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

这是一个极具创新性和真正开创性的研究项目，由巴西EmbRapa小麦、大豆、遗传资源和生物技术及生物信息学和英国Rothamsted Research的科学家设计。需要解决的问题是控制一种名为镰刀菌赤霉病的真菌病害，它是世界上最严重和最危险的作物病害之一。FHB的主要后果是毛霉烯类真菌毒素，如脱氧雪腐镰刀菌烯醇(DON)，在谷物中积累，对人类和动物的健康构成风险。在巴西南部，90%的巴西小麦种植在那里，FHB严重流行年至少每4或5年发生一次。不同用途的收获谷物中允许的DON水平已经设定了法律限制。然而，即使是中等的FHB年份也是非常有问题的，导致缺乏可用于农场或销售到市场的安全谷物。对于低收入的巴西农民来说，FHB病降低了农民家庭及其当地社区的生活水平。迫切需要开发新的和有效的赤霉病防治方法。在这个项目中，我们打算采用一种新的全真菌基因组和疾病模拟指导的方法来开发一个含有T-DNA结构的转基因小麦基因型管道，该管道可以通过宿主诱导的基因沉默(HIGS)来沉默与小麦侵染有关的镰刀菌基因。我们还打算确定控制HIGS现象的植物和真菌机制。HIGS可以用来控制多种病原菌。本项目有六个主要研究步骤。探索使用下一代测序技术对巴西南部引起FHB的五个物种的基因组进行测序。确定FHB物种复合体(FGSC)的核心蛋白质组和物种特异性蛋白质组，以开发分子诊断工具和选择HIGS靶标。监测3年内可能发生的基因组变化。加强赤霉病风险预测，建立能够检测和量化镰刀菌种类的孢子采样器网络。设计和使用诊断分析来确定和监测FGSC的多样性。采集大气中的镰刀菌孢子样本，以确定潜在的接种源和种群结构。将空气传播的镰刀菌孢子种群动态数据纳入现有的区域赤霉病风险模型。3.构建多种基于T-DNA的载体，通过HIGS沉默镰刀菌基因的表达，从而控制镰刀菌的侵染。使用一种新的小麦分蘖切割法(瞬时试验)和通过稳定转化拟南芥或生菜来评估单基因HIGS构建。两个针对多个镰刀菌基因的Lead HIGS构建体将被转化到巴西小麦品种中抗赤霉病。在由此产生的转基因植物种群中，FHB的严重程度和DON水平将被量化。4.为了探索HIGS的潜在机制，将完成三个前沿实验。我们将确定长的或短的RNA分子是否在真菌和小麦细胞之间移动，确定一旦启动基因沉默现象是否系统地运作，并调查运输的RNA分子是否利用囊泡运输到植物细胞表面以输送到真菌细胞。利用四个最好的HIGS品系和三个适当的对照品系，在巴西的两个地点进行为期两年的转基因小麦试验。评估现场FHB症状、空气传播接种量、谷物质量、DON污染和真菌孢子生产。收集能够在HIGS品系上引起任何疾病的镰刀菌分离物，并完成全基因组分析。研究小组将与巴西和英国的农民、农民合作社、谷物购买者、植物育种者和全球学者一起解释该项目、研究结果，并讨论如何实施新的建模/预测技术、使用新的转基因特征和进一步了解HIGS现象。

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.