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Abstract • 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. graminicola Tandem 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 8–10days 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 ∼6kb 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The protein-glycogen particle preparation isolated from rabbit skeletal muscle extracts by mild acidification and differential centrifugation shows eight major protein-staining bands when examined by polyacrylamide gel electrophoresis, five of which have previously been identified as the glycogen debranching enzyme, the α and β subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthase [Taylor, C., Cox, A. J., Kernohan, J. C. & Cohen, P. (1975) Eur. J. Biochem. 51, 105–115]. The other three major proteins in this preparation have now been identified. Two of the proteins were identified as the heavy chain of myosin and actin respectively, based on their electrophoretic migration, their insolubility in buffers of low ionic strength, and their solubility in buffers containing 0.6 M KCI. The third protein has been found to be calsequestrin, a major calcium-binding protein of the sarcoplasmic reticulum. This conclusion was based on the unusual chromatographic behaviour of the protein on DEAE-cellulose at pH 7.5, its electrophoretic mobility, amino acid composition, and immunological cross reactivity. A simple method for the isolation of calsequestrin as a by-product of the purification of glycogen synthase has been developed, by which 70–80 mg of the protein were routinely isolated from 5000 g of muscle (6 rabbits) within five days. The protein was homogeneous by the criterion of poly- acrylamide gel electrophoresis and sedimentation equilibrium centrifugation. The molecular weight determined by the latter technique was 41000 and the absorbance index A280nm,1% was 11.7. The protein had a very low sedimentation coefficient of 1.97 S indicating that it is either very asymmetric or a largely unfolded protein, in the buffers used in these experiments, a conclusion supported by its abnormal gel filtration behaviour. Since the three hitherto unidentified components of the protein-glycogen particle preparation are not enzymes of glycogen metabolism and merely constituents of particulate material (actomyosin and fragments of the sarcoplasmic reticulum) which have coprecipitated with the protein-glycogen complex, over 95% of the protein attached to glycogen is accounted for by just four enzymes. [ABSTRACT FROM AUTHOR]

Proteins associated with the hypersensitive response (b-proteins) were purified from various Nicotiana species and compared biochemically and serologically. The method developed to purify proteins b, b and b of N. tabacum cv. Xanthi-nc was used to purify b-proteins present in N. sylvestris (b, b and b) and N. tomentosiformis (b), the parental species of N. tabacum, and b″ from both N. glutinosa and N. debneyi. Ultracentrifugation and amino acid analysis of some of these proteins has shown that they are very similar and that they are all monomers in their native form (mol wt = 15 700 for b, b, b and b; mol wt = 13 800 for b″). Based on their reactions to an antiserum produced against protein b of N. tabacum cv. Xanthi-nc, 3 serological groups can be recognized which are independent of the source species (I) b and b, (II) b″ and b, (III) b. Thus, proteins in the same serological group but from different species are more closely related than the b-proteins in different serological groups but present in the same species. The implication of this site on the possible phylogeny of b-proteins is discussed. Serological tests confirmed the b-protein present as a constitutive component in the virus resistant interspecific hybrids of N. glutinosa × N. debneyi as protein b″. [ABSTRACT FROM AUTHOR]