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

Lead Research Organisation: Rothamsted Research
Department Name: Biointeractions and Crop Protection

Abstract

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.

Technical Summary

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.

Planned Impact

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

Publications

10 25 50
 
Title A collection of postcards 
Description A collection of postcard highlighting the importance of Fusarium and the research on-going at Rothamsted were prepared for the Fusarium one-day event held at Rothamsted in July 2016. These postcards were disseminated to the wider public and key stakeholders through this event and follow-on activities. 
Type Of Art Artwork 
Year Produced 2016 
Impact These postcards have made the Fusarium disease problems on crop plants and the subsequent detrimental affects on food and feed industries farm far more accessible to the general public, potiticians and the Agri-Industry. 
 
Title Effector discover timeline, HIGS, BSMV-VOX and protein-protein interactions 
Description The VCU team at Rothamsted Research was commissioned to provide the following artwork to improve knowledge transfer to less specialist audiences - A timeline showing the progression of filamentous plant pathogen effector prediction and identification from the pregenomic era to the present day. - A simplified version of how host-induced gene silencing (HIGS) is thought works in plants - A simplified version of how the transient Barley Stripe Mosaic Virus - protein overexpression - BSMV-VOX technology works in plants - A simplified version of three different protein-protein interaction techniques. This artwork has already appeared in the review article by Kanja and Hammond-Kosack (2020) Proteinaceous effector discovery and characterization in filamentous plant pathogens. Molecular Plant Pathology 21, 1353-1376 
Type Of Art Artwork 
Year Produced 2020 
Impact This artwork will be used in future scientific outreach activities to non-specialist audiences . 
URL https://doi.org/10.1111/mpp.12980
 
Title Fusarium and cereal cartoons 
Description A Fusarium cartoon was produced that portrayed the risk of Fusarium-borne diseases to cereals and the problems associated with current control strategies. The cartoon progresses to explain new GM and non-GM mediated approaches to stop Fusarium. In addition, the cartoon depicts the mechanism behind how host-induced gene silencing can be used to combat fungal disease, in a simplified manner assessable to the general public. This cartoon has now been translated into Portuguese and is to be used by Embrapa-Trigo at a national agricultural show in March 2017. 
Type Of Art Artwork 
Year Produced 2016 
Impact The cartoon was used at the Fusarium event for the general public in 2016 and at the Cereals 2016 event. It has helped the general public and the Agri-Industry understand the huge Fusarium disease problem globally and the potential for using Host induced gene silencing (HIGS) for disease control. 
URL http://www.rothamsted.ac.uk/system/files/How%20to%20sustainably%20control%20Fusarium.pdf
 
Title Fusarium science cartoons and infographics text 
Description An eight page infographic text with ten cartoons was devised to explain the Fusarium disease risk in cereal production, how 10 years of related Fusarium - Arabidopsis research had lead to several major scientific breakthroughs as well as identify new ways to control this serious disease problem 
Type Of Art Artwork 
Year Produced 2015 
Impact The cartoons and other images devised have been used in various scientific outreach events including Cereal in 2016 and the Fusarium event in 2016. 
 
Description 2018 entry
The moderately FHB resistant wheat cultivar Guarmirin has been successfully stably transformed with two different Host Induced Gene Silencing (HIGS) constructs using Agrobacterium mediated transformation. The sequencing of the full genomes of 24 Brazilian fusarium isolates from 5 species which cause FHB disease has been completed. The 16 F. graminearum genomes have been annotated and inter-compared with the seven other Fg genomes available for other global isolates to create the pan-genome.

2019 entry
- The pan-genome of Fusarium graminearum has been thoroughly investigated and completed using full genome sequence information from 24 isolates sequenced (16 Brazilian, 8 North American). The pangenome is predicted to contain 14,798 genes, whilst the core genome contains 13,000 genes, i.e. genes found in all isolates. In total, 653 genes were found in one or more other strains but not in the global reference PH-1 genome. The secretome is predicted to be encoded by 902 genes, of which 784 genes represent the core. Within the predicted effector subset, 161 genes were assigned to the core and 35 to the variable part of the genome. Also predicted in the pan-genome was the presence of 9 new secondary metabolites cluster not found in the PH-1 genome.

- Using two different Arabidopsis leaf infection bioassays, we have revealed that the vesicle trafficking gene Min7 when mutated in two different ways leads to dramatically enhanced susceptibility to Fusarium graminearum. Whereas the independent mutation of 12 other vesicle trafficking genes did not alter the interaction outcome. Min7 is an immunity-associated vesicle traffic regulator which was previous only known to be targeted by the bacterial effector HopM1. Several different cellular mechanisms are proposed to be controlled by MIN7 including plant plasma membrane integrity, extracellular (apoplastic) water content levels and the cargoeing of various metabolites and molecules to the plant cell surface. The wheat homologue of Min7 has been identified and BSMV-VIGS constructs have been produced to silence A, B and D genome MIN7 homoeologues in wheat and test for altered susceptibility to Fusarium is underway.

- Additional transgenic wheat HIGS lines were generated in the cultivar Bobwhite by co-bombardment. In total, 50 independent lines harbour the chitin synthetase RNAi silencing construct and 36 independent lines harbour the cytochrome P450 sterol 14a-demethylase/ Tri6 /Tri5 RNAi silencing construct have been generated. Transformation lines harbouring just the antibiotic selection gene were also selected. Gene copy numbers in the T0 generation ranged from zero to ~35, with a high proportion harbouring < 5 copies. T1 plants from each line have been tested using a newly devised high throughput wheat hypocotyl - F. graminearum infection bioassay. A wide range of disease outcomes were observed. The most promising reduced disease severity lines with low to moderate gene copy numbers have been selected for re-testing in the T2 generation.

- In July 2018 a visit was made to the EMBRAPA Trigo in Brazil, to discuss the forthcoming GM field trial. A trial site was chosen close by the main buildings which will permit the use of irrigation if required.

- The permits to export GM Arabidopsis and wheat seeds from the UK to Brazil are now in place. This took a full year to complete.

- 35S promoter driven chitin synthetase RNAi silencing construct and cytochrome P450 sterol 14a-demethylase/ Tri6 /Tri5 RNAi silencing constructs were transformed into Arabidopsis Col-0 and T3 homozygous plants testing for responses to F. graminearum infection commenced in October 2018 and is still underway using the leaf bioassay.

- Ten F. graminearum genes either required either for virulence or considered to be essential for life were carefully selected. HIGS constructs were generated by direct DNA synthesised and transformed into Arabidopsis Columbia erecta. A newly available seed coat specific dsRED T-DNA vector was used to simplify the recovery of the primary transformants. Testing of the T2 generation commenced in December 2018 using the leaf bioassay and replicated experiments are still ongoing.

2020 entry
- Evaluating T1, T2 and homozygous T3 wheat HIGS lines for resistance to Fusarium
Transgenic wheat HIGS lines generated in the cultivar Bobwhite were inoculated with F. graminearum using the coleoptile assay. A total of 41 independent lines harbouring the chitin synthetase (Chs) RNAi silencing construct and 31 independent lines harbouring the cytochrome P450 sterol 14a-demethylase/ Tri6 /Tri5 (Cyp) RNAi silencing construct were tested in the T1 generation. Twenty-one T1 lines (nine Chs and 12 Cyp) showed reduced F. graminearum lesions in wheat coleoptile bioassay. The presence of the transgene was checked for all selected lines by PCR. T2 seeds from selected T1 lines were harvested and homozygous lines were selected in the T2 generation. In the subsequent generation, twelve homozygous T3 lines (6 for Chs and 6 for Cyp) were then tested by point inoculating F. graminearum spores into floral tissue. Although two lines showed reduced F. graminearum infection in the first experiment, the same phenotype was not seen in the replicated experiment. Therefore, at the time of writing we have no direct evidence that HIGS could be used effectively for controlling Fusarium infections in wheat. A small RNA-seq experiment and small RNA Northern blot analyses will be carried out next with these homozygous lines to confirm the presence of small RNA.

- Arabidopsis results
Transgenic Arabidopsis Col-0 carrying 35S promoter driven chitin synthetase RNAi silencing construct and cytochrome P450 sterol 14a-demethylase/ Tri6 /Tri5 RNAi silencing constructs were tested. A total of 11 T3 homozygous lines (4 Cyp/Tri, 4 Chs and 3 Empty vector) were selected and tested using the detached leaf assay. Each line was tested at least twice. Results revealed no differences between the levels of F. graminearum infections in either the HIGS lines or the controls. In addition, six constructs were generated using a vector backbone previously published as efficient in generating siRNA. Each construct targeted a single gene either important for Fusarium virulence or considered to be 'essential for life'. This new series of HIGS lines were generated by transforming with the dsRED-expressing T-DNA vector, which labels the seed testa with DsRED. Using this approach, the selection of the original transformants and the subsequent homozygous T3 lines had been very straightforward with the aid of a UV lamp. A total of 55 T2 independent lines were tested using the detached leaf assay. Seven lines showed some reduction in F. graminearum infection in the preliminary screen, and therefore these infection assays were repeated two more times. However, in these follow up experiments when the proportions of infected leaf area were compared, no difference were detected between control WT and the various HIGS lines. Fusarium infected leaves of some of the tested lines were collected to detect expression of the target gene. No significant difference in gene expression was detected between control and HIGS lines, indicating absence of efficient silencing of target genes.

- In vitro RNA uptake results
To directly assess the uptake of environmental dsRNA by F. graminearum, germinating spores were co-incubated with FAM-labelled 250 nt long dsRNA derived from a GFP gene for 24 hours. The necrotrophic fungus Botrytis cinerea was used as a positive control. Germinated spores were monitored using fluorescent microscopy. Fluorescent signals were readily detected in the germinating B. cinerea spores. However, fluorescent signals were rarely detected in germinated F. graminearum conidiospores. Propidium iodide (PI), a cell viability dye, was used to assess the viability of F. graminearum conidia and spore germlings co-incubated with FAM-labelled dsRNA. PI is only taken up by dead cells with damaged plasma membranes and leads to cells fluorescing red. Most of the F. graminearum germinated conidia that fluoresced green due to the FAM-labelled dsRNA uptake were stained with PI, indicating that these conidia were dead. This indicates that the small amount of dsRNA uptake present in F. graminearum germlings and conidia was due to a passive uptake via the damaged plasma membrane.

- Conclusion
Collectively, these results from the wheat and Arabidopsis HIGS, and in vitro dsRNA uptake experiments provide minimal evidence that host induced gene silencing is working efficiently to control Fusarium infections in plants. Our results do not match the results for the identical gene targets published in other peer reviewed publications. The precise reasons for this are unknown and need to be investigated, but it is possible that some Fusarium isolates may be particularly susceptible to HIGS. Either way this raises questions about the general utility of HIGS or spray-induced gene silencing for control of natural Fusarium infections under field conditions. These new negative results will be presented to and discussed with the UK and international plant pathology community in 2020 at research conferences and also submitted for peer review as a research publication.
Exploitation Route Transformation of a commercial Brazilian wheat cultivar opens up for the first time the possibility to transform additional traits into wheat for testing under commercial agronomic conditions in various locations in Brazil.

The F. graminearum pan-genome developed for Brazil ill be very useful in future global pan-genome analyses for this and closely related Fusarium species able to cause FHB disease. The diagnostics industry sector will find these data sets particularly useful.

2021 entry
Our overall vision was to develop a novel way to control Fusarium Head blight (FHB) disease in wheat fields in Southern Brazil. However, based on all the negative HIGS results obtained in both the UK and Brazil, it is highly unlikely this initial aspiration will be possible to fulfil via the use of this HIGS approach.

However, several other large data set and new knowledge generated in this project can be taken forward and put to use by others

The genome sequence for the various Fusarium species causing FHB disease on wheat in Brazil, will be exceptionally useful for the development of Fusarium species specific PCR based diagnostic tests.

The genome sequence for 16 Fusarium graminearum strains from Brazil has been a major contribution to the development of a global pan- genome for this species. Manuscript in the advances stages of preparation. As a result, it has been possible to define the core and variable parts of the predicted proteome. Therefore control measures developed in the future should focus on targeting and disrupting the function of the core proteome.
Sectors Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Education,Environment

 
Description In 2017 / 2018 the team wrote a comprehensive review : Machado, A. K., Brown, N. A., Urban, M., Kanyuka, K., Hammond-Kosack, K.E. (2018) RNAi as an emerging approach to control Fusarium Head Blight disease and mycotoxin contamination in cereals Pest Management Science 74:790-799 doi: 10.1002/ps.4748 This review has been very well cited since the article was published in 2018 (17 times - Web of Science and 33 Google Scholar). In addition, several AgTech companies in Europe and the USA have approached the team at Rothamsted to discuss this new technology further with us. These discussions with industry are continuing. 2021 entry The above review has been very well cited since the article was published in 2018 (51 times - Web of Science and 71 Google Scholar). We have interacted with several AgTech companies and they have confirmed to us that they also have only obtained negative data sets when testing the siRNA and HIGS technology against Fusarium graminearum. This is why we are pursuing the publication of our negative data sets from this project . In 2020 we were invited to write a review article on new ways to achieve durable control of FHB disease in cereal crops via genomic and cell biology inspired investigations which was published in 2021. The post-doc hired on this project was a part of the writing team. In the light of our findings from the project, we have therefore primarily devised and discussed other non-HIGs/ non-siRNA ways to achieve FHB control. This review article is now published Kanja, C., Machado Wood, A.K., Baggaley, L., Walker, C. and Hammond-Kosack K. E. 2021 Cereal-Fusarium interactions: Improved fundamental insights into Fusarium pathogenomics and cereal host resistance reveals new ways to achieve durable disease control. Chapter 5 In 'Achieving durable disease resistance in cereals' Ed R. Oliver, Burleigh Dodds Science Publishing, ISBN-13: 9781786766014
Sector Agriculture, Food and Drink,Education
Impact Types Economic

 
Description BBSRC Institute Strategic Programme Grant
Amount £16,000,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 03/2022
 
Description Bilateral BBSRC-EMBRAPA - Disease risk forecasting, NGS and HIGS
Amount £500,000 (GBP)
Funding ID BB/N018095/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 06/2019
 
Description Development of new tools to detect and quantify five wheat infecting Fusarium species for use in Southern Brazil
Amount £9,965 (GBP)
Funding ID Alliance for Sustainable Agriculture Previously: UK-Brazil Partnership for Yield Stability & Protection in a Changing Climate (PYSP - N8085) 
Organisation Rothamsted Research 
Sector Academic/University
Country United Kingdom
Start 08/2018 
End 03/2019
 
Description The Genetics Society (UK) - Travel award to attend the IS-MPMI Congress Glasgow 14-18 th July 2019
Amount £750 (GBP)
Organisation Rothamsted Research 
Sector Academic/University
Country United Kingdom
Start 06/2019 
End 07/2019
 
Description Understanding the mechanisms underlying Host Induced Gene Silencing (HIGS) in Fusarium graminearum (BSPP) Michael Grimwade-Mann (summer bursary project)
Amount £2,000 (GBP)
Organisation Rothamsted Research 
Sector Academic/University
Country United Kingdom
Start 05/2018 
End 09/2018
 
Title Ensembl Invertebrates, especially Ensembl Fungi, Ensembl Protist and Ensembl bacteria 
Description Ensembl Invertebrates provides annotated genome and a range of simple and advanced query tools to explore the genomes of numerous pathogenic micro-organisms. The genes curated into the Pathogen Host Interactions database (PHI-base), are directly available within the genome browsers of individual species. This data is colour coded to provide the phenotypic outcome from wet biology experimentation and is linked back to the full curated data sets available within PHI-base. The PHI genes can also be searched for within the BioMart Tool across multiple species and by using nine published high level phenotype terms. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact Improved comparative genomic analysis of multiple pathogenic and non-pathogenic species. Hypothesis testing. Providing up-to-date novel functional data into poorly annotated genomes. 
URL https://fungi.ensembl.org/index.html
 
Title PHI-base: Pathogen-Host Interactions Database 
Description PHI-base (www.phi-base.org) is a knowledge database accessed by researchers in over 125 countries. PHI-base contains expertly curated molecular and biological information on genes proven to affect the outcome of pathogen-host interactions reported in peer reviewed research articles. Genes not affecting the disease interaction phenotype are also curated. PHI-base data is linked to the genome browsers and advanced query tools in ENSEMBL and FungiDB. The data content provided comes from >3000 manually curated references and reports information on 6438 genes from 263 pathogens tested on 194 hosts (plant, animal, others) in 11340 interactions. Direct targets of pathogen effector proteins are also included. Recently the PHI-base team in collaboration with the PomBase team based at the University of Cambridge have developed an online author curation tool called PHI-Canto 
Type Of Material Database/Collection of data 
Provided To Others? Yes  
Impact Over 250 peer reviewed publications have cited PHI-base use in their article and cites one or more of the PHI-base references 
URL http://www.phi-base.org
 
Description EMBRAPA Brazil - Bioinformatics Laboratory, Cenargen Brasilia 
Organisation Brazilian Agricultural Research Corporation
Country Brazil 
Sector Public 
PI Contribution The genomes of 16 well-characterised Fusarium graminearum (Fg) (15-ADON) isolates, eight each from Parana and Rio Grande du Sul states, were sequenced by Illumina paired end reads. The highly virulent isolate CML3066, with the best sequence coverage (x180), was nominated as the Brazilian reference isolate. We have subsequently created the pan genome for Fg using this data and an additional six global Fg stains including the global reference strain PH-1 originally from the USA. The focus at Rothamsted was then the characterisation of genes predicted to code for small secreted proteins or predicted to reside with discrete secondary metabolite clusters. The sequence variation in the known Fg pathogenicity and virulence genes documented in the PHI-base database has also been explored. To complement these comparative genome analyses, the relative disease causing ability of the 16 Brazilian isolates compared to the global reference strain has been explored in detail.
Collaborator Contribution The EMBRAPA bioinformatics team have applied their expertise in transmembrane spanning proteins to explore the predicted G-protein coupled receptor, 7 transmembrane spanning protein superfamiliy. This superfamiliy contains > 100 genes and some of there are now know to be required for the disease causing ability of Fg.
Impact Three publications have already arisen from the initial joint genome data analysis on the Fg PH-1 genome, which was done in preparation for the main project.. Bresso, E., Leroux, V., Urban, M., Hammond-Kosack, K.E., Maigret, B.. and Martins, N.F. (2016) Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase, a possible target to reduce Fusarium head blight disease. Journal of Molecular Modeling 22, 1-13. Martins, N.F., Bresso, E., Togawa, R. C., Urban, M., Antoniw, J., Maigret, B. and Hammond-Kosack, K.E. (2016) Searching for novel targets to control wheat head blight disease. I- Protein identification, 3D modeling and virtual screening. Advances in Microbiology 6 (11), 811-830. Doi 10.4236/aim.2016.611079. Bresso, E., Togawa, R. C., Hammond-Kosack, K.E., Urban, M., Maigret, B. and Martins, N.F (2016). GPCRs from Fusarium graminearum, detection, modeling and virtual screening - the search for new routes to control head blight disease. BMC Bioinformatics 17 (18), 39. PMID: 28105916. These joint studies were multi-disciplinary and involved bioinformatics and protein modelling.
Start Year 2013
 
Description EMBRAPA Brazil - Passo fundo - Trigo (wheat) team 
Organisation Embrapa Trigo
Country Brazil 
Sector Private 
PI Contribution The Rothamsted Team has sequenced the genomes of 16 Brazilian strains of the Fusarium head blight disease causing strain F. graminearum (Fg) and has now created a pan -genome for this species by comparing with the available Fg genomes for 6 six additional global isolates including the reference isolate PH-I. This has indicated that the Fg pan genome is relatively closed. The Rothamsted Team has stably transformed the Brazilian commercial wheat cultivar Guaramin for the first time
Collaborator Contribution The EMBRAPA Trigo team have generated various transgenic Arabidopsis harboring different Fg HIGS constructs. The EMBRAPA Team have also screened and identified lettuce cultivars that are fully susceptible to the nominated reference Brazilian strain.
Impact A major display at the annual Cereals event held in Cambridgeshire in June 2016 on the new HIGS and SIGS technologies for the control of FHB disease in wheat . A open evening public event entitled @ Healthy Crops- Healthy Food done at Rothamsted Research in July 2016.
Start Year 2014
 
Description EMBRAPA LabEx Common Interest Project 
Organisation Brazilian Agricultural Research Corporation
Country Brazil 
Sector Public 
PI Contribution Different members of the Rothamsted Research team trained the visiting senior EMBRAPA LabEx scientists in bioinformatics and molecular genetics skills relating to fungal pathogens which infect wheat. The Rothamsted team and especially Professor John Lucas advised on European research labs and companies to visit to obtain relevant new knowledge and techniques and initiate new collaborations for EMBRAPA.
Collaborator Contribution EMBRAPA Brazil established a Common Interest project and an office at Rothamsted. In addition to conducting and publishing original research the visiting Senior Fellow, Dr Alex Amaral established an extensive collaborative network at Rothamsted, throughout the UK and Europe.
Impact Visiting senior fellow Dr Amaral whilst at Rothamsted helped to facilitate the joint BBSRC -EMBRAPA wheat workshop held in Londrinas Brazil in May 2011 which subsequently lead to the joint wheat research pump priming call in 2014/2015 and the subsequent main call
Start Year 2010
 
Description Investigating HIGS and SIGS using the F. graminearum - wheat interaction 
Organisation Justus Liebig University Giessen
Country Germany 
Sector Academic/University 
PI Contribution The Rothamsted Research team has discussed in depth the data and techniques in the Giessen group's various publications at the BSPP autumn conference at the University of Nottingham. The post-doc on the bilateral BBSRC-EMPRAPA project has visited the Giessen group for 2 weeks in late 2017 to be taught the various techniques. The post doc returned with specific F. graminaerum strains and Arabidopsis transgenic lines to establish these HIGS and SIGS techniques at Rothamsted Research during 2018. A series of joint experiments are underway.
Collaborator Contribution The head of the Guissen group gave an oral presentation at the BSPP autumn 2017 conference at the University of Nottingham and then discussed in depth their results and techniques with the Rothamsted Resarch. The Guissen group hosted the Rothamsted Research post - doc for two weeks in late 2017 and trained them in specialist in vitro and in planta HIGS and SIGS techniques involving F. graminearum, Arabidopsis and barley leaves. A series of joint experiments are underway.
Impact Specialist training. Sharing of experimental protocols. Exchange of published and unpublished Fusarium strains and Arabidopsis lines.
Start Year 2017
 
Description Molecular and biological characterisation of Fusarium species and isolates collected from infected wheat fields in Southern Brazil 
Organisation Federal University of Viçosa
Country Brazil 
Sector Academic/University 
PI Contribution Rothamsted team has used a next generation sequencing approach to explore the genomes of the five Fusarium Head Blight causing species in Southern Brazil, namely F. graminearum, F. meridionale, F.cortaderiae, F. austroamericanum and F. asiaticum.. The Rothamsted team are currently focussing on investigating and defining the core and variable parts of the pan genome of F. graminearum. The Rothamsted team also assembled and annotated the F. meridionale genome and has given this data to the University team for further analysis.
Collaborator Contribution The University team had collected field isolates during 2009 - 2012 and provided the 24 Fusarium isolates covering the five required species. The University team had also characterised the disease causing ability of each isolate on the floral spikes of various Brazilian wheat genotypes. Currently, the University team is exploring the genomes of the various F. meridionale,
Impact The two main output delivered so far has been (1) the biological characterisation of the 24 / 5 species collection for disease causing ability on both Brazilian and non-Brazilian wheat genotypes and (2) the 24 newly assembled and annotated genomes covering the five most important FHB causing species in Southern Brazil.
Start Year 2014
 
Description siRNA oligo-mediated silencing of genes in plant pathogenic fungi and their cereal hosts - Royal Holloway - Dr Laurence Bindschedler 
Organisation Royal Holloway, University of London
Department School of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution The Rothamsted Research team has designed and generated single gene deletion mutants for a conserved effector in two wheat infecting fungal species, namely Fusarium graminearum and Zymoseptoria tritici. The conserved effector was selected because this small secreted protein is required for pathogenicity by the wheat and the barley attacking species of powdery mildew, respectively Blumeria graminis f sp tritici and Bg fsp hordei . Characterisation of the two sets of mutants both in vitro and in planta has also been carried out at Rothamsted Research. Our collaboartors at Royal Holloway have neither the facilities nor required government licences to carry out these types of experiments. The PhD student visits Rothamsted for a few days each month to complete specific experiments.
Collaborator Contribution Our collaborator has been silencing the same effector in trhe wheat powdery mildew fungus Blumeria graminis f sp tritici, using synthetic oligos. Thus confirming that this effector which is predicted to encode a specialist zinc metal protease is required for the infection of both the wheat and the barley infecting powdery mildew species. The Royal Holloway team are also exploring how the expression of this effector is regulated.
Impact So far only joint conference posters , eg BSPP Warwick Dec 2018, MBPP Norwich March 2019 and IS-MPMI Glasgow July 2019
Start Year 2016
 
Description Cereal Show 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Cereals
A Fusarium Head Blight exhibit was situated at the Rothamsted Research stand (Cereals 2016). This exhibit portrayed the impact of Fusarium on wheat production and the associated risk of mycotoxin contamination. It highlighted the need for new approaches to tackle this hazardous fungal disease. During the two day event, knowledge of the approaches taken at Rothamsted, including those within the associated fellowship, were described to farmers, agronomists, the press and industry. This exhibit commonly promoted the discussion of the use of GM and non-GM mediated approaches to control fungal diseases.

This two day event is attended by > 25,000 visitors (approximately 10% from overseas) from the AgIndustries, AgriFood and Farming sectors, as well as the media, politicians and NGOs.
Year(s) Of Engagement Activity 2016
 
Description Cereals 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Demonstration plots and posters
Year(s) Of Engagement Activity 2016
URL http://www.bbsrc.ac.uk/news/events/2016/1506-cereals/
 
Description Cereals 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Demonstration plots and posters
Year(s) Of Engagement Activity 2017
URL https://www.rothamsted.ac.uk/events/cereals-2017
 
Description Dissemination of project and results to date to EMBRAPA Trigo, Brazilian Farmers, Agronomists, AgIndustry and their Milling and Baking companies + links to many University Campus in Brazil as well as Kansas State University, USA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Kim Hammond-Kosack gave a 30 min invited plenary presentation at an EMBRAPA Trigo organised event in Passo Fundo, Southern Brazil in June 2018. The event was the 12th Meeting of the Brazilian Wheat and Triticale Research Commission (RCBPTT) . This 3 day event had an audiences of ~ 350 attendees. The meeting was attended by EMBRAPA scientists from many of their centres, numerous Brazilian Farmers, Agronomists, various AgIndustry companies both breeding and Ag chemical and several Brazilian Milling, Baking companies, and wheat importers and exports. Also present were government regulators involved in determining the levels of grain and food contamination by mycotoxins. The entire event was linked to many University Campus in Brazil as well as Kansas State University, USA. The second invited plenary speaker was Professor Barbara Valent, Kansas State University, USA.

After both plenary presentations there was a 30 minute discussion and Q and A session hosted by Professor Mauricio Fernandez (Embrapa Trigo) focussing on the control of Fusarium Head blight and Wheat Blast in wheat by breeding , cultural and emerging GM and GE technologies.

During the event Kim Hammond-Kosack visited the private wheat breeding company Biotrigo to see the facilities available for wheat improvement, especially the control of fungal pathogens. Folow up conversations have continued with Biotrigo since this visit.
Year(s) Of Engagement Activity 2018
 
Description Fusarium event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This one day Fusarium event was hosted at the Rothamsted Research (July 2016) educated the general public and interested stakeholders in the impact of Fusarium-borne diseases and the associated risk of mycotoxin contamination. It highlighted deficiencies is current approaches to prevent Fusarium-borne diseases and the need for new approaches to tackle this hazardous fungal disease. During this event, knowledge of the approaches taken at Rothamsted, including those within the associated fellowship, were described to the general public, farmers and agronomists. This exhibit commonly promoted the discussion of the use of GM and non-GM mediated approaches to control fungal diseases, and also described the background behind the use of host-induced gene silencing as a GM approach to fight fungal disease.

This event was from 4 to 8 pm and included a wheat and soybean field tour to visit and discuss three experiments, the running of a virtual laboratory for fungal pathogen transformation and analysis, a bioinformatics display and game to analyse sequenced fungal genomes, poster and live exhibits on the effect of fungal pathogens on various crops plant species and post harvest fruits and a poster display and talk on using GM and non-GM approaches to fight fungal diseases.
Year(s) Of Engagement Activity 2016
 
Description Government Open Access article (Jan 2020), page 370 entitled ' Fighting infectious diseases: Protecting the global wheat crop with big data analysis and knowledge networks' 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact This article was written to increase awareness of the importance of crop plant health amongst UK and European politicians, leaders of industry and leaders of third sector organisations. The article had a focus on the effects of disease causing pathogens on the No 1 arable crop in Europe namely wheat and how experimentation is increasing becoming predictive through using big data sets and network analyses. The two BBSRC funded resources highlighted in this online and hard copy article are the Pathogen-Host Interactions database (PHI-base) and the knowledge graphical visualisation tool Knetminer.
Year(s) Of Engagement Activity 2020
URL https://edition.pagesuite-professional.co.uk/html5/reader/production/default.aspx?pubname=&edid=e7e6...
 
Description Invited guest Lecture - University of Bath - Healthy humans, healthy animals, healthy crops, healthy food and healthy natural ecosystems. What's the problem ? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Invited lecture focussing on the importance of different beneficial and detrimental fungi on society and the global economy. Examples were given for the major crop species in particular fungi that cause diseases on wheat and maize crops, how fungicide resistance emerges in plant, human and farmed animals and how fungi can be used to produce a wide range of high value metabolites and proteins. The PHI-base database was also explained as away to keep track of the genes required by fungi to cause disease. This special lecture concluded with a lively debate with the students many of which were international MSc students on how to improve plant healthy and make crop yield more resilient. We also discussed career pathways in science and the differences and similarities between doing research in a company, for a charity or at an academic institution.
Year(s) Of Engagement Activity 2020
 
Description Rothamsted Festival of Ideas 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact It was our Institute's (Rothamsted Research) 175th birthday and we were celebrating with a Festival of Ideas. The event was free and open to all with something for everyone, from young to old. Our Festival brought together activities, exhibitions and demonstrations to immerse general public in the science of feeding the world. Numerous live plant displays and demonstration have been organised, as well as posters, interactive displays, tractor rides, talks, games and a trove of other astonishing exhibits describing our proud history of ground-breaking discoveries, from crop treatment to crop protection, from statistical interpretation to soils management.
Year(s) Of Engagement Activity 2018
URL https://www.rothamsted.ac.uk/events/rothamsted-festival-ideas