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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: Crop Improvement and Protection Research

Project Number: 2038-22000-013-05-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 16, 2013
End Date: Mar 15, 2017

1. Perform global transcriptome analysis on B. tabaci upon infection by the begomovirus, Tomato yellow leaf curl virus (TYLCV) and the criniviruses, Tomato chlorosis virus (ToCV) and Cucurbit yellow stunting disorder virus (CYSDV), identify genes of interest, and compare whitefly gene expression. 2. Determine the genome sequence of virus vector, Bemisia tabaci. 3. Identify whether virus infection of a host plant alters gene expression in a whitefly vector. 4. Determine if the same physiological pathways are activated in different whiteflies with and without virus infection on tomato and melon. 5. What biological pathways are differentially activated when whiteflies feed on preferred or palatable hosts vs. not preferred or less palatable hosts. 6. Develop and evaluate RNAi technology using selected functional genes with key biological functions on whiteflies.

• Using established Bemisia tabaci whitefly colonies raised on Brassica oleracea, and a Trialeurodes vaporariorum whitefly colony raised on Physalis wrightii, whiteflies will be transferred virus-infected and healthy tomato plants, respectively, as well as young and old blackberry and Nicotiana benthamiana plants. All experiments will be conducted in triplicate. • RNA will be prepared at Salinas and sent to BTI for preparation RNA-seq and sRNA libraries. • RNA-seq and sRNA sequencing will be multiplexed and conducted on HiSeq at the Cornell Genomic Service Center by BTI. • Bioinformatics analysis on RNA-seq and sRNA (miRNA) will be performed at BTI and genes differentially expressed under comparative conditions will be identified. • Selected RNA expression levels will be evaluated at the USDA-ARS in Salinas from whitefly extract to validate results of RNAseq analysis. • 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 to artificial diet or allowed uptake into tomato cuttings for ingestion by whiteflies to evaluate impact of each treatment on whitefly mortality and/or impact on reducing virus acquisition and transmission by the whitefly. This work will be conducted at USDA-ARS in Salinas and Charleston. • USDA/ARS-Charleston will provide purified whitefly DNA to BTI for use in genome sequencing. BTI will conduct sequencing, assembly, annotation, and genome evolution and comparative genomics analysis. USDA-ARS-Salinas will contribute funds to partially support a postdoctoral position to be located at BTI for this work.

Last Modified: 09/21/2017
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