Interaction between host RNA silencing and viral infection in plants

现代分子生物学中最令人兴奋的进展之一是最近发现小RNA通过被称为RNA沉默的机制来调节包括发育和病毒感染在内的许多细胞活动。RNA沉默是一种古老而广泛的调控真核基因表达的机制，也被用作一种人为降低多种重要动植物基因表达的手段。根据对植物和动物病毒的非常有限的发现，宿主小RNA可以被篡夺以帮助病毒复制或针对病毒进行破坏。尽管做出了相当大的努力，但关于来自病毒基因组的小RNA是否也可以控制宿主基因的表达，这可能会对疾病的产生产生实质性的后果，人们知之甚少。该项目将结合研究得最好的病毒复制系统芜菁皱缩病毒(TCV)及其卫星RNA SATC和发展最好的寄主沉默模型拟南芥。初步实验已经证明，宿主酶将TCV和SATC切成小RNA，这些小RNA主要来自它们基因组的“热点”区域。一个测试的TCV小RNA能够减少至少六个宿主mRNAs的表达，这与症状产生有关，可能为成功的感染提供了更宽松的环境。为了了解(1)TCV/SATC靶向的全部基因；(2)产生病毒小RNA的酶如何选择其目标序列，以及(3)自然抑制宿主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与sRNAs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RNAs的水平。这项研究将有助于将目前分离的RNA病毒复制和RNA沉默结合在一起，消除关于RNA病毒在植物中复制的错误假设，并为充分发挥病毒RNA沉默抑制因子的作用提供新的思路。这些实验的成功完成将大大促进我们对宿主RNA沉默途径和入侵病毒之间相互作用的理解，并应用于动物和植物病毒发病，并可能在植物RNA沉默的广泛领域提供基本的新信息。该项目的广泛影响(教育和推广)：研究和知识获取与促进乔治王子县的教师培训相结合，乔治王子县是毗邻华盛顿特区的一个基本上是少数族裔的学区。2005年，国际和平研究所发起了一项努力，以提高杜瓦尔高中教师对尖端生物学/病毒学的理解。教师报告说，他们的学生对生物的理解和热情都有所提高，年中生物评估提高了100%。通过每周与AP生物学学生一起学习，所有学生都参加了AP考试(去年只有2人)，所有人都申请并被录取了研究型大学，这不是他们最初的选择(包括两名将加入我们马里兰大学的学生-这在这个学区是罕见的)。这一试点方案促成了一个为期两周的暑期讲习班，与从全县挑选的30名高中教师一起工作(收到了50份申请)。讲习班的最后一课将纳入导致这一研究项目的想法的发展，并将在今后计划的夏季讲习班上介绍进展情况。计划在秋季与AP生物专业的学生继续互动，每期课程都以“一周的结果”开始，这将使学生能够跟踪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(其中许多人拥有NSF资助的研究)配对，他们的愿望是通过描述尖端研究是如何进行的，以及NSF在推动知识获取和科学教育方面所起的作用，来推广这一模式，并激发全县的非生物学学生。

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.