Defining the predicted protein secretome of the fungal wheat leaf pathogen Mycosphaerella graminicola.

The Dothideomycete fungus Mycosphaerella graminicola is the causal agent of Septoria tritici blotch, a devastating disease of wheat leaves that causes dramatic decreases in yield. Infection involves an initial extended period of symptomless intercellular colonisation prior to the development of visible necrotic disease lesions. Previous functional genomics and gene expression profiling studies have implicated the production of secreted virulence effector proteins as key facilitators of the initial symptomless growth phase. In order to identify additional candidate virulence effectors, we re-analysed and catalogued the predicted protein secretome of M. graminicola isolate IPO323, which is currently regarded as the reference strain for this species. We combined several bioinformatic approaches in order to increase the probability of identifying truly secreted proteins with either a predicted enzymatic function or an as yet unknown function. An initial secretome of 970 proteins was predicted, whilst further stringent selection criteria predicted 492 proteins. Of these, 321 possess some functional annotation, the composition of which may reflect the strictly intercellular growth habit of this pathogen, leaving 171 with no functional annotation. This analysis identified a protein family encoding secreted peroxidases/chloroperoxidases (PF01328) which is expanded within all members of the family Mycosphaerellaceae. Further analyses were done on the non-annotated proteins for size and cysteine content (effector protein hallmarks), and then by studying the distribution of homologues in 17 other sequenced Dothideomycete fungi within an overall total of 91 predicted proteomes from fungal, oomycete and nematode species. This detailed M. graminicola secretome analysis provides the basis for further functional and comparative genomics studies.

The predicted secretome of the plant pathogenic fungus Fusarium graminearum: a refined comparative analysis.

The fungus Fusarium graminearum forms an intimate association with the host species wheat whilst infecting the floral tissues at anthesis. During the prolonged latent period of infection, extracellular communication between live pathogen and host cells must occur, implying a role for secreted fungal proteins. The wheat cells in contact with fungal hyphae subsequently die and intracellular hyphal colonisation results in the development of visible disease symptoms. Since the original genome annotation analysis was done in 2007, which predicted the secretome using TargetP, the F. graminearum gene call has changed considerably through the combined efforts of the BROAD and MIPS institutes. As a result of the modifications to the genome and the recent findings that suggested a role for secreted proteins in virulence, the F. graminearum secretome was revisited. In the current study, a refined F. graminearum secretome was predicted by combining several bioinformatic approaches. This strategy increased the probability of identifying truly secreted proteins. A secretome of 574 proteins was predicted of which 99% was supported by transcriptional evidence. The function of the annotated and unannotated secreted proteins was explored. The potential role(s) of the annotated proteins including, putative enzymes, phytotoxins and antifungals are discussed. Characterisation of the unannotated proteins included the analysis of Pfam domains and features associated with known fungal effectors, for example, small size, cysteine-rich and containing internal amino acid repeats. A comprehensive comparative genomic analysis involving 57 fungal and oomycete genomes revealed that only a small number of the predicted F. graminearum secreted proteins can be considered to be either species or sequenced strain specific.

OmniMapFree: a unified tool to visualise and explore sequenced genomes.

BACKGROUND: Acquiring and exploring whole genome sequence information for a species under investigation is now a routine experimental approach. On most genome browsers, typically, only the DNA sequence, EST support, motif search results, and GO annotations are displayed. However, for many species, a growing volume of additional experimental information is available but this is rarely searchable within the landscape of the entire genome. • RESULTS: We have developed a generic software which permits users to view a single genome in entirety either within its chromosome or supercontig context within a single window. This software permits the genome to be displayed at any scales and with any features. Different data types and data sets are displayed onto the genome, which have been acquired from other types of studies including classical genetics, forward and reverse genetics, transcriptomics, proteomics and improved annotation from alternative sources. In each display, different types of information can be overlapped, then retrieved in the desired combinations and scales and used in follow up analyses. The displays generated are of publication quality. • CONCLUSIONS: OmniMapFree provides a unified, versatile and easy-to-use software tool for studying a single genome in association with all the other datasets and data types available for the organism.

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

A partial chromosomal deletion caused by random plasmid integration resulted in a reduced virulence phenotype in Fusarium graminearum.

Fusarium graminearum (teleomorph: Gibberella zeae) is an Ascomycete fungal plant pathogen which infects a range of agriculturally important crops, including wheat, barley, and maize. A random plasmid insertion mutagenesis approach was used to analyze the pathogenicity of the PH-1 strain, for which full genomic information is available. Fungal transformants were initially screened for their ability to infect wheat ears. From a total of 1,170 transformants screened, eight were confirmed to be highly reduced in pathogenicity toward wheat ears and roots. These were designated disease-attenuated F. graminearum (daf) mutants. The in vitro growth rate and appearance of each daf mutant was equivalent to the parental strain. Deoxynivalenol (DON) was not detected in threshed grain recovered from ears inoculated with the daf10 mutant. Plasmid rescue and sequencing of the mutant daf10 revealed a deletion of approximately 350 kb from one end of chromosome 1. This chromosome segment is predicted to contain 146 genes. Microarray analysis of daf10 gene expression during growth in DON-inducing conditions confirmed the large deletion. The identities of the genes deleted and their potential role in DON production, pathogenesis, and other life processes are discussed.

Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing.

RNA interference is a natural defense against viruses in plants. To date, the only viral siRNAs characterized have been those for positive-sense RNA viruses with one or two genome components. Here, we characterized siRNAs derived from rice stripe virus (RSV), a member of the genus Tenuivirus with four genomic RNAs and an ambisense coding strategy. Deep sequencing of small RNAs from infected rice leaves showed that siRNAs were derived almost equally from virion and complementary RNA strands and were mostly 20-22 nucleotides long. Most viral siRNAs were produced within the coding sequences and 5' termini of the RSV genome. RSV siRNAs had a higher G and lower C content than the viral genome but a strong A/U bias at the first nucleotide and a U bias at the final one, suggesting preferential targeting of such sequences by rice Dicer-like proteins.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Identification and characterisation of Mycosphaerella graminicola secreted or surface-associated proteins with variable intragenic coding repeats.

Pathogenic micro-organisms have been suggested to vary the number of intragenic repeats present within secreted or cell membrane/cell wall-associated proteins in order to manipulate host immune responses. We have identified a number of genes predicted to encode secreted proteins possessing internal tandem repeats in the genome sequence of Mycosphaerella graminicola (isolate IPO323), a wheat leaf-specific fungal pathogen and causal agent of Septoria tritici blotch disease. Twenty-three M. graminicolaTandem Repeat Proteins (MgTRPs) were subject to further analysis. Many MgTRPs varied in the number of intragenic repeats between isolates and almost all were expressed. Peak gene expression was frequently observed towards the end of the symptomless phase of wheat leaf colonisation which typically lasts for 8-10 days after inoculation. In contrast, with one exception, increased expression of the majority of MgTRPs was not detected during interactions with resistant host genotypes. Repeat number differences detected in genomic DNA were retained in different transcript sizes produced during plant infection by different isolates. One in planta expressed MgTRP was found to reside within a approximately 6 kb region that appears to be absent from a number of tested isolates and also from individual members of a modern field population. Sequence analysis of another in planta expressed MgTRP from six isolates highlighted the potential for structural changes which may occur as a consequence of varying internal repeat numbers and provided support for repeat variation occurring as a consequence of intragenic recombination. These data provide new insights into the genetic variation which exists within M. graminicola populations at the level of in planta expressed secreted/surface-associated proteins which are candidate effectors in the host-pathogen interaction.

Virgaviridae: a new family of rod-shaped plant viruses.

The new plant virus family Virgaviridae is described. The family is named because its members have rod-shaped virions (from the Latin virga = rod), and it includes the genera Furovirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus and Tobravirus. The chief characteristics of members of the family are presented with phylogenetic analyses of selected genes to support the creation of the family. Species demarcation criteria within the genera are examined and discussed.

The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization.

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

Transcriptional adaptation of Mycosphaerella graminicola to programmed cell death (PCD) of its susceptible wheat host.

Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue, followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke or tolerate it. We are studying a major host-specific fungal pathogen of cultivated wheat, Septoria tritici (teleomorph Mycosphaerella graminicola). Here, we describe the host responses of wheat leaves infected with M. graminicola during the development of disease symptoms and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response, a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease-resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell-membrane integrity and dramatic increases in apoplastic metabolites and the rate of fungal growth. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms, and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.

Transcriptome profiling of the response of Mycosphaerella graminicola isolates to an azole fungicide using cDNA microarrays.

SUMMARY Resistance to azole antifungals is a major problem in the control of diseases caused by fungal pathogens of both humans and plants. Potential for the development of azole resistance in the wheat leaf blotch pathogen Mycosphaerella graminicola, the causal agent of the most economically significant foliar disease of wheat in north-western Europe, is now of particular concern after the recent emergence of widespread resistance to quinone outside inhibitor fungicides. Using a cDNA microarray representing around 25% of the genome, we have profiled the transcriptional response of M. graminicola to epoxiconazole, currently the most widely used azole fungicide on cereal crops. By comparing the transcription profiles of two M. graminicola isolates with contrasting sensitivities to epoxiconazole we show qualitative and quantitative differences in differentially expressed genes, including those involved in ergosterol biosynthesis, mitochondrial respiration and transport mechanisms. This represents the first study investigating the response of a plant pathogenic fungus to a fungicide using cDNA microarray technology.

A combinatorial pattern discovery approach for the prediction of membrane dipping (re-entrant) loops.

MOTIVATION: Membrane dipping loops are sections of membrane proteins that reside in the membrane but do not traverse from one side to the other, rather they enter and leave the same side of the membrane. We applied a combinatorial pattern discovery approach to sets of sequences containing at least one characterised structure described as possessing a membrane dipping loop. Discovered patterns were found to be composed of residues whose biochemical role is known to be essential for function of the protein, thus validating our approach. TMLOOP (http://membraneproteins.swan.ac.uk/TMLOOP) was implemented to predict membrane dipping loops in polytopic membrane proteins. TMLOOP applies discovered patterns as weighted predictive rules in a collective motif method (a variation of the single motif method), to avoid inherent limitations of single motif methods in detecting distantly related proteins. The collective motif method applies several, partially overlapping patterns, which pertain to the same sequence region, allowing proteins containing small variations to be detected. The approach achieved 92.4% accuracy in sensitivity and 100% reliability in specificity. TMLOOP was applied to the Swiss-Prot database, identifying 1392 confirmed membrane dipping loops, 75 plausible membrane dipping loops hitherto uncharacterised by topology prediction methods or experimental approaches and 128 false positives (8.0%).

Genome and EST analyses and expression of a gene family with putative functions in insect chemoreception.

Odorant-binding proteins (OBPs) are thought to be responsible for the transport of semiochemicals across hydrophobic interfaces to olfactory receptors. In insects, a second class of OBPs with four conserved cysteines has been variously named as sensory appendage proteins, olfactory segment-D proteins, and chemosensory proteins (CSPs). The physiological functions of these proteins have remained elusive. Here we report a comprehensive survey of both genome and expressed sequence tags (EST) databases. This showed that CSPs are apparently only present in the phylum, Arthropoda, and in two subphyla, Crustacea and Uniramia. This is the first report of a putative CSP in Crustacea and suggests that the origin of these genes predates the divergence of Uniramia and Crustacea. For the Uniramia, we identified 74 new genes encoding putative CSPs of insect species from 10 different orders. Using tissue-specific EST libraries, we have examined the relative expression of putative CSP genes in many tissues from 22 insect species suggesting that the genes are expressed widely. One Drosophila CSPs is expressed sixfold higher in head than other CSPs. One Bombyx mori CSPs was found at a very high level in pheromone gland, and for the first time, six CSPs were identified in B. mori compound eyes. The different frequencies of CSP transcripts were observed between solitary and gregarious EST libraries of Locusta migratoria.

DPVweb: a comprehensive database of plant and fungal virus genes and genomes.

DPVweb (http://www.dpvweb.net/) provides a central source of information about viruses, viroids and satellites of plants, fungi and protozoa. Comprehensive taxonomic information, including brief descriptions of each family and genus, and classified lists of virus sequences are provided. The database also holds detailed, curated, information for all sequences of viruses, viroids and satellites of plants, fungi and protozoa that are complete or that contain at least one complete gene (currently, n approximately 9000). For comparative purposes, it also contains a single representative sequence of all other fully sequenced virus species with an RNA or single-stranded DNA genome. The start and end positions of each feature (gene, non-translated region and the like) have been recorded and checked for accuracy. As far as possible, nomenclature for genes and proteins are standardized within genera and families. Sequences of features (either as DNA or amino acid sequences) can be directly downloaded from the website in FASTA format. The sequence information can also be accessed via client software for PC computers (freely downloadable from the website) that enable users to make an easy selection of sequences and features of a chosen virus for further analyses.

Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis.

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.

Analysis of expressed sequence tags from the wheat leaf blotch pathogen Mycosphaerella graminicola (anamorph Septoria tritici).

Mycosphaerella graminicola is a major fungal pathogen of wheat as the causal agent of Septoria leaf blotch disease. As a first step toward a greater understanding of the mechanism of host infection we have generated, sequenced, and analyzed three M. graminicola EST libraries from conditions predicted to resemble independent phases of the host infection process, including one library generated from the fungus during interaction with its host. A total of 5180 ESTs were sequenced and clustered into 886 contigs and 2039 singletons to give a set of 2925 unique sequences (unisequences). BLASTX analysis revealed 33% of the unknown M. graminicola unisequences to be orphans. Very limited inter-library overlap of expression was seen with the majority of unisequences (contigs and singletons) being library-specific. Analysis of EST redundancy between libraries demonstrated a significant difference in gene expression in the three conditions. Comparisons made against fully sequenced genomes revealed most M. graminicola sequences to be homologous to genes present in both pathogenic and non-pathogenic Ascomycete filamentous fungi. A range of sequences having significant homology to verified pathogenicity/virulence genes (HvPV-genes) of either plant or mammalian fungal and Oomycete pathogens were also identified (<1e-20). The generation of, and the diversity present within, this EST collection will facilitate future efforts aimed at a more detailed study of the transcriptome of the fungus during host infection.

Overview and analysis of the polyprotein cleavage sites in the family Potyviridae.

SUMMARY The genomes of plant viruses in the family Potyviridae encode large polyproteins that are cut by virus-encoded proteases into ten mature proteins. Three different types of protease have been identified, each of which cuts at sites with a distinctive sequence pattern. The experimental evidence for this specificity is reviewed and the cleavage site patterns are compiled for all sequenced species within the family. Seven of the nine cleavage sites in each species are cut by the viral NIa-Pro and patterns around these sites are related where possible to the active site-substrate interactions recently deduced following the resolution of the crystal structure of Tobacco etch virus (TEV) NIa-Pro (Phan et al., 2002. J. Biol. Chem. 277, 50564-50572). In particular, a revised series of cleavage sites for Sweet potato mild mottle virus (genus Ipomovirus) is proposed with a conserved His at the P1 position. This is supported by homology modelling studies using the TEV structure as a template. The data also provide a standard to correct the annotation of some other published sequences and to help predict these sites in further virus sequences as they become available. Comprehensive data for all sequences of each virus in the family, together with some summaries, have been made available at http://www.rothamsted.bbsrc.ac.uk/ppi/links/pplinks/potycleavage/index.html.

Metabolic and stress adaptation by Mycosphaerella graminicola during sporulation in its host revealed through microarray transcription profiling.

SUMMARY Pathogenic microbes must successfully adapt to the host environment, acquiring nutrients and tolerating immune/defence responses. Studies on host-pathogen interactions at the transcriptome level have predominantly investigated host responses. Here we present a broad-scale transcriptional analysis on a fungal pathogen during sporulation within its host environment. Septoria leaf blotch is an important fungal disease of cultivated wheat and is caused by the ascomycete fungus Septoria tritici (teleomorph Mycosphaerella graminicola). A cDNA microarray containing 2563 unigenes was generated and then used to compare fungal nutrition and development in vitro under nutrient-rich and nutrient-limiting conditions and in vivo at a late stage of plant infection. The data obtained provided clear insights into metabolic adaptation in all three conditions and an elevated stress adaptation/tolerance specifically in the host environment. We conclude that asexual sporulation of M. graminicola during the late stage of plant infection occurs in a rich nutritional environment involving adaptation to stresses imposed in part by the presence of reactive oxygen species.

Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling.

Vitamin C deficiency in the Arabidopsis mutant vtc1 causes slow growth and late flowering. This is not attributable to changes in photosynthesis or increased oxidative stress. We have used the vtc1 mutant to provide a molecular signature for vitamin C deficiency in plants. Using statistical analysis, we show that 171 genes are expressed differentially in vtc1 compared with the wild type. Many defense genes are activated, particularly those that encode pathogenesis-related proteins. Furthermore, transcript changes indicate that growth and development are constrained in vtc1 by the modulation of abscisic acid signaling. Abscisic acid contents are significantly higher in vtc1 than in the wild type. Key features of the molecular signature of ascorbate deficiency can be reversed by incubating vtc1 leaf discs in ascorbate. This finding provides evidence that many of the observed effects on transcript abundance in vtc1 result from ascorbate deficiency. Hence, through modifying gene expression, vitamin C contents not only act to regulate defense and survival but also act via phytohormones to modulate plant growth under optimal conditions.