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United States Department of Agriculture

Agricultural Research Service

Research Project: Transcriptome Analysis in Whiteflies to Identify Target Genes That Are Useful for Interfering Rna (Rnai) Technology

Location: Vegetable Research

Project Number: 6659-22000-025-11
Project Type: Specific Cooperative Agreement

Start Date: Sep 01, 2013
End Date: Sep 30, 2014

1. Perform global transcriptome analysis on B. tabaci upon infection by Tomato yellow leaf curl virus (TYLCV) and identify genes of interest. 2. Comparative analysis of siRNA and miRNA profiles upon TYLCV infection on whiteflies and identify candidate genes that may be useful for RNAi technology. 3. Develop and evaluate RNAi technology using selected functional genes with key biological functions on whiteflies.

Using established whitefly (Bemisia tabaci) colonies on collard green, over 2,000 whiteflies will be transferred on TYLCV-infected and healthy tomato plants, respectively. After feeding on the test plants for specific time intervals at 0 hr, 6 hr, 24 hr and 48 hr, 500 whiteflies will be aspirated at each time point and immediately frozen at -80C until use. We plan to perform 3 biological replications for each set of whiteflies. In total, the number of whiteflies needed is 12,000, including 6,000 viruliferous and 6,000 feeding on healthy tomato plants. In each subsample, total RNA preparations will be prepared using the TRIzol method. A half volume of RNA preparation will be used for RNA-seq library construction (paired-end). Another half volume of RNA preparation will be used for sRNA library construction. Sequencing of RNA-seq and sRNA will be multiplexed and conducted on HiSeq at Cornell genomic service center. Bioinformatics analysis on RNA-seq and sRNA (miRNA) will be performed at BTI and those genes highly regulated upon TYLCV infection will be selected. Once whitefly functional genes or their predicted precursor sequences are identified, dsRNAs will be designed and synthesized. These dsRNAs in varied concentration will be added on artificial diet or in nutrient solution for tomato cuttings for whitefly feeding. The effect of RNAi on whiteflies will be evaluated in replicated experiments for their mortality rate relative to controls. Upon initial screening, those promising dsRNAs will be tested again in replicated tests to evaluate the effects on whitefly survivability and impact on TYLCV transmission.

Last Modified: 9/10/2014
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