BREAD: Engineering Broad-spectrum Resistance Against Multiple Geminiviruses and Cassava Brown Streak Viruses Using Synthetic Trans-acting Small Interfering RNA (tasiRNA)

PI: Vincent Fondong（特拉华州立大学）合作PI: Oumar Doungous（特拉华州立大学），Kone Mongomake（特拉华州立大学），Stephan Winter（德国微生物和细胞培养物收集）木薯是全球超过5亿人的重要主食作物，其中大多数人生活在撒哈拉以南非洲。在非洲的热带和亚热带地区，木薯作物受到双病毒科和波病毒科病毒的感染，这两种病毒是世界上最重要的两种植物病毒。双病毒科的成员引起木薯花叶病，而波蒂病毒科引起木薯褐条病。迄今为止，已在非洲各地确定了7种双病毒科病毒和2种痘病毒科病毒。这些病毒越来越多地发生在混合感染中，造成协同相互作用，导致严重症状和几乎全部作物损失。目前，这些病毒的控制是通过使用抗性植物品种以及各种管理策略来实现的。然而，利用传统育种方法进行抗性转移受到几个因素的限制，包括木薯核心种质中缺乏有用基因、杂合性和异源多倍体、开花不规则和低育性。因此，尽管传统育种努力已被证明有助于开发抗病毒木薯品种，但它是复杂和缓慢的，并且不太可能为快速传播的病毒疾病大流行提供持久的解决方案。这强调需要开发新的方法来遏制这些病毒。该项目的目标是从木薯病毒的保守区域设计合成的反式作用小干扰rna (tasiRNAs)，具有靶向在非洲引起木薯疾病的病毒家族成员的能力。TasiRNAs已被证明可以有效地靶向RNA转录物，包括病毒转录物，因此是控制植物病毒的潜在强大工具。由于超过21个核苷酸的合成tasirna足以有效地沉默同源基因，这项技术为使用小的转基因构建物靶向许多病毒提供了前所未有的机会。这种方法比目前基于长发夹转基因结构的策略更有效，后者是不稳定的，其特点是siRNA不能有效地传播到适当的靶标上。该项目将对非洲的木薯病毒防治工作作出重大贡献。该项目还将为两名非洲合作项目负责人提供独特的分子生物学培训机会，他们将在本国机构中使用这些技术。本项目将培养2名博士后研究员和6名本科生。预期的结果包括转基因结构和具有控制木薯病毒潜力的DNA序列。科学界可以通过特拉华州立大学网站（http://www.appliedplantbiotech.com/index.html）和德国的Co-PI网站（http://www.dsmz.de/）获得这项研究的结果和材料。

PI: Vincent Fondong (Delaware State University)Co-PIs: Oumar Doungous (Delaware State University), Kone Mongomake (Delaware State University), Stephan Winter (German Collection of Microorganisms and Cell Cultures Braunschweig, Germany)Cassava is an important staple food crop to more than 500 million people worldwide, most of whom live in sub-Saharan Africa. In tropical and subtropical regions of Africa, the cassava crop is infected by viruses belonging to Geminiviridae and Potyviridae families, which are the two most important plant viruses in the world. Members of the Geminiviridae family cause the cassava mosaic disease while the Potyviridae cause the cassava brown streak disease. So far, seven virus species of the Geminiviridae family and two species of the Potyviridae have been identified across Africa. These viruses are increasingly occurring in mixed infections, causing synergistic interactions that result in severe symptoms and almost total crop loss. Currently, control of these viruses is through the use of resistant plant varieties coupled with various management strategies. However, transfer of resistance using conventional breeding is limited by several factors including lack of useful genes in core cassava germplasm collections, heterozygosity and allopolyploidy, irregular flowering, and low fertility rates. Thus, although classical breeding efforts have proved to be instrumental in developing virus resistant cassava varieties, it is complicated and slow, and is unlikely to provide a lasting solution to the fast spreading virus disease pandemics. This emphasizes the need for the development of new approaches to contain these viruses. The goal of this project is to design synthetic trans-acting small interfering RNAs (tasiRNAs) from conserved regions of cassava viruses with the capability of targeting members of virus families causing cassava diseases across Africa. TasiRNAs have been shown to be efficient in targeting RNA transcripts, including virus transcripts, and is thus a potentially powerful tool for the control of plant viruses. Because synthetic tasiRNAs of greater than 21 nucleotides are sufficient to efficiently silence cognate genes, this technology provides an unprecedented opportunity to target many viruses using small transgene constructs. This approach is more efficient than the current strategy based on long hairpin transgene constructs, which are unstable and are characterized by inefficient spread of the siRNA to appropriate targets. This project will contribute significantly to cassava virus control efforts in Africa. The project will also provide unique training opportunities in molecular biology to two African co-PIs who will use the technologies in their home institutions. Two postdoctoral researchers and six undergraduate students will be trained in the project. Anticipated outcomes include transgene constructs and DNA sequences with the potential to control cassava viruses. Results and materials generated from this research will be accessible to the scientific community through the Delaware State University website (http://www.appliedplantbiotech.com/index.html) and at the Co-PI website in Germany (http://www.dsmz.de/).