite absence of clearly identified mechanisms driving gene transfer in fungi, genome analysis has provided evidence for a number of fungal genes derived from foreign genomes by HGT. We briefly summarise current approaches to identifying HGT using genome data and make the case that phylogenetic analysis is the best approach to find and test potential examples of HGT. By applying this approach we have collected as many datasets as we could find for which phylogenetic analyses have been used as evidence of HGT and re-tested all 340 examples using updated taxon sampling. This approach enabled us to provide further supporting evidence for 323 examples of HGT, representing a significant pattern of transfer from both prokaryotes (mainly bacteria) and fungi into fungal genomes. Annotation of the HGTs suggests that these transfers have added to the core nutrient-processing metabolic network of many fungi, expanding the sugar, nitrogen, amino acid, nucleobase, and macromolecule metabolism of fungal microbes. Furthermore, these transfers appear to have added a significant number of new genes to the secretome and transporter repertoire of fungi, implying that gene transfer has added to the osmotrophic capacity of many fungal species.

An insertional mutagenesis screen in the rice blast fungus, Magnaporthe oryzae, identified a novel mutant, A2-12-3, which is defective in infection-related morphogenesis and pathogenicity. Analysis of the mutation confirmed an insertion into MoLDB1, which putatively encodes an 806-amino-acid protein with a predicted LIM binding domain. Targeted gene deletion mutants of MoLDB1 were unable to produce asexual or sexual spores and were significantly impaired in vegetative growth and fungal virulence. The Δmoldb1 mutants also showed reduced expression of genes coding hydrophobic proteins (e.g. MPG1 and MHP1), resulting in an easily wettable phenotype in vegetative culture. Moreover, the expression of four genes encoding LIM proteins predicted from the M. oryzae genome was significantly downregulated by deletion of MoLDB1. Analysis of an M. oryzae strain expressing a MoLbd1-green fluorescent protein gene fusion was consistent with the protein being nuclear localized. When considered together, MoLdb1 appears to be involved in regulation of cell wall proteins, including hydrophobins and LIM proteins, and is essential for conidiation, sexual development, appressorium formation, and pathogenicity in M. oryzae.

Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries and is an important evolutionary phenomenon in the ancestry of many microbes. The role of HGT in plant evolutionary history is, however, largely unexplored. Here, we compare the genomes of six plant species with those of 159 prokaryotic and eukaryotic species and identify 1689 genes that show the highest similarity to corresponding genes from fungi. We constructed a phylogeny for all 1689 genes identified and all homolog groups available from the rice (Oryza sativa) genome (3177 gene families) and used these to define 14 candidate plant-fungi HGT events. Comprehensive phylogenetic analyses of these 14 data sets, using methods that account for site rate heterogeneity, demonstrated support for nine HGT events, demonstrating an infrequent pattern of HGT between plants and fungi. Five HGTs were fungi-to-plant transfers and four were plant-to-fungi HGTs. None of the fungal-to-plant HGTs involved angiosperm recipients. These results alter the current view of organismal barriers to HGT, suggesting that phagotrophy, the consumption of a whole cell by another, is not necessarily a prerequisite for HGT between eukaryotes. Putative functional annotation of the HGT candidate genes suggests that two fungi-to-plant transfers have added phenotypes important for life in a soil environment. Our study suggests that genetic exchange between plants and fungi is exceedingly rare, particularly among the angiosperms, but has occurred during their evolutionary history and added important metabolic traits to plant lineages.

Rispail, Nicolas et al., Mitogen-activated protein kinase (MAPK) cascades and the calcium–calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium–calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches., This analysis was carried out by members of the SIGNALPATH Marie Curie training network (MRTN-CT-2005-019277), which provided financial support for N.R., C.A., R.C., A.G., R.H., E.P.N., A.P., E.S., V.V. and M.Y.

Plant pathogenic microbes secrete proteins known as effectors, which enter the cytoplasm of plant cells and suppress host defences. Known effectors in oomycete pathogens possess an RXLR-EER motif in their amino acid sequence that is necessary for transport of the effector into a host plant cell. A large number of putative effectors have now been identified in oomycete genomes, the sequences of which show evidence of diversifying selection at their C terminus. Here, we describe recent progress in characterizing RXLR-EER effectors and discuss why so many of these rapidly evolving proteins are encoded by the genomes of plant pathogenic oomycetes.

Fungi and oomycetes are the causal agents of many of the world's most serious plant diseases and are unique among the microbial pathogens in being able to breach the intact surfaces of host plants, rapidly establishing infections that can have disastrous consequences for large-scale agricultural

The rice blast fungus Magnaporthe grisea infects plants by means of specialized infection structures known as appressoria. Turgor generated in the appressorium provides the invasive force that allows the fungus to breach the leaf cuticle with a narrow-penetration hypha gaining entry to the underlying epidermal cell. Appressorium maturation in M. grisea involves mass transfer of lipid bodies to the developing appressorium, coupled to autophagic cell death in the conidium and rapid lipolysis at the onset of appressorial turgor generation. Here, we report identification of the principal components of lipid metabolism in M. grisea based on genome sequence analysis. We show that deletion of any of the eight putative intracellular triacylglycerol lipase-encoding genes from the fungus is insufficient to prevent plant infection, highlighting the complexity and redundancy associated with appressorial lipolysis. In contrast, we demonstrate that a peroxisomally located multifunctional, fatty acid β-oxidation enzyme is critical to appressorium physiology, and blocking peroxisomal biogenesis prevents plant infection. Taken together, our results indicate that, although triacylglycerol breakdown in the appressorium involves the concerted action of several lipases, fatty acid metabolism and consequent generation of acetyl CoA are necessary for M. grisea to complete its prepenetration phase of development and enter the host plant.

Magnaporthe grisea, the causal agent of rice blast disease, invades plant tissue due to the action of specialized infection structures called appressoria, which are used to breach the leaf cuticle and allow development of intracellular, infectious hyphae. In this report we demonstrate that peroxisomal carnitine acetyl transferase (CAT) activity is necessary for appressorium function, and in particular, for the elaboration of primary penetration hyphae. The major CAT activity in M. grisea is encoded by the PTH2 gene, which shows elevated expression in response to acetate and lipid, and is regulated by the cyclic AMP response pathway. Furthermore, a Pth2-GFP fusion protein colocalizes with a peroxisomal marker protein. Targeted deletion of PTH2, generated mutants that were completely non-pathogenic, lacked CAT activity and were unable to utilize a range of lipid substrates. The impairment of appressorium function in Deltapth2 was associated with a delay in lipid reserve mobilization from germ tubes into developing infection cells, and abnormal chitin distribution in infection structures. Addition of glucose to Deltapth2 mutants partially restored the ability to cause rice blast disease and lipid reserve mobilization. Taken together, our findings provide evidence that Pth2 plays a role in the generation of acetyl CoA pools necessary for appressorium function and rapid elaboration of penetration hyphae during host infection.

SUMMARY We describe the analysis of 57 727 unique expressed sequence tags (ESTs) from 15 species of phytopathogenic and three species of saprophytic fungi. This resource is held within the COGEME phytopathogen EST database (http://cogeme.ex.ac.uk/). Comparative analysis was performed to investigate the differences between pathogenic and free-living fungi based on a substantial collection of expressed gene sequences and available, completed fungal genome sequences. We report that the expressed gene inventories of pathogenic fungi were not significantly more similar to each other than to those of free-living filamentous fungi. As expected, however, filamentous fungi as a group share more sequences in common than with the free-living yeast species Saccharomyces cerevisiae. Interestingly, ESTs of the obligate biotrophic fungus Blumeria graminis f. sp. hordei were more dissimilar to those of all other fungal species assessed, having a lower number of sequences in common with filamentous ascomycetes studied to date and also possessing a larger proportion of unisequences of unknown function. Our analysis of ESTs in the COGEME database enabled identification of a set of functional groups of genes that are more highly represented in the genomes of pathogenic fungi than non-pathogenic species.

The rice blast fungus Magnaporthe grisea develops specialized infection structures known as appressoria, which develop enormous turgor pressure to bring about plant infection. Turgor is generated by accumulation of compatible solutes, including glycerol, which is synthesized in large quantities in the appressorium. Glycogen, trehalose and lipids represent the most abundant storage products in M. grisea conidia. Trehalose and glycogen are rapidly degraded during conidial germination and it is known that trehalose synthesis is required for virulence of the fungus. Lipid bodies are transported to the developing appressoria and degraded at the onset of turgor generation, in a process that is cAMP-dependent. A combined biochemical and genetic approach is being used to dissect the process of turgor generation in the rice blast fungus.

Fungal phytopathogens continue to cause major economic impact, either directly, through crop losses, or due to the costs of fungicide application. Attempts to understand these organisms are hampered by a lack of fungal genome sequence data. A need exists, however, to develop specific bioinformatics tools to collate and analyse the sequence data that currently is available. A web-accessible gene discovery database (http://cogeme.ex.ac.uk/biosynthesis.html) was developed as a demonstration tool for the analysis of metabolic and signal transduction pathways in pathogenic fungi using incomplete gene inventories. Using Bayesian probability to analyse the currently available gene information from pathogenic fungi, we provide evidence that the obligate pathogenBlumeria graminispossesses all amino acid biosynthetic pathways found in free-living fungi, such asSaccharomyces cerevisiae. Phylogenetic analysis was also used to deduce a gene history of succinate-semialdehyde dehydrogenase, an enzyme in the glutamate and lysine biosynthesis pathways. The database provides a tool and methodology to researchers to direct experimentation towards predicting pathway conservation in pathogenic microorganisms.

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%–61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than β-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease., Author summary Blastocystis are unicellular eukaryotic organisms related to algae and some plant pathogens. They are common constituents of the human gut microbial community, colonizing approximately 1 billion humans worldwide. Whether their presence is harmful or not continues to be hotly debated. Part of the uncertainty stems from the fact that at least 17 subtypes have been identified from various mammalian hosts, including 9 from humans. To better characterize and understand Blastocystis, we have sequenced and annotated the genome for subtype 1 and compared it with previous genomic results for subtypes 7 and 4. The comparisons revealed considerable differences between the 3 sequenced subtypes for a number of genomic features like DNA base composition, size of genome, number of genes, and number of introns. We also examined various biochemical pathways and cellular systems in the context of the full gene complement to better understand the biology of Blastocystis, including some of its more unusual features like a mitochondrion related organelle. We also identified subtype-specific gene family expansions that may be related to virulence. Finally, we showed that Blastocystis appears to have most of the genes necessary for sexual reproduction. This study provides resources and hypotheses for future investigations into the biology and potential pathogenicity of these common gut microbes.

Biosciences, University of Exeter, Exeter EX4 4QD, UKSummaryBackground: Many microbial eukaryotes have evolved anaer-obic alternatives to mitochondria known as mitochondrion-related organelles (MROs). Yet, only a few of these havebeen experimentally investigated. Here we report an RNA-seq-based reconstruction of the MRO proteome of Pygsuiabiforma, an anaerobic representative of an unexplored deep-branching eukaryotic lineage.Results: Pygsuia’s MRO has a completely novel suite offunctions, defying existing ‘‘function-based’’ organelle classi-fications.Mostnotableisthereplacementofthemitochondrialiron-sulfur cluster machinery by an archaeal sulfur mobiliza-tion (SUF) system acquired via lateral gene transfer (LGT).Using immunolocalization in Pygsuia and heterologousexpressioninyeast,weshowthattheSUFsystemdoesindeedlocalize to the MRO. The Pygsuia MRO also possesses aunique assemblage of features, including: cardiolipin, phos-phonolipid, amino acid, and fatty acid metabolism; a partialKreb’s cycle; a reduced respiratory chain; and a laterallyacquired rhodoquinone (RQ) biosynthesis enzyme. The latterobservation suggests that RQ is an electron carrier of a fuma-rate reductase-type complex II in this MRO.Conclusions:TheuniquefunctionalprofileofthisMROunder-scores the tremendous plasticity of mitochondrial functionwithin eukaryotes and showcases the role of LGT in forgingmetabolic mosaics of ancestral and newly acquired organellarpathways.IntroductionMitochondria of modern-day eukaryotes evolved from ana-proteobacterial endosymbiont that was integrated as anorganellewithinahostcellpriortothelasteukaryoticcommonancestor [1]. In aerobic eukaryotes, mitochondria carry out anumber of important functions, including pyruvate decarbox-ylation, oxygen-dependent ATP production, amino acid meta-bolism, and iron-sulfur (Fe-S) cluster biosynthesis. Over thepast 20 years, investigations into the mitochondria orhomologous organelles of anaerobic organisms (mito-chondrion-related organelles, MROs) have revealed a varietyof different metabolic phenotypes.Classical‘‘aerobic’’mitochondriagenerateATPbyoxidativephosphorylation using ATP synthase coupled to the electrontransport chain, ultimately reducing O

The dematiaceous (melanized) fungus Scedosporium prolificans is an emerging and frequently fatal pathogen of immunocompromised humans and which, along with the closely related fungi Pseudallescheria boydii, Scedosporium apiospermum and S. aurantiacum in the Pseudallescheria-Scedosporium complex, is a contributing aetiology to tsunami lung and central nervous system infections in near-drowning victims who have aspirated water laden with spores. At present, the natural habitat of the fungus is largely unknown, and accurate detection methods are needed to identify environmental reservoirs of infectious propagules. In this study, we report the development of a monoclonal antibody (mAb) (CA4) specific to S. prolificans, which does not cross-react with closely related fungi in the Pseudallescheria-Scedosporium complex or with a wide range of mould and yeast species pathogenic to humans. Using genome sequencing of a soil isolate and targeted gene disruption of the CA4 antigen-encoding gene, we show that mAb CA4 binds to the melanin-biosynthetic enzyme tetrahydroxynaphthalene reductase. Enzyme-deficient mutants produce orange-brown or green-brown spore suspensions compared with the black spore suspension of the wild-type strain. Using mAb CA4 and a mAb (HG12) specific to the related fungi P. boydii, P. apiosperma, S. apiospermum and S. aurantiacum, we demonstrate how the mAbs can be used in combination with a semiselective isolation procedure to track these opportunistic pathogens in environmental samples containing mixed populations of human pathogenic fungi. Specificity of mAb CA4 was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of fungi isolated from estuarine muds.

Microsporidia are obligate intracellular parasites with the smallest known eukaryotic genomes. Although they are increasingly recognized as economically and medically important parasites, the molecular basis of microsporidian pathogenicity is almost completely unknown and no genetic manipulation system is currently available. The fish-infecting microsporidian Spraguea lophii shows one of the most striking host cell manipulations known for these parasites, converting host nervous tissue into swollen spore factories known as xenomas. In order to investigate the basis of these interactions between microsporidian and host, we sequenced and analyzed the S. lophii genome. Although, like other microsporidia, S. lophii has lost many of the protein families typical of model eukaryotes, we identified a number of gene family expansions including a family of leucine-rich repeat proteins that may represent pathogenicity factors. Building on our comparative genomic analyses, we exploited the large numbers of spores that can be obtained from xenomas to identify potential effector proteins experimentally. We used complex-mix proteomics to identify proteins released by the parasite upon germination, resulting in the first experimental isolation of putative secreted effector proteins in a microsporidian. Many of these proteins are not related to characterized pathogenicity factors or indeed any other sequences from outside the Microsporidia. However, two of the secreted proteins are members of a family of RICIN B-lectin-like proteins broadly conserved across the phylum. These proteins form syntenic clusters arising from tandem duplications in several microsporidian genomes and may represent a novel family of conserved effector proteins. These computational and experimental analyses establish S. lophii as an attractive model system for understanding the evolution of host-parasite interactions in microsporidia and suggest an important role for lineage-specific innovations and fast evolving proteins in the evolution of the parasitic microsporidian lifecycle., Author Summary Microsporidia are unusual intracellular parasites that infect a broad range of animal cells. In comparison to their fungal relatives, microsporidian genomes have shrunk during evolution, encoding as few as 2000 proteins. This minimal molecular repertoire makes them a reduced model system for understanding host-parasite interactions. A number of microsporidian genomes have now been sequenced, but the lack of a system for genetic manipulation makes it difficult to translate these data into a better understanding of microsporidian biology. Here we present a deep sequencing project of Spraguea lophii, a fish-infecting microsporidian that is abundantly available from environmental samples. We use our sequence data combined with germination protocols and complex-mix proteomics to identify proteins released by the cell at the earliest stage of germination, representing potential pathogenicity factors. We profile the RNA expression pattern of germinating cells and identify a set of highly transcribed hypothetical genes. Our study provides new insight into the importance of uncharacterized, lineage-specific and/or fast evolving proteins in microsporidia and provides new leads for the investigation of virulence factors in these enigmatic parasites.