Interaction between host RNA silencing and viral infection in plants

现代分子生物学最令人兴奋的进展之一是最近发现小RNA通过被称为RNA沉默的机制调节许多细胞活动，包括发育和病毒感染。RNA沉默是一种古老而广泛的调节真核生物基因表达的机制，也被用作人工降低多种重要植物和动物基因表达的手段。基于对植物和动物病毒的非常有限的发现，宿主小rna可以被篡夺来帮助病毒复制或靶向病毒进行破坏。尽管付出了巨大的努力，但人们对来自病毒基因组的小rna是否也能控制宿主基因表达知之甚少，这可能对疾病的产生产生重大影响。本项目将结合目前研究最完善的病毒复制系统——芜菁皱缩病毒（Turnip crcrcrv， TCV）及其卫星RNA satC，以及目前开发最完善的宿主沉默模型拟南芥（Arabidopsis thaliana）。初步实验已经证明，宿主酶将TCV和satC切割成主要来自其基因组“热点”区域的小rna。测试的一种TCV小RNA能够降低至少六种宿主mrna的表达，这与症状的产生有关，可能为成功感染提供了更有利的环境。为了了解(1)TCV/satC靶向的全范围基因；(2)生成病毒小RNA的酶如何选择它们的靶序列；(3)天然抑制宿主RNA沉默的TCV外壳蛋白如何在抑制satC复制的同时促进TCV复制。本研究将确定TCV和satC vsRNAs的其他细胞靶点，确定病毒小RNA切除的序列和结构要求，并分析外壳蛋白是否负责改变特定小RNA的水平。本研究将有助于将目前分离的RNA病毒复制和RNA沉默领域融合在一起，消除关于RNA病毒在植物中复制的错误假设，并为病毒RNA沉默抑制因子的充分作用提供新的思路。这些实验的成功完成将极大地促进我们对宿主RNA沉默途径与入侵病毒之间相互作用的理解，并将其应用于动物和植物病毒的发病机制，并可能为植物RNA沉默的广泛领域提供新的基础信息。项目的广泛影响（教育和推广）：在乔治王子县，研究和知识获取与促进教师培训相结合，这是一个毗邻华盛顿特区的少数民族学区。2005年，PI发起了一项努力，以提高DuVal高中教师对前沿生物学/病毒学的理解。老师们报告说，他们的学生提高了对生物的理解和热情，年中生物评估提高了100%。通过每周与AP生物学学生一起工作，所有学生都参加了AP考试（去年只有2人），所有学生都申请并被录取了研究型大学，而不是他们最初的选择（包括两名学生将加入我们马里兰大学-这在这个学区很少见）。这一试点项目促成了为期两周的暑期讲习班的发展，从全县选出30名高中教师（已收到50份申请）。研讨会的最后一讲将包括导致本研究项目的想法的发展，并将在未来计划的夏季研讨会上介绍进展情况。计划在秋季继续与AP生物学学生进行互动，每次会议以“本周结果”开始，这将使学生了解这项研究是如何计划的，以及如何产生新的想法。通过将研讨会上的所有30名教师与化学与生命科学学院的其他研究人员（其中许多是由美国国家科学基金会资助的研究）配对，我们的愿望是扩展这种模式，并通过描述前沿研究是如何进行的以及美国国家科学基金会在推进知识获取和科学教育方面所起的作用来激发整个县的ap -生物学学生。

One of the most exciting advances in modern molecular biology was the recent discovery that small RNAs regulate numerous cellular activities including development and virus infection through mechanisms referred to as RNA silencing. RNA silencing is an ancient, widespread mechanism for regulating eukaryotic gene expression, and is also used as a means of artificially reducing gene expression in a wide variety of important plants and animals. Based on very limited findings with plant and animal viruses, host small RNAs can be usurped to aid in virus replication or target viruses for destruction. Despite considerable efforts, little is understood about whether small RNAs derived from viral genomes can also control host gene expression, which could have substantial consequences for disease production. This project will combine the best studied viral replication system, Turnip crinkle virus (TCV)and its satellite RNA satC, with the best developed host silencing model, Arabidopsis thaliana. Preliminary experiments have already demonstrated that host enzymes dice TCV and satC into small RNAs that are mainly derived from "hotspot" regions of their genomes. One TCV small RNA tested was able to reduce the expression of at least six host mRNAs, which was related to symptom production and may be providing a more amenable environment for a successful infection. To gain an understanding of (1) the full range of genes targeted by TCV/satC; (2) how the enzymes that generate the viral small RNAs chose their target sequences, and (3) how the TCV coat protein, which naturally suppresses host RNA silencing, contributes to enhancing TCV replication while suppressing satC replication, this research will identify additional cellular targets of TCV and satC vsRNAs, determine the sequence and structural requirements for viral small RNA excision and analyze if the coat protein is responsible for altering the levels of specific small RNAs. This research will help blend together the currently separate fields of RNA virus replication and RNA silencing, dispel incorrect assumptions about RNA virus replication in plants and provide new ideas for the full role of viral RNA silencing suppressors. Successful completion of these experiments should significantly advance our understanding of the interplay between the host RNA silencing pathway and invading viruses with applications to both animal and plant viral pathogenesis, and will likely provide fundamental new information in the broad area of RNA silencing in plants.Broader Impact of the Project (education and outreach):Research and knowledge acquisition is being paired with promotion of teacher training in Prince George's County, a substantially minority school district adjacent to Washington DC. An effort was initiated by the PI in 2005 to improve teacher understanding of cutting edge Biology/Virology at DuVal High School. Teachers reported that their students improved both their understanding and enthusiasm for biology and midyear biology assessments improved by 100%. By also working weekly with AP Biology students, all students took the AP exam (compared with only 2 last year) and all applied to and were admitted to research-oriented universities, which were not their original choices (including two students who will join us at the University of Maryland- which is rare for this school district). This pilot program has led to the development of a two week summer workshop to work with 30 high school teachers selected from throughout the county (50 applications were received). The final lecture of the workshop will incorporate the development of the ideas that led to this research project and progress will also be presented at future planned summer workshops. Continued interaction is planned with AP biology students in the Fall, with each session leading off with the "results of the week", which will allow students to follow how this research is planned and how new ideas are generated. By pairing up all 30 teachers at the workshop with other investigators in the College of Chemical and Life Sciences, many with NSF funded research, the aspiration is to expand this model and excite AP-biology students throughout the county by describing how cutting-edge research is conducted and the role that NSF plays in advancing knowledge acquisition and science education.