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

Bisaro 9723744双生病毒亚群(双病毒科)由单一种烟粉虱以持续、循环的方式传播。传播途径要求病毒粒子在昆虫取食过程中与植物汁液一起进入昆虫的肠道，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，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。向寄主植物韧皮部的传播是通过口针分泌的唾液进行的。这种通过媒介的复杂旅程要求病毒粒子被昆虫细胞吸收和释放，并跨越几个传播障碍。这些屏障中的每一个都涉及病毒外壳蛋白(CP)和粉虱蛋白(传播因子/受体)之间的特定相互作用。目前，还没有鉴定出参与关键识别事件的CP序列，也没有关于粉虱蛋白参与传播的信息。在与亚利桑那大学的朱迪思·布朗博士合作的一个项目中，分子生物学、生物化学和酵母遗传学的强大组合将被用来解决这些问题。此外，还将使用一种新的、敏感的基于聚合酶链式反应的生物检测方法来监测烟粉虱对病毒的获取和传播。本研究的具体目的是：1.鉴定双生病毒CP中粉虱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)和不可传播的阿布蒂翁花叶病毒(AbMV)。确定的序列交换和氨基酸替换将被引入到AbMV CP基因中，以努力恢复传递性(功能获得)，并被引入SqLCV CP基因，以取消传递性(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.鉴定粉虱编码的cDNA，确定与双生病毒CP相互作用的蛋白，以加强病毒的获取和传播。酵母双杂交系统和在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，以确定特定烟粉虱蛋白相互作用所涉及的CP结构域，并检查这些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。粉虱传播双生病毒(Geminiviridae；亚群III)是蔬菜和纤维作物的重要病原。这些病原体在农业生态系统中的传播依赖于三个相互作用的组成部分：双生病毒病原体、粉虱媒介以及作为病毒和媒介宿主的植物物种。在这个项目中，描述了一些实验，这些实验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。确定粉虱传播双生病毒的分子机制对于理解生物媒介与媒介传播的病原体以及动植物之间的相互作用具有广泛的意义，而在大多数情况下，对这些相互作用的研究很少。

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.