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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Research Project #431572

Research Project: Comparative Genomics to Determine the Mechanism of Curtovirus Transmission by the Beet Leafhopper, Circulifer tennellus

Location: Crop Improvement and Protection Research

Project Number: 2038-22000-018-03-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 15, 2016
End Date: Sep 30, 2020

Objective:
1) Complete initial annotation of the genome of the beet leafhopper (Circulifer tennellus). 2) Compare the transcriptome and C. tennellus after feeding on healthy source plants, and source plants infected with Beet curly top virus to elucidate gene expression changes triggered by feeding on virus-infected plants using RNA-seq and small RNA analysis. 3) Examine tissue-specific gene expression differences associated with the leafhopper gut-wall, which virus must pass through to become transmissible, as well as salivary glands, where virus accumulates prior to transmission. Results will improve understanding of virus-vector interactions, possible identification of virus receptors in the insect vector, and identification of new targets for RNAi or CRISPR/CAS9 based control of leafhopper and prevention of virus infection. This will provide new methods for control of leafhoppers and curly top in an era in which insecticide use is under increasing scrutiny.

Approach:
1) Propagate beet leafhopper on healthy sugarbeet for use in transcriptome analysis. 2) Feed beet leafhopper on healthy and diseased sugarbeet for select periods of time (24, 48, 72 hours) under standard conditions in a growth chamber for transcriptome and proteome studies. 3) Extract DNA and RNA from leafhoppers described above, and develop libraries for use in genome and transcriptome studies using methods used previously by Wintermantel Lab for whitefly studies (Chen et al., 2016; in prep). Extract protein from leafhoppers using methods previously used by Webb and Wintermantel (Physiol. Mol. Plant Path. 91: 96-105). 4) Sequence, assemble and annotate the genome of the beet leafhopper (Circulifer tenellus). 5) Compare the transcriptome of C. tennellus after feeding on healthy source plants, and source plants infected with Beet curly top virus over 72 hours to elucidate gene expression changes triggered by feeding on virus-infected plants using RNA-seq and small RNA analysis. 6) Compare the transcriptome of C. tennellus after feeding on healthy source plants, and source plants infected with the phytoplasma that causes big vein disease of tomato over 72 hours to elucidate gene expression changes triggered by feeding on virus-infected plants using RNA-seq. 7) Additional studies will examine tissue-specific gene expression differences associated with the leafhopper gut-wall, which virus must pass through to become transmissible, as well as salivary glands, where virus accumulates prior to transmission. Note: All experiments will be conducted in growth chambers under tightly regulated conditions, and will be evaluated at distinct time points to selectively identify key changes. 8) Develop a predictive model to determine probability of curly top infection based on previous and new studies on curly top epidemiology.