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

主要研究者：Vincent Fondong（特拉华州州立大学）联合PI：Oumar Doungous（特拉华州州立大学），Kone Mongomake（特拉华州州立大学），Stephan Winter（德国微生物和细胞培养物保藏中心，布伦瑞克，德国）木薯是全球5亿多人的重要主食作物，其中大部分生活在撒哈拉以南非洲。在非洲的热带和亚热带地区，木薯作物被属于双生病毒科和马铃薯Y病毒科的病毒感染，这是世界上两种最重要的植物病毒。双生病毒科的成员引起木薯花叶病毒病，而马铃薯Y病毒科引起木薯褐条病。到目前为止，在非洲各地已经确定了双生病毒科的七种病毒和马铃薯Y病毒科的两种病毒。这些病毒越来越多地发生在混合感染中，引起协同作用，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加上各种管理策略。然而，使用常规育种的抗性转移受到几个因素的限制，包括核心木薯种质资源中缺乏有用的基因、杂合性和异源多倍体、不规则开花和低生育率。因此，尽管传统的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，不太可能为快速传播的病毒病大流行提供持久的解决方案。这就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（tasiRNA），该RNA能够靶向引起非洲木薯疾病的病毒家族成员。TasiRNA已被证明在靶向RNA转录物（包括病毒转录物）方面是有效的，并且因此是用于控制植物病毒的潜在有力工具。由于大于21个核苷酸的合成tasiRNA足以有效地沉默同源基因，因此该技术提供了使用小转基因构建体靶向许多病毒的前所未有的机会。这种方法比目前基于长发夹转基因构建体的策略更有效，长发夹转基因构建体是不稳定的，并且特征在于siRNA向适当靶标的低效传播。该项目将大大有助于非洲的木薯病毒控制工作。该项目还将为两名非洲共同首席研究员提供独特的分子生物学培训机会，他们将在本国机构使用这些技术。该项目将培养两名博士后研究人员和六名本科生。预期的成果包括转基因结构和DNA序列与控制木薯病毒的潜力。科学界可通过特拉华州州立大学网站（http：//www.appliedplantbiotech.com/index.html）和德国的Co-PI网站（http：//www.dsmz.de/）获取本研究产生的结果和材料。

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/).