Collaborative Project: Coat Protein-Whitefly Protein Interactions Necessary for Geminivirus Transmission.

合作项目：双生病毒传播所需的外壳蛋白-粉虱蛋白相互作用。

• 批准号: 9729397
• 负责人: Judith Brown
• 金额: $ 25.09万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9729397&HistoricalAwards=false
• 关键词: Collaborative; Project; Coat; Protein; Whitefly

Brown 9729397 The subgroup lII geminiviruses (Geminiviridae) are transmitted by a single species of whitefly, Bemisia tabaci, in a persistent, circulative fashion. The transmission pathway requires that virions, taken up during insect feeding along with plant sap, enter the insect gut and cross the hindgut epithelium into the hemocoel. Virions then pass through the hemocoel in the hemolymph, and must traverse both the basal lamina and plasmalemma of the accessory salivary gland secretory cells to reach the salivary canal. Transmission to the host plant phloem occurs through saliva delivered by the stylet. This complex journey through the vector demands that virions be taken up and released by insect cells, and that several transmission barters be crossed. Each of these barriers involves a specific interaction between vital coat protein (CP) and whitefly proteins (transmission factors/receptors). At this time, CP sequences that participate in key recognition events have not been identified, and there is no information concerning whitefly proteins involved in transmission. To address these questions, a powerful combination of molecular biology, biochemistry, and yeast genetics will be employed. In addition, a novel and sensitive PCR-based bioassay for monitoring virus acquisition and transmission by the whitefly will be used. The specific objectives of this research are: 1. To identify specific amino acids in geminivirus CP that are necessary for whitefly-mediated transmission. These studies will employ the transmissible squash leaf curl virus (SqLCV) and the non-transmissible Abutilon mosaic virus (AbMV). Defined sequence exchanges and amino acid substitutions will be introduced into the AbMV CP gene in an effort to restore transmissibility (gain of function), and into the SqLCV CP gene to abolish transmissibility (loss of function). These experiments should identify CP amino acids involved in transmission, and in particular those responsible for the loss of AbMV transmissibility. 2. To identify whitefly-encoded cDNAs specifying proteins which interact with geminivirus CP to potentiate virus acquisition and transmission. The yeast two-hybrid system and expression cloning in COS cells will be used to identify cDNAs encoding whitefly proteins capable of interacting with geminivirus CP. Genetic analysis will be carried out to identify CP domains involved in specific whitefly protein interactions, and to examine the relevance of the interactions to transmission. Immunolocalization of interacting whitefly proteins will also be performed as a prelude to functional analysis. The whitefly-transmitted (WFT) geminiviruses (Geminiviridae; subgroup III) are important pathogens of vegetable and fiber crops. The dissemination of these pathogens through agoecosystems depends on three interacting components: the geminivirus pathogen, the whitefly vector, and plant species that serve as hosts for both the virus and the vector. In this project experiments are described which are designed to extend the current understanding of the molecular mechanisms of transmission, and the molecular basis of transmission specificity. Defining the molecular mechanisms of geminivirus transmission by whiteflies has broad implications for understanding the interactions that occur between biological vectors and vector-borne pathogens of animals and plants, which in most cases are poorly studied.

Brown 9729397 第 III 亚组双生病毒（双生病毒科）由单一种粉虱（烟粉虱）以持久、循环的方式传播。传播途径要求病毒体在昆虫进食过程中与植物汁液一起进入昆虫肠道并穿过后肠上皮进入血腔。然后病毒粒子穿过血淋巴中的血腔，并且必须穿过副唾液腺分泌细胞的基底层和质膜才能到达唾液管。通过管心针输送的唾液传播到宿主植物韧皮部。这个通过载体的复杂旅程要求病毒颗粒被昆虫细胞吸收和释放，并且需要交叉几个传播易货贸易。这些屏障中的每一个都涉及重要外壳蛋白（CP）和粉虱蛋白（传输因子/受体）之间的特定相互作用。目前，参与关键识别事件的CP序列尚未确定，也没有有关参与传播的粉虱蛋白的信息。为了解决这些问题，将采用分子生物学、生物化学和酵母遗传学的强大组合。此外，还将使用一种新颖、灵敏的基于 PCR 的生物测定法来监测粉虱的病毒获取和传播。 本研究的具体目标是： 1. 鉴定双生病毒 CP 中粉虱介导的传播所必需的特定氨基酸。这些研究将采用传染性南瓜曲叶病毒（SqLCV）和非传染性苘麻花叶病毒（AbMV）。确定的序列交换和氨基酸取代将被引入到 AbMV CP 基因中，以努力恢复可传递性（功能获得），并引入到 SqLCV CP 基因中，以消除可传递性（功能丧失）。这些实验应该鉴定出参与传播的 CP 氨基酸，特别是那些导致 AbMV 传播能力丧失的氨基酸。 2. 鉴定粉虱编码的 cDNA，该 cDNA 特异与双生病毒 CP 相互作用以增强病毒获取和传播的蛋白质。酵母双杂交系统和 COS 细胞中的表达克隆将用于鉴定编码能够与双生病毒 CP 相互作用的粉虱蛋白的 cDNA。将进行遗传分析，以确定参与特定粉虱蛋白相互作用的 CP 结构域，并检查相互作用与传播的相关性。相互作用的粉虱蛋白的免疫定位也将作为功能分析的前奏进行。 粉虱传播的 (WFT) 双生病毒（双生病毒科；III 亚组）是蔬菜和纤维作物的重要病原体。这些病原体通过前生态系统的传播取决于三个相互作用的组成部分：双生病毒病原体、粉虱载体以及作为病毒和载体宿主的植物物种。在该项目中描述的实验旨在扩展目前对传播分子机制和传播特异性的分子基础的理解。定义烟粉虱双生病毒传播的分子机制对于理解生物载体与动植物媒介传播病原体之间发生的相互作用具有广泛的意义，而在大多数情况下，这种相互作用的研究很少。