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强劲的食物供应至关重要。这种反应的关键是RNA，一种遗传信息，从植物的一个部分移动到另一个部分。然而，由于过渡RNA的数量很少，对RNA在植物中如何运动的详细了解还不是很清楚。此外，我们几乎所有的知识都来自对短命植物的研究，而对长寿树的了解很少，因为生长和研究它们需要时间。一种新的类病毒RNA(称为CyVaV)的发现几乎感染了所有柑橘品种，为探索树木生物学和RNA在植物中的运动提供了一个无与伦比的机会。CYVaV在树脉中积聚到高水平，并在没有特定病毒产生的“移动”蛋白的情况下移动。由于CYVaV RNA必须利用树木的分子机械来移动其RNA，因此它是研究RNA运动的理想系统。这个项目使用CyVaV来开发技术来了解RNA在植物中的运动。此外，CYVaV可能使研究人员能够在树木中引入遗传元素。一个重要的更广泛的影响是，有可能利用CYVaV作为一种媒介，通过靶向细菌和存放细菌的昆虫来抗击细菌疾病柑橘绿化。这可能有助于将美国柑橘产业从柑橘绿色中拯救出来。最近有消息称，50%的伴生细胞(CC)转录组正在通过筛子元件(SE)进行长距离移动，这引发了许多关于mRNAs如何以及为什么发生这种迁移的问题。要回答有关mRNAs细胞内和细胞间运动的关键问题，需要一个由丰富的移动RNA组成的模型系统，其位置可以在不同细胞条件下在活组织中追踪。新近发现的柑橘黄脉相关病毒是一种非常罕见的类似病毒的RNA，它在缺乏编码运动蛋白(MPS)、外壳蛋白或沉默抑制因子的情况下，通过所有商品化品种的柑橘和实验室寄主本氏烟草的韧皮部进行双向运输。该项目利用MS2 CP和&amp；lambda；N22系统开发了一个转导mRNAs的模型，并将其作为一个VIGS载体来研究柑橘韧皮部基因表达变化的影响。尽管在受感染植物的乙锭染色凝胶上可以检测到的水平，但在柑橘上几乎不会引起任何症状。这应该允许小RNA的高水平表达，最初的目标是下调CC痂蛋白合成酶，该酶在应激或病原体入侵期间导致SE中的痂蛋白沉积。由于CYVaV不应该有寄主-MP特异性不亲和性，它也将在其他木本植物和藤本植物上进行扩展寄主范围的检查，这些植物和藤本植物目前还没有可用的载体。通过研究CYVaV的运动和作为VIGS媒介的能力，这项工作的一个更广泛的影响是使用CYVaV来阻止柑橘绿色的破坏性影响。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.