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

lII双病毒亚群（双病毒科）由一种白蝇，即烟粉虱，以持续、循环的方式传播。这种传播途径需要在昆虫取食过程中携带病毒粒子与植物汁液一起进入昆虫肠道，并穿过后肠上皮进入血液。然后病毒粒子穿过血淋巴中的血腔，必须穿过副唾液腺分泌细胞的基底层和质膜到达唾液管。通过花柱传递的唾液传播到寄主植物韧皮部。这种通过载体的复杂旅程要求病毒粒子被昆虫细胞吸收和释放，并且需要跨越几个传播障碍。每种屏障都涉及病毒外壳蛋白（CP）和粉虱蛋白（传递因子/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，也没有关于粉虱蛋白参与传播的信息。在与亚利桑那大学朱迪思·布朗博士的合作项目中，将采用分子生物学、生物化学和酵母遗传学的有力结合来解决这些问题。此外，还将使用一种新的、灵敏的基于pcr的生物测定方法来监测白蝇的病毒获取和传播。本研究的具体目标是：1。鉴定双病毒CP中白蛉介导传播所必需的特定氨基酸。这些研究将采用传染性南瓜卷叶病毒（SqLCV）和非传染性阿布蒂隆花叶病毒（AbMV）。定义的序列交换和氨基酸替换将被引入AbMV CP基因以恢复可传递性（功能获得），并被引入SqLCV CP基因以消除可传递性（功能丧失）。这些实验应该确定参与传播的CP氨基酸，特别是那些导致AbMV传播性丧失的氨基酸。2. 鉴定与双病毒CP相互作用以增强病毒获取和传播的白蛉编码的特定蛋白质的cdna。酵母双杂交系统和COS细胞的表达克隆将用于鉴定编码能与双病毒CP相互作用的白蝇蛋白的cdna。遗传学分析将用于鉴定涉及特定白蝇蛋白相互作用的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。白蛉传播的双病毒（双病毒科；III亚群）是蔬菜和纤维作物的重要病原体。这些病原体通过农业生态系统传播取决于三个相互作用的组成部分：双病毒病原体、粉虱病媒和同时作为病毒和病媒宿主的植物物种。在这个项目中，实验描述了旨在扩展目前对传播的分子机制的理解，以及传播特异性的分子基础。确定白蝇传播双病毒的分子机制对理解生物媒介和媒介传播的病原体以及动物和植物之间发生的相互作用具有广泛的意义，在大多数情况下，这方面的研究很少。

Bisaro 9723744 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 barriers be crossed. Each of these barriers involves a specific interaction between viral 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. In a collaborative project with Dr. Judith Brown of the University of Arizona, a powerful combination of molecular biology, biochemistry, and yeast genetics will be employed to address these questions. 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, an d 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 agroecosystems 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 and animals and plants, which in most cases are poorly studied.