Location: Pest Management and Biocontrol Research2012 Annual Report
1a. Objectives (from AD-416):
Insect transmission of plant viruses is a major problem in agriculture that requires proactive research to limit its occurrence. The objective of the proposed cooperative research is to better understand the genes expressed in Bemisia tabaci salivary gland and midgut tissues in whiteflies harboring the begomovirus, Squash leaf curl virus. Next-generation massively-parallel DNA sequencing of Bemisia tabaci expressed sequence tag (EST) libraries will provide a more comprehensive representation of genes that are expressed in these tissues than is currently available and how vectoring this begomovirus affects gene expression. These ESTs could represent novel targets for disrupting normal homeostasis of the insect pest and/or viral disease transmission. As many plant disease causing begomoviruses must cross the epithelial barrier within salivary glands or digestive tracts of their arthropod vector, these tissues represent logical targets for understanding the processes involved in virus transmission. How whiteflies respond to viral infection will yield important new information about how begomoviruses enter their insect hosts and are transmitted to plants.
1b. Approach (from AD-416):
Next-generation sequencing will be used to provide a view of gene expression (e.g. transcriptome) in tissues of Bemisia tabaci vectoring Squash leaf curl virus. The approach follows standard procedures adapted for next-generation sequencing of transcriptomes, including tissue dissection from B. tabaci (naïve and infected with Squash leaf curl virus), RNA extraction, cDNA library construction, massively parallel DNA sequencing of ESTs, annotation, and organization in a PAVE database for mining. Post-sequencing analysis includes verification of changes in gene expression and functional analysis of proteins encoded by transcripts. Documents SCA with U of AZ. Formerly 5347-22620-020-01S (11/10).
3. Progress Report:
This Specific Cooperative Agreement is in support of objective 1 – Develop knowledge and control tactics based on the physiology, biochemistry, genetics and vector-pathogen interactions of insect pests, Subobjective 1.4 – Pathogen-mediated changes in host plants and their impact on vector populations, of the approved in-house project. The whitefly, Bemisia tabaci, is a major pest of cotton and other crops. In addition to injury caused by its feeding, the whitefly transmits, or vectors, many economically important plant viruses among or within different crops. Included in these is the squash leaf curl virus, which is a representative of a larger class of viruses know as begomoviruses. Two specific organ systems, the salivary glands and the midgut of the digestive tract, are intimately involved in virus infection of the whitefly and subsequent virus transmission to host plants. Observing gene expression in the whitefly salivary glands or midgut during virus infection and transmission, may lead to identification of genetic or biochemical targets to disrupt growth, development, or survival of the whitefly, or to interfere with transmission of the virus. Salivary glands and midguts were dissected from uninfected whiteflies, and from whiteflies infected by the squash leaf curl virus. These tissues were used to create respective transcriptomes, or DNA libraries that specifically represent the sequences of genes being expressed at the time of dissection. In all, more than 200,000 expressed sequences were obtained. The sequences corresponding to the four DNA libraries will be compared with each other, and also with previously assembled sequence databases obtained from the midguts of uninfected whiteflies and from whole whiteflies. Differences in the sequences that are expressed in the various transcriptomes will provide information about how the whitefly responds to begomovirus infection, and may identify tissue-specific targets involved in virus transmission. Sequences from the DNA libraries are currently maintained in the University of Arizona PAVE database and are made publicly available as the research results are published. This information contributes to a better understanding of the whitefly and its role as a disease vector, and facilitates the identification of genes targeted for direct control of this pest and associated viruses.