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United States Department of Agriculture

Agricultural Research Service

Title: Methods for effective real-time RT-PCR analysis of virus-induced gene silencing

Authors
item Rotenberg, Dorith - UNIV OF WI - MADISON
item Thompson, Thea - UNIV OF WI - MADISON
item German, Thomas - UNIV OF WI - MADISON
item WILLIS, DAVID

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 19, 2006
Publication Date: December 1, 2006
Citation: Rotenberg, D., Thompson, T.S., German, T.L., Willis, D.K. 2006. Methods for effective real-time RT-PCR analysis of virus-induced gene silencing. Journal of Virological Methods. 138:49-59.

Interpretive Summary: Plant diseases caused by viruses cause billions of dollars of damage to agronomically important crops each year. The problem is compounded by the lack of effective control of viral disease in plants by chemical means or breeding for resistance. Arthropod vectors play an essential role in dissemination of viruses that cause important diseases in humans, animals, and plants. Developing a better understanding of insect–virus interactions leading to successful transmission is vital to developing effective control strategies. Tomato spotted wilt virus (TSWV) is a important pathogen of many crops including tomato, lettuce, and pineapple and is transmitted by thrips insect vectors. In this paper, we show that feeding the thrips vector a protein located on the surface of the virus inhibits the ability of the thrips to transmit the virus to uninfected host plants. This work identifies a novel stategy for the control of virus diseases by preventing the essential insect vector from speading the disease.

Technical Abstract: Arthropod vectors play an essential role in dissemination of viruses that cause important diseases in humans, animals, and plants. Developing a better understanding of insect–virus interactions leading to successful transmission is vital to developing effective control strategies. Tomato spotted wilt virus (TSWV) is transmitted in a persistent propagative manner by its thrips vectors. The two viral glycoproteins, GN and GC, mediate virus entry into the insect vector midgut. Previously, we found that a recombinant, soluble form of the tomato spotted wilt virus (TSWV) GN protein (GN-S) inhibits virus acquisition by the arthropod vector, but because TSWV replicates in the vector, an initial reduction in amount of virus at the acquisition level may not result in transmission inhibition. Therefore, we assayed GN-S for the ability to inhibit TSWV transmission. GN-S and TSWV were fed concomitantly and sequentially to larval thrips and transmission inhibition was assayed by insect transmission to Datura stramonium leaf discs. In concomitant and sequential feeding experiments, GN-S significantly reduced TSWV transmission by thrips. This is the first demonstration of inhibition of insect transmission of an enveloped virus. Quantitative real time-PCR analysis of virus titer in insects revealed that non-transmitters did not contain detectable amounts of virus, underscoring the finding that reduced acquisition lead to reduced transmission of the virus to plant tissue. Furthermore, the GN-S protein did not reduce TSWV infectivity on the local lesion plant-host Nicotiana glutinosa, indicating that transmission inhibition was due to a block in acquisition rather than a direct and lethal interaction between virions and the recombinant glycoprotein. These results indicate that GN-S reduces TSWV infection of thrips midgut epithelial cells and inhibits TSWV transmission.

Last Modified: 9/10/2014
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