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

Dissecting the molecular interactions between wheat and the fungal pathogen Zymoseptoria tritici

The Dothideomycete fungus Zymoseptoria tritici (previously known as Mycosphaerella graminicola and Septoria tritici) is the causative agent of Septoria tritici leaf blotch (STB) disease of wheat (Triticum aestivum L.). In Europe, STB is the most economically damaging disease of wheat, with an estimated ∼€1 billion per year in fungicide expenditure directed toward its control. Here, an overview of our current understanding of the molecular events that occur during Z. tritici infection of wheat leaves is presented. On the host side, this includes the contribution of (1) the pathogen-associated molecular pattern-triggered immunity (PTI) layer of plant defense, and (2) major Stb loci for resistance against Z. tritici. On the pathogen side of the interaction, we consolidate evidence from recent bioinformatic, transcriptomic and proteomic studies that begin to explain the contribution of Z. tritici effector proteins to the biphasic lifestyle of the fungus. This includes the discovery of chitin-binding proteins in the Z. tritici secretome, which contribute to evasion of immune surveillance by this pathogen, and the possible existence of ‘necrotrophic’ effectors from Z. tritici, which may actively stimulate host recognition in a manner similar to related necrotrophic fungal pathogens. We finish by speculating on how some of these recent fundamental discoveries might be harnessed to help improve resistance to STB in the world’s second largest food crop

Characterisation of major genes mediating resistance to Septoria tritici blotch disease in wheat.

The fungus Zymoseptoria tritici is one of the most destructive wheat (Triticum aestivum) pathogens in Europe and worldwide, causing crop losses of up to 50% in high risk climates (Goodwin, 2007). Traditionally this disease has been controlled with widely used resistance genes and fungicides, but the high selection pressures placed on the fungi result in a serious risk of these protections being overcome, particularly when heavily relied upon. Some major resistances have already been widely broken – for example, the Stb6 resistance gene present in most European wheat cultivars is now ineffective against many Septoria strains in the field. It is therefore important that new, more diverse sources of resistance be identified and utilised in elite wheat lines. These will allow us to prepare for the breaking of currently common resistances but may also extend their lifetimes – Chartrain et al. (2004) found that many wheat lines with highly durable Septoria resistance contain multiple resistance (Stb) genes, suggesting that gene pyramiding may be a viable method for enhancing the longevity of resistances in this pathosystem. The research described here will screen currently known Stb genes against an array of recent Septoria field isolates to identify resistances still effective in the field and potentially interesting combinations of resistances that in combination could provide protection against most or all isolates tested. Such resistances will then be fine mapped using KASP markers to enable breeders to more easily integrate them into elite lines. In light of the recent identification of the Stb6 gene as a wall-associated receptor-like kinase (WAK) (Saintenac et al., 2018), WAK genes in the regions identified will be further investigated using Virus-induced gene silencing to identify individual resistance genes where possible, aiding in further investigations that may establish the methods through which these resistances function

Virus-mediated transient expression techniques enable gene function studies in black-grass

Even though considerable progress has been made in weed ecology, weed molecular biology has been hindered by an inability to genetically manipulate weeds. Genetic manipulation is essential to demonstrate a causative relationship between genotype and phenotype. Herein we demonstrate that virus-mediated transient expression techniques developed for other monocots can be used in black-grass (Alopecurus myosuroides) for loss- and gain-of-function studies. We not only use virus induced gene silencing (VIGS) to create the black-grass exhibiting reduced PHYTOENE DESATURASE expression and virus-mediated overexpression (VOX) to drive GREEN FLUORESCENT PROTEIN, we demonstrate these techniques are applicable to testing hypotheses related to herbicide resistance in black-grass. We use VIGS to demonstrate that AmGSTF1 is necessary for the resistant biotype Peldon to survive fenoxaprop application and show the heterologous expression of the bialaphos resistance gene with VOX is sufficient to confer resistance to an otherwise lethal dose of glufosinate. Black-grass is the most problematic weed for winter-cereal farmers in the UK and Western Europe as it has rapidly evolved adaptions that allow it to effectively avoid current integrated weed management practices. Black-grass also reduces yields and therefore directly threatens food security and productivity. Novel disruptive technologies which mitigate resistance evolution and enable better control over this pernicious weed are therefore required. These virus-mediated protocols offer a step change in our ability to alter genes of interest under controlled laboratory conditions and therefore to gain a molecular-level understanding of how black-grass can survive in the agri-environment

A large bioassay identifies Stb resistance genes that provide broad resistance against Septoria tritici blotch Disease in the UK

Septoria tritici blotch (STB) is one of the most damaging fungal diseases of wheat in Europe, largely due to the paucity of effective resistance genes against it in breeding materials. Currently dominant protection methods against this disease, e.g. fungicides and the disease resistance genes already deployed, are losing their effectiveness. Therefore, it is vital that other available disease resistance sources are identified, understood and deployed in a manner that maximises their effectiveness and durability. In this study, we assessed wheat genotypes containing nineteen known major STB resistance genes (Stb1 through to Stb19) or combinations thereof against a broad panel of 93 UK Zymoseptoria tritici isolates. Five infection symptom components (days post infection to the development of first symptoms and pycnidia, percentage coverage of the infected leaf area with chlorosis/necrosis and pycnidia and spore counts from spore wash) were measured and average disease levels calculated for each genotype. The different Stb genes were found to vary greatly in the levels of protection they provided, with no Z. tritici isolate found to be virulent against all tested resistance genes. Disease resistance controlled by different Stb genes was associated with different levels of chlorosis, with high levels of early chlorosis in some genotypes correlated with high resistance to fungal pycnidia development. Stb10, Stb11, Stb12, Stb16q, Stb17, and Stb19 were identified as contributing broad spectrum disease resistance, and synthetic hexaploid wheat lines were identified as particularly promising sources of broadly effective STB resistances. Wheat genotypes carrying multiple Stb genes were found to provide higher levels of resistance than expected given their historical levels of use. The knowledge obtained here will aid UK breeders in prioritising Stb genes for future breeding programmes. In addition, this study identified the most interesting Stb genes for cloning and detailed functional analysis

Efficient CRISPR/Cas-Mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties

CRISPR/Cas technology has recently become the molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. Primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. While BAK1 is an important regulator of plant immunity and development, Ta-eIF4E and Ta-eIF(iso)4E act as susceptibility (S) factors required for plant viruses from the Potyviridae family to complete their life cycle. We anticipate the resultant homozygous tabak1-2 mutant lines will facilitate studies on the involvement of BAK1 in immune responses in wheat, while ta-eif4e and ta-eif(iso)4e mutant lines have the potential to become a source of resistance to wheat spindle streak mosaic virus (WSSMV) and wheat yellow mosaic virus (WYMV), both of which are important pathogens of wheat. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop