Bilateral BBSRC-Embrapa: Using disease risk forecasting, NGS and HIGS to explore and control Fusarium Head Blight disease in wheat fields

This is a highly innovative and truly ground breaking research project devised by scientists based at Embrapa Wheat, Soybean, Genetic Resources and Biotechnology and Bioinformatics in Brazil and Rothamsted Research in the UK. The problem to be addressed is the control of a fungal disease called Fusarium head blight (FHB) which is one of the most serious and hazardous crop diseases worldwide. The main consequence of FHB is that trichothecene mycotoxins, such as deoxynivalenol (DON), accumulate in the grain, presenting a health risk to humans and animals. In Southern Brazil, where 90% of Brazilian wheat is grown, severe FHB epidemic years occur at a minimum of every 4 or 5 years. Legal limits have been set on the DON levels permitted in harvested grain used for different purposes. However, even moderate FHB years are highly problematic causing the lack of available safe grain for use either on farms or for sale into the market. For low income Brazilian farmers, FHB disease reduces the standards of living of farmer's families and that of their local communities. There is a pressing need to develop novel and effective FHB control options.
In this project, we intend to take a novel whole fungal genome and disease modelling guided approach to develop a pipeline of genetically modified wheat genotypes harbouring T-DNA constructs, which can silence Fusarium genes critical for wheat infection via host-induced gene silencing (HIGS). We also intend to determine the plant and fungal mechanisms that control the HIGS phenomenon. HIGS could be used to control multiple pathogens.
This project has six main research steps.
1. To explore using next generation sequencing the genomes of the five FHB causing species in Southern Brazil. Define the core and species-specific proteome of the FHB species complex (FGSC) for the development of molecular diagnostic tools and the selection of HIGS targets. Monitor for possible genome alterations over 3 years.
2. To enhance FHB disease risk forecasting, by establishing a spore sampler network that can detect and quantify Fusarium species. Devise and use diagnostic assays to identify and monitor FGSC diversity. Sample atmospheric Fusarium spores to identify potential inoculum sources and population structure. Incorporate data on the dynamics of airborne Fusarium spore populations into the existing regional FHB risk model.
3. To develop various T-DNA based constructs to silence Fusarium gene expression by HIGS, thereby controlling Fusarium infections. Evaluate single gene HIGS constructs using a novel cut wheat tillers (transient assay) and via stable transformation into Arabidopsis or lettuce. Two lead HIGS constructs targeting multiple Fusarium genes will be transform into a moderately FHB resistance Brazilian wheat cultivar. In the resulting transgenic plant populations, FHB severity and DON levels will be quantified.
4. To explore the underlying mechanisms of HIGS three cutting edge experiments will be completed. We will establish if long or short RNA molecules move between fungal and wheat cells, determine if the gene silencing phenomena once initiated operates systemically and investigate if transported RNA molecules are cargoed using vesicular transport to the plant cell surface for delivery into fungal cells.
5. To perform two years of GM wheat trial in two locations in Brazil using the four best HIGS lines and three appropriate control lines. Assess in-field FHB symptoms, airborne inoculum, grain quality, DON contamination and fungal spore production. To collect Fusarium isolates able to cause any disease on the HIGS lines and complete a full genome analyse.
6. The research team will engage with farmers, farmer co-operatives, grain purchasers, plant breeders in Brazil and the UK and academics globally to explain the project, the research findings and discuss ways to implement the new modelling/forecasting technologies, to use the novel GM trait and further understand the HIGS phenomenon.

WP1 Illumina and PacBio sequencing and bespoke bioinformatics pipelines used to generate, assemble and annotate the genomes of five Fusarium species, and identify the sequence variations (SNPS, Indels). Reciprocal BLAST and OrthoMCL analyses used to define the core/ species specific Fusarium proteomes and eliminate Fusarium sequences present in non-target organisms.
WP2 Two-arm Rotorod spore sampler mounted on ground vehicles and unmanned aerial vehicle used to sample fungal spore at different altitudes/ locations. Selective culture medium used to culture Fusarium isolates for molecular diagnostics using newly devised LAMP or TwistDX assays. A ground network of spore samplers will be established across Southern Brazil in association with automatic weather stations. Current Bayesian modelling framework improved by combining inferences from different crop/ disease/weather cycle processes.
WP3 Functional cut wheat tiller feeding bioassay used to evaluate 30 single HIGS gene constructs using a GFP expressing F. graminearum (Fg) strain. Ten single Fg gene silencing constructs tested by stably transforming Arabidopsis and lettuce, and evaluating disease symptoms on leaves in T2/T3 plants. Five lead Fg genes used to make 2 HIGS constructs targeting different 2 or 3 Fg gene combinations. Stably transform by co-bombardment into wheat cultivar Guamirim. FHB control evaluated from the T1 generation onwards.
WP4 Dicer and Argonaute mutants in Fg and Arabidopsis used to establish if long dsRNA or siRNA moves trans-kingdom. Systemic travel of silencing signal tested using Fg resistant Arabidopsis HIGS line either in wild-type or dicer mutant backgrounds and reciprocal scion and stock grafting.
WP5. Replicated GM field trials done over two years in two locations in Brazil using the four best HIGS lines and three control lines. Assess FHB symptoms, grain quality, DON contamination and fungal spore production. Full genome analyse of isolates causing disease on the HIGS lines.

This project will deliver fundamental research impacts as well as social, economic and political impacts. There is a pressing need to develop novel, effective Fusarium Head Blight (FHB) management options for wheat crops globally. To effectively control FHB disease in Southern Brazil a new multifaceted approach has been devised by EMBRAPA and UK scientists, which is underpinned by novel knowledge and mechanistic insight.

The project aims to deliver: (1) the completed and annotated genomes of the five Fusarium species causing FHB disease in Southern Brazil, knowledge on gene/genome variation and on emerging FHB causing isolates; (2) a pipeline of Fusarium target genes for intervention via the HIGS technology that will not affect gene expression in wheat, humans or other non-target organisms; (3) a network of fungal spore traps combined with adapted unmanned aerial vehicles to accurately monitor the dynamics of wheat infecting Fusarium populations in Southern Brazil using species-specific diagnostics; (4) an improved Bayesian modelling framework achieved by combining inferences from different crop/ disease/weather cycle processes; (5) a pipeline to test single gene HIGS constructs that can then be multiplexed in various combinations and introduced by stable transformation into wheat in the absence of any vector sequences; (6) an increased understanding of Fusarium and host genes and pathways controlling the trans-kingdom gene silencing phenomenon in several plant species and (7) an established and validated GM wheat field trials system in two locations in Brazil that can be used to evaluate HIGS constructs to control FHB disease, lower mycotoxin levels, improve grain quality, reduce Fusarium spore production and assess whether the composition of the natural Fusarium complex has altered in the presence of each HIGS construct.

The partnership has identified the key beneficiaries for each of the five research objectives. These key beneficiaries include academics in Brazil, the UK and globally; farmers, farmer co-operatives, farm advisors, grain purchasers, grain processers, grain importers / exporters, supply chain managers in Brazil and the UK; multi-national Agri-Tech industry and small-medium enterprises in South America, other EMBRAPA crop institutes which focus on other fungal induced biotic stresses, the wheat breeders in Passo Fundo, the Brazilian and UK governments and Brazilian society. Low income farmers in Brazil should greatly benefit from the new multifaceted approach to be implemented. By involving three Brazilian Universities in the project, the training of next generation of Brazilian scientists will be greatly enhanced.

Various impact activities have been designed to maximise this project's outputs through engagement across the three years. Some public engagement and/or press release activities envisaged will require further training of the more junior research team members. The various impact activities to be completed include developing and maintaining a project website within the RRes domain linked to the FHB forecasting system hosted in Brazil, a regular review of intellectual property and invention disclosures (note - a humanitarian clause targeting the poorest developing countries will be included in all patent applications to ensure free use of the new technology by those countries in greatest need), the quarterly release of completed Fusarium genome datasets, visits to and continuing engagement with Brazilian farming co-operatives, a project dissemination workshop in Brazil, presenting project findings at Agri-Tech events such as Cereals in the UK and Showrural and Expodireto in Brazil, a targeted seminar to industry in the UK, attending and presenting research findings at national and international conferences, publishing high impact articles available immediately via open access, preparing press release materials, and helping to train and enthuse the next generation of scientists