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

布朗 9729397 III亚组双生病毒（双生病毒科）由单一种粉虱烟粉虱以持续的循环方式传播。传播途径要求病毒粒子在昆虫取食期间沿着植物汁液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膜和质膜才能到达唾液管。传播到寄主植物韧皮部发生通过唾液提供的口针。这种通过载体的复杂旅程需要病毒粒子被昆虫细胞吸收和释放，并且需要交叉几种传播交换。这些障碍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。此时，参与关键识别事件的CP序列尚未被鉴定，并且没有关于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，还将使用一种新的、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。 这项研究的具体目标是： 1.确定双生病毒CP中对粉虱介导的传播所必需的特定氨基酸。这些研究将使用可传播的南瓜曲叶病毒（SqLCV）和非可传播的Abutilon花叶病毒（AbMV）。将限定的序列交换和氨基酸取代引入AbMV CP基因中以恢复可传递性（功能获得），并引入SqLCV CP基因中以消除可传递性（功能丧失）。这些实验应该确定参与传播的CP氨基酸，特别是那些负责AbMV传播性丧失的氨基酸。 2. 鉴定粉虱编码的cDNA，确定与双生病毒CP相互作用以增强病毒获得和传播的蛋白质。利用酵母双杂交系统和COS细胞表达克隆技术，筛选能与双生病毒CP相互作用的烟粉虱蛋白基因。将进行遗传分析以识别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质的免疫定位也将作为功能分析的前奏进行。 粉虱传双生病毒属（Geminiviridae; subgroup III）是蔬菜和纤维作物的重要病原。这些病原体通过农业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，植物物种，作为病毒和载体的宿主。在这个项目中描述的实验，旨在扩大目前的理解的分子机制的传输，和传输特异性的分子基础。确定由粉虱传播双生病毒的分子机制对于理解生物载体与动物和植物的载体传播的病原体之间发生的相互作用具有广泛的意义，在大多数情况下，这些相互作用的研究很少。

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.