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Background: Studying genomic variation in rapidly evolving pathogens potentially enables identification of genes supporting their "core biology", being present, functional and expressed by all strains or "flexible biology", varying between strains. Genes supporting flexible biology may be considered to be "accessory", whilst the "core" gene set is likely to be important for common features of a pathogen species biology, including virulence on all host genotypes. The wheat-pathogenic fungus Zymoseptoria tritici represents one of the most rapidly evolving threats to global food security and was the focus of this study. Results: We constructed a pangenome of 18 European field isolates, with 12 also subjected to RNAseq transcription profiling during infection. Combining this data, we predicted a "core" gene set comprising 9807 sequences which were (1) present in all isolates, (2) lacking inactivating polymorphisms and (3) expressed by all isolates. A large accessory genome, consisting of 45% of the total genes, was also defined. We classified genetic and genomic polymorphism at both chromosomal and individual gene scales. Proteins required for essential functions including virulence had lower-than average sequence variability amongst core genes. Both core and accessory genomes encoded many small, secreted candidate effector proteins that likely interact with plant immunity. Viral vector-mediated transient in planta overexpression of 88 candidates failed to identify any which induced leaf necrosis characteristic of disease. However, functional complementation of a non-pathogenic deletion mutant lacking five core genes demonstrated that full virulence was restored by re-introduction of the single gene exhibiting least sequence polymorphism and highest expression. Conclusions: These data support the combined use of pangenomics and transcriptomics for defining genes which represent core, and potentially exploitable, weaknesses in rapidly evolving pathogens. [ABSTRACT FROM AUTHOR]

Background: The soil dwelling saprotrophic non-pathogenic fungus Fusarium venenatum, routinely used in the commercial fermentation industry, is phylogenetically closely related to the globally important cereal and non-cereal infecting pathogen F. graminearum. This study aimed to sequence, assemble and annotate the F. venenatum (strain A3/5) genome, and compare this genome with F. graminearum. Results: Using shotgun sequencing, a 38,660,329 bp F. venenatum genome was assembled into four chromosomes, and a 78,618 bp mitochondrial genome. In comparison to F. graminearum, the predicted gene count of 13,946 was slightly lower. The F. venenatum centromeres were found to be 25% smaller compared to F. graminearum. Chromosome length was 2.8% greater in F. venenatum, primarily due to an increased abundance of repetitive elements and transposons, but not transposon diversity. On chromosome 3 a major sequence rearrangement was found, but its overall gene content was relatively unchanged. Unlike homothallic F. graminearum, heterothallic F. venenatum possessed the MAT1–1 type locus, but lacked the MAT1–2 locus. The F. venenatum genome has the type A trichothecene mycotoxin TRI5 cluster, whereas F. graminearum has type B. From the F. venenatum gene set, 786 predicted proteins were species-specific versus NCBI. The annotated F. venenatum genome was predicted to possess more genes coding for hydrolytic enzymes and species-specific genes involved in the breakdown of polysaccharides than F. graminearum. Comparison of the two genomes reduced the previously defined F. graminearum-specific gene set from 741 to 692 genes. A comparison of the F. graminearum versus F. venenatum proteomes identified 15 putative secondary metabolite gene clusters (SMC), 109 secreted proteins and 38 candidate effectors not found in F. venenatum. Five of the 15 F. graminearum-specific SMCs that were either absent or highly divergent in the F. venenatum genome showed increased in planta expression. In addition, two predicted F. graminearum transcription factors previously shown to be required for fungal virulence on wheat plants were absent or exhibited high sequence divergence. Conclusions: This study identifies differences between the F. venenatum and F. graminearum genomes that may contribute to contrasting lifestyles, and highlights the repertoire of F. graminearum-specific candidate genes and SMCs potentially required for pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Accurate genome assembly and gene model annotation are critical for comparative species and gene functional analyses. Here we present the completed genome sequence and annotation of the reference strain PH-1 of Fusarium graminearum, the causal agent of head scab disease of small grain cereals which threatens global food security. Completion was achieved by combining (a) the BROAD Sanger sequenced draft, with (b) the gene predictions from Munich Information Services for Protein Sequences (MIPS) v3.2, with (c) de novo whole-genome shotgun re-sequencing, (d) re-annotation of the gene models using RNA-seq evidence and Fgenesh, Snap, GeneMark and Augustus prediction algorithms, followed by (e) manual curation. Results: We have comprehensively completed the genomic 36,563,796 bp sequence by replacing unknown bases, placing supercontigs within their correct loci, correcting assembly errors, and inserting new sequences which include for the first time complete AT rich sequences such as centromere sequences, subtelomeric regions and the telomeres. Each of the four F. graminearium chromosomes was found to be submetacentric with respect to centromere positioning. The position of a potential neocentromere was also defined. A preferentially higher frequency of genetic recombination was observed at the end of the longer arm of each chromosome. Within the genome 1529 gene models have been modified and 412 new gene models predicted, with a total gene call of 14,164. The re-annotation impacts upon 69 entries held within the Pathogen-Host Interactions database (PHI-base) which stores information on genes for which mutant phenotypes in pathogen-host interactions have been experimentally tested, of which 59 are putative transcription factors, 8 kinases, 1 ATP citrate lyase (ACL1), and 1 syntaxin-like SNARE gene (GzSYN1). Although the completed F. graminearum contains very few transposon sequences, a previously unrecognised and potentially active gypsy-type long-terminal-repeat (LTR) retrotransposon was identified. In addition, each of the sub-telomeres and centromeres contained either a LTR or MarCry-1_FO element. The full content of the proposed ancient chromosome fusion sites has also been revealed and investigated. Regions with high recombination previously noted to be rich in secretome encoding genes were also found to be rich in tRNA sequences. This study has identified 741 F. graminearum species specific genes and provides the first complete genome assembly for a Sordariomycetes species. Conclusions: This fully completed F. graminearum PH-1 genome and manually curated annotation, available at Ensembl Fungi, provides the optimum resource to perform interspecies comparative analyses and gene function studies. [ABSTRACT FROM AUTHOR]

Background: Accurate genome assembly and gene model annotation are critical for comparative species and gene functional analyses. Here we present the completed genome sequence and annotation of the reference strain PH-1 of Fusarium graminearum, the causal agent of head scab disease of small grain cereals which threatens global food security. Completion was achieved by combining (a) the BROAD Sanger sequenced draft, with (b) the gene predictions from Munich Information Services for Protein Sequences (MIPS) v3.2, with (c) de novo whole-genome shotgun re-sequencing, (d) re-annotation of the gene models using RNA-seq evidence and Fgenesh, Snap, GeneMark and Augustus prediction algorithms, followed by (e) manual curation. Results: We have comprehensively completed the genomic 36,563,796 bp sequence by replacing unknown bases, placing supercontigs within their correct loci, correcting assembly errors, and inserting new sequences which include for the first time complete AT rich sequences such as centromere sequences, subtelomeric regions and the telomeres. Each of the four F. graminearium chromosomes was found to be submetacentric with respect to centromere positioning. The position of a potential neocentromere was also defined. A preferentially higher frequency of genetic recombination was observed at the end of the longer arm of each chromosome. Within the genome 1529 gene models have been modified and 412 new gene models predicted, with a total gene call of 14,164. The re-annotation impacts upon 69 entries held within the Pathogen-Host Interactions database (PHI-base) which stores information on genes for which mutant phenotypes in pathogen-host interactions have been experimentally tested, of which 59 are putative transcription factors, 8 kinases, 1 ATP citrate lyase (ACL1), and 1 syntaxin-like SNARE gene (GzSYN1). Although the completed F. graminearum contains very few transposon sequences, a previously unrecognised and potentially active gypsy-type long-terminal-repeat (LTR) retrotransposon was identified. In addition, each of the sub-telomeres and centromeres contained either a LTR or MarCry-1_FO element. The full content of the proposed ancient chromosome fusion sites has also been revealed and investigated. Regions with high recombination previously noted to be rich in secretome encoding genes were also found to be rich in tRNA sequences. This study has identified 741 F. graminearum species specific genes and provides the first complete genome assembly for a Sordariomycetes species. Conclusions: This fully completed F. graminearum PH-1 genome and manually curated annotation, available at Ensembl Fungi, provides the optimum resource to perform interspecies comparative analyses and gene function studies. [ABSTRACT FROM AUTHOR]

Background: Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes. Results: We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations. Conclusions: This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants. [ABSTRACT FROM AUTHOR]

Background: Research into psychotherapy for binge-eating disorder (BED) has focused mainly on cognitive behavioural therapies, but efficacy, failure to abstain, and dropout rates continue to be problematic. The experience of negative emotions is among the most accurate predictors for the occurrence of binge eating episodes in BED, suggesting benefits to exploring psychological treatments with a more specific focus on the role of emotion. The present study aimed to explore the feasibility of individual emotion-focused therapy (EFT) as a treatment for BED by examining the outcomes of a pilot randomised wait-list controlled trial. Methods: Twenty-one participants were assessed using a variety of feasibility measures relating to recruitment, credibility and expectancy, therapy retention, objective binge episodes and days, and binge eating psychopathology outcomes. The treatment consisted of 12 weekly one-hour sessions of EFT for maladaptive emotions over 3 months. A mixed model approach was utilised with one between effect (group) using a one-way analysis of variance (ANOVA) to test the hypothesis that participants immediately receiving the EFT treatment would demonstrate a greater degree of improvement on outcomes relating to objective binge episodes and days, and binge eating psychopathology, compared to participants on the EFT wait-list; and one within effect (time) using a repeated-measures ANOVA to test the hypothesis that participation in the EFT intervention would result in significant improvements in outcome measures from pre to post-therapy and then maintained at follow-up. Results: Recruitment, credibility and expectancy, therapy retention outcomes indicated EFT is a feasible treatment for BED. Further, participants receiving EFT demonstrated a greater degree of improvement in objective binge episodes and days, and binge eating psychopathology compared to EFT wait-list control group participants. When participants in the EFT wait-list control group then received treatment and outcomes data were combined with participants who initially received the treatment, EFT demonstrated significant improvement in objective binge episodes and days, and binge eating psychopathology for the entire sample. Conclusions: These findings provide further preliminary evidence for the feasibility of individual EFT for BED and support more extensive randomised control trials to assess efficacy. Trial registration: The study was retrospectively registered with the Australian New Zealand Clinical Trials Registry (ACTRN12620000563965) on 14 May 2020. [ABSTRACT FROM AUTHOR]

Background: Free asparagine is the precursor for acrylamide formation during cooking and processing of grains, tubers, beans and other crop products. In wheat grain, free asparagine, free glutamine and total free amino acids accumulate to high levels in response to sulphur deficiency. In this study, RNA-seq data were acquired for the embryo and endosperm of two genotypes of bread wheat, Spark and SR3, growing under conditions of sulphur sufficiency and deficiency, and sampled at 14 and 21 days post anthesis (dpa). The aim was to provide new knowledge and understanding of the genetic control of asparagine accumulation and breakdown in wheat grain. Results: There were clear differences in gene expression patterns between the genotypes. Sulphur responses were greater at 21 dpa than 14 dpa, and more evident in SR3 than Spark. TaASN2 was the most highly expressed asparagine synthetase gene in the grain, with expression in the embryo much higher than in the endosperm, and higher in Spark than SR3 during early development. There was a trend for genes encoding enzymes of nitrogen assimilation to be more highly expressed in Spark than SR3 when sulphur was supplied. TaASN2 expression in the embryo of SR3 increased in response to sulphur deficiency at 21 dpa, although this was not observed in Spark. This increase in TaASN2 expression was accompanied by an increase in glutamine synthetase gene expression and a decrease in asparaginase gene expression. Asparagine synthetase and asparaginase gene expression in the endosperm responded in the opposite way. Genes encoding regulatory protein kinases, SnRK1 and GCN2, both implicated in regulating asparagine synthetase gene expression, also responded to sulphur deficiency. Genes encoding bZIP transcription factors, including Opaque2/bZIP9, SPA/bZIP25 and BLZ1/OHP1/bZIP63, all of which contain SnRK1 target sites, were also expressed. Homeologues of many genes showed differential expression patterns and responses, including TaASN2. Conclusions: Data on the genetic control of free asparagine accumulation in wheat grain and its response to sulphur supply showed grain asparagine levels to be determined in the embryo, and identified genes encoding signalling and metabolic proteins involved in asparagine metabolism that respond to sulphur availability. [ABSTRACT FROM AUTHOR]