Exploring the diversity of promoter and 5’UTR sequences in ancestral, historic and modern wheat

A dataset of promoter and 5’UTR sequences of homoeo-alleles of 495 wheat genes that contribute to agriculturally important traits in 95 ancestral and commercial wheat cultivars is presented here. The high stringency myBaits technology used made individual capture of homoeo-allele promoters possible, which is reported here for the first time. Promoters of most genes are remarkably conserved across the 82 hexaploid cultivars used with <7 haplotypes per promoter and 21% being identical to the reference Chinese Spring. InDels and many high-confidence SNPs are located within predicted plant transcription factor binding sites, potentially changing gene expression. Most haplotypes found in the Watkins landraces and a few haplotypes found in T. monococcum, germplasms hitherto not thought to have been used in modern wheat breeding, are already found in many commercial hexaploid wheats. The full dataset which is useful for genomic and gene function studies and wheat breeding is available at https://rrescloud.rothamsted.ac.uk/index.php/s/3vc9QopcqYEbIUs/authenticate

Proteinaceous effector discovery and characterisation in filamentous plant pathogens

The complicated interplay of plant-pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens which produce small proteins called effectors to manipulate the host and aid infection/colonisation. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterisation of the effectors of filamentous plant pathogens and identification of their host targets

Apoplastic and vascular defences

The apoplast comprises the intercellular space between cell membranes, includes the xylem, and extends to the rhizoplane and the outer surfaces of the plant. The apoplast plays roles in different biological processes including plant immunity. This highly specialised space is often the first place where pathogen recognition occurs, and this then triggers the immune response. The immune response in the apoplast involves different mechanisms that restrict pathogen infection. Among these responses, secretion of different molecules like proteases, proteins related to immunity, small RNAs and secondary metabolites play important and often additive or synergistic roles. In addition, production of reactive oxygen species occurs to cause direct deleterious effects on the pathogen as well as reinforce the plant’s immune response by triggering modifications to cell wall composition and providing additional defence signalling capabilities. The pool of available sugar in the apoplast also plays a role in immunity. These sugars can be manipulated by both interactors, pathogens gaining access to nutrients whilst the plant’s responses restrict the pathogen’s access to nutrients. In this review, we describe the latest findings in the field to highlight the importance of the apoplast in plant – pathogen interactions and plant immunity. We also indicate where new discoveries are needed

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue

The Fusarium graminearum MAP1 gene is essential for pathogenicity and development of perithecia

Fusarium, graminearum is the causal agent of ear blight disease of cereals. Infection occurs at anthesis when ascospores and/or conidia directly penetrate exposed anther and ovary tissue. The hemibiotrophic hyphae colonize floral tissues and developing grains to cause premature ear senescence. During infection, Fusarium hyphae can also produce hazardous trichothecene mycotoxins, thereby posing a threat to human and animal health and safety. The Fusarium MAP1 gene was identified using a PCR approach by its homology to a known pathogenicity gene of Magnaporthe grisea, the mitogen-activated protein kinase gene PMK1. Gene replacement F. graminearum map1 mutants were non-pathogenic on wheat flowers and roots, and also could not infect wounded wheat floral tissue or tomato fruits. Unlike the wild-type strain, map1 mutant inoculations did not compromise grain yield. Map1 mutants lost their ability to form perithecia in vitro, but their rate of asexual conidiation was unaffected. DON mycotoxin production in planta was still detected. Collectively, the observed phenotypes suggest that the Map1 signalling protein controls multiple events in disease establishment and propagation. Novel approaches to control Fusarium ear blight disease by blocking perithecial development are discussed

Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users

The completed genome sequence of the pathogenic ascomycete fungus Fusarium graminearum

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