EAGER: Developing a Novel Virus-like RNA as a Model for mRNA Systemic Movement and as a Vector for Citrus and Other Trees and Vines

了解植物对病原体的反应对于维持稳定的食物供应至关重要。 这种反应的关键是RNA的移动，一种遗传信息，从植物的一个部分到另一个部分。 然而，由于转运RNA的数量很少，对RNA在植物中如何移动的详细了解还没有得到很好的理解。 此外，我们几乎所有的知识都来自于对短命植物的研究，而由于生长和研究它们所需的时间，我们对长寿树木知之甚少。一种新型病毒样RNA（称为CYVaV）感染几乎所有柑橘品种的发现为探索树木生物学和植物中RNA的运动提供了无与伦比的机会。 CYVaV在树脉中积累到高水平，并在缺乏特定病毒产生的“运动”蛋白的情况下移动。 由于CYVaV RNA必须利用树状分子机器来移动其RNA，因此它是研究RNA运动的理想系统。 该项目使用CYVaV开发技术来了解RNA在植物中的运动。 此外，CYVaV可以使研究人员在树木中引入遗传元素。 一个重要的更广泛的影响是使用CYVaV作为一种工具，通过靶向细菌和昆虫沉积的细菌来对抗细菌性疾病柑橘绿化的可能性。 这可能有助于拯救美国柑橘产业从柑橘绿化。最近的启示，50%的伴随细胞（CC）转录组是移动长距离通过筛元件（SE）引起了许多问题，关于如何以及为什么这种迁移的mRNA正在发生。 要解决mRNA在细胞内和细胞间运动的关键问题，需要一个由丰富的移动的RNA组成的模型系统，该模型系统的位置可以在不同的细胞条件下在活组织中追踪。柑橘黄脉相关病毒（Citrus yellow vein associated virus，CYVaV）是一种非常罕见的病毒样RNA，在不编码运动蛋白（MPs）、外壳蛋白或沉默抑制因子的情况下，通过所有商业品种的柑橘和实验室宿主烟草的韧皮部双向传播。 该项目开发CYVaV作为使用MS 2 CP和λ;N22系统转运mRNA的模型，并作为VIGS载体来研究柑橘韧皮部中改变的基因表达的影响。 CYVaV在柑橘上引起很少的症状，如果有的话，尽管它在感染植物中积累到在乙锭染色的凝胶上可检测到的水平。 这应该允许小RNA的高水平表达，其初始目标是下调CC胼胝质合酶，其在胁迫或病原体入侵期间引起SE中的胼胝质沉积。 由于CYVaV应该没有宿主-MP特异性不相容性，因此还将检查它在其他木本植物和藤本植物中的扩展宿主范围，这些植物和藤本植物目前没有可用的载体。 通过研究CYVaV运动和作为VIGS载体的能力，这项工作的一个更广泛的影响是使用CYVaV来阻止柑橘绿化的破坏性影响。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Understanding how plants respond to pathogens is critical for maintaining a robust food supply. Key to this response is the movement of RNA, a type of genetic information, from one part of the plant to another. However, detailed understanding of how RNA moves in plants is not well understood due to the small amounts of transiting RNAs. In addition, nearly all of our knowledge comes from research on short-lived plants and very little is known about long-lived trees due to the time it takes to grow and study them. The discovery of a novel virus-like RNA (called CYVaV) that infects virtually all citrus varieties provides an unparalleled opportunity to explore the biology of trees and the movement of RNA in plants. CYVaV accumulates to high levels in tree veins and moves in the absence of specific virus-produced "movement" proteins. Since CYVaV RNA must use the trees molecular machinery to move its RNA, it is an ideal system to study RNA movement. This project uses CYVaV to develop technologies to understand the movement of RNAs in plants. In addition, CYVaV may enable researchers to introduce genetic elements in trees. An important broader impact is the possibility of using CYVaV as a vehicle to combat the bacterial disease Citrus Greening by targeting the bacteria and the insect depositing the bacteria. This could help save the US citrus industry from Citrus Greening.Recent revelations that 50% of the companion cell (CC) transcriptome is moving long distances through sieve elements (SE) have elicited numerous questions concerning how and why this migration of mRNAs is occurring. Answering key questions on intracellular and intercellular movement of mRNAs requires a model system consisting of an abundant mobile RNA, whose location can be traced in living tissue under different cellular conditions. The recently discovered Citrus yellow vein associated virus (CYVaV) is a highly unusual virus-like RNA that traffics bi-directionally through the phloem of all commercial varieties of citrus and laboratory host Nicotiana benthamiana in the absence of encoded movement proteins (MPs), coat protein or silencing suppressors. This project develops CYVaV as a model for transiting mRNAs using MS2 CP and λN22 systems and as a VIGS vector to study effects of altered gene expression in citrus phloem. CYVaV causes few if any symptoms on citrus although it accumulates to levels detectable on ethidium-stained gels in infected plants. This should allow for high level expression of small RNAs, with the initial goal of down regulating CC callose synthase, which causes callose deposition in SE during stress or pathogen invasion. Since CYVaV should have no host-MP specific incompatibility, it will also be examined for an extended host range in other woody plants and vines, which currently have no useable vectors. By studying CYVaV movement and ability to serve as a VIGS vector, one broader impact of this work is using CYVaV to block the devastating effects of Citrus Greening.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.