BREAD: Functional Analysis of DNA Satellites Associated with Cassava Mosaic Disease

PI: Linda K. Hanley-Bowdoin（北卡罗来纳州立大学）科普i: Joseph Ndunguru[坦桑尼亚米科切尼农业研究所（MARI）]主要合作者：Peter Sseruwagi[乌干达国家作物资源研究所（NaCCRI-NARO）]木薯是非洲和亚洲重要的主食作物，每天有7亿多人食用木薯。它是由最贫穷村庄的自给自足的农民种植的，当其他作物歉收或被冲突摧毁时，它往往是唯一的食物来源。木薯可以在干旱、高温和贫瘠的土壤条件下生长，但其产量受到病毒病的严重限制。木薯花叶病是由包括7种双病毒的DNA病毒复合体引起的。两个卫星dna与该复合体相关，可以打破耐药性并提高疾病的严重程度。这些卫星病毒与其他双病毒卫星病毒不同，其作用机制尚不清楚。该项目的第一个目标是研究卫星如何与木薯双病毒和寄主植物相互作用，以增强疾病进程。该研究将表征卫星的转录产物，并询问它们是否对增强症状和抗性打破是必要的。这项分析将作为研究的基础，以确定卫星是否通过小RNA或蛋白质起作用，以及它们在感染期间如何发挥作用。这两个卫星只显示有限的同源性，导致不同的表型，并且可能通过不同的机制起作用，这将需要不同的方法来克服它们的活性并恢复抗病能力。如果卫星产生靶向宿主基因的小rna，这一信息可以作为开发表达抗切割靶点的转基因木薯的基础。相反，基因沉默策略可以针对卫星蛋白编码区的转录本进行部署。该项目的第二个目标是确定这些卫星是像木薯双子星病毒一样被白蝇包裹并传播，还是在感染期间从木薯基因组中释放出来。如果卫星从宿主基因组中释放出来，这一信息将影响未来筛选不含卫星序列的抗性木薯品种，这些品种不能作为释放卫星序列的储存库。一个多世纪以来，木薯花叶病一直制约着非洲的木薯生产，但在过去20年里，这种疾病性质的变化导致了前所未有的大规模损失。若干因素促成了这一流行病，包括木薯双病毒之间的重组导致毒性增强和出现破药卫星。2005年，坦桑尼亚、乌干达、卢旺达和布隆迪因木薯花叶病造成的作物总损失为400万吨/年。卫星对木薯产量的影响尚未有文献记载，但如果不采取措施遏制其影响，它们很可能会大大增加损失，并导致非洲的粮食不安全。这个过程的第一步是了解卫星如何加强疾病进程，以及如何在受感染的木薯田里传播或释放。该项目将提供有关卫星作用机制和传播机制的重要知识，使人们能够合理制定战略，克服它们对防治木薯花叶病工作的有害影响。该项目还将为美国的一名博士后研究员（部分资金来自美国国家科学基金会国际科学与工程办公室）和坦桑尼亚的一名研究生提供独特的培训机会，包括国际合作以及实验室和实地经验。该项目将使受训者做好准备，成为参与全球事务的独立科学家，能够有效地解决未来由快速进化的植物病毒引起的疾病问题。这些研究将转移关于双子星病毒复制和宿主相互作用的专门知识，以促进今后对木薯双子星病毒及其卫星的研究，并增加坦桑尼亚这类研究的资源。最后，研究结果将通过项目网站MARI主页（http://cloud2.gdnet.org/cms.php?id=organization_details&organization_id=3273）提供，并发表在诸如《非洲作物科学学会新闻》（http://www.acss.ws）等开源期刊和在线通讯上，以确保公众和发展中国家的科学家能够获得这些成果。作为项目的一部分创建的材料将根据要求提供给科学研究界。

PI: Linda K. Hanley-Bowdoin (North Carolina State University)CoPI: Joseph Ndunguru [Mikocheni Agricultural Research Institute (MARI), Tanzania]Key Collaborator: Peter Sseruwagi [National Crops Resource Research Institute (NaCCRI-NARO), Uganda]Cassava is an important staple crop in Africa and Asia, where it is eaten by over 700 million people every day. It is grown by subsistence farmers in the poorest villages and is often the only food source when other crops fail or are destroyed by conflict. Cassava can grow under drought, high temperature and poor soil conditions, but its production is severely limited by viral diseases. Cassava mosaic disease is caused by a DNA virus complex that includes seven geminivirus species. Two satellite DNAs are associated with the complex and can break resistance and enhance disease severity. The satellites do not resemble other geminivirus satellites and their mechanisms of action are not known. The first goal of the project is to examine how the satellites interact with cassava geminiviruses and the host plant to enhance disease processes. The research will characterize the transcription products of the satellites and ask if they are necessary for enhanced symptoms and resistance breaking. This analysis will serve as the basis of studies that determine if the satellites act through a small RNA or a protein and how they function during infection. The two satellite show only limited homology, cause different phenotypes, and may act via different mechanisms that will require different approaches to overcome their activities and restore disease resistance. If the satellites produce small RNAs that target host genes, this information could serve as the basis for developing transgenic cassava that express cleavage-resistant targets. Conversely, a gene silencing strategy could be deployed against the transcripts of a satellite protein coding region. The second goal of the project will determine if the satellites are encapsidated and transmitted by whiteflies like cassava geminiviruses or are released from the cassava genome during infection. If the satellites are released from the host genome, this information will impact future screens for resistant cassava cultivars that do not contain satellite sequences and cannot serve as reservoirs for their release.Cassava mosaic disease has constrained cassava production in Africa for more than a century, but changes in the nature of the disease have led to losses on an unprecedented scale in the last 20 years. Several factors have contributed to the pandemic, including recombination between cassava geminiviruses resulting in enhanced virulence and the emergence of resistance breaking satellites. In 2005, the total crop losses due to cassava mosaic disease were 4M metric tons/year in Tanzania, Uganda, Rwanda and Burundi. The impact of the satellites on cassava yields has not yet been documented, but it is very likely that they will significantly increase losses and lead to food insecurity in Africa if measures are not taken to contain their effects. The first step in this process is to understand how the satellites enhance disease processes and are transmitted or released in infected cassava fields. The project will contribute important knowledge about the mechanism(s) of action and transmission of the satellites and enable the rational development of strategies to overcome their detrimental effects on efforts to combat cassava mosaic disease. The project will also provide unique training opportunities for a postdoctoral researcher in the US (funded in part through the NSF Office of International Science and Engineering) and a graduate student in Tanzania that include international collaboration as well as bench and field experience. The project will prepare the trainees to become globally engaged, independent scientists who can effectively address future disease problems caused by rapidly evolving plant viruses. The studies will transfer expertise on geminivirus replication and host interactions to facilitate future studies on cassava geminiviruses and their satellites and increase resources for these types of studies in Tanzania. Finally, the results of the research will be accessible through a project website, the MARI homepage (http://cloud2.gdnet.org/cms.php?id=organization_details&organization_id=3273), and published in open source journals and online newsletters such as the African Crop Science Society News (http://www.acss.ws) to ensure access to the public and to scientists in developing countries. Materials created as part of the project will be provided to the scientific research community upon request.