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.
