With the help of a US Department of Agriculture grant, a Kansas State University doctoral student is using RNAi to combat an insect that acts as a vector for a plant disease responsible for an estimated $1 billion in crop damage worldwide each year.
According to Ismael Badillo-Vargas, who received the $71,000 award from the USDA's National Institute of Food and Agriculture, the project stems from an earlier effort to elucidate the interactions between tomato spotted wilt virus, which affects more than 1,000 plant species including tomato, potato, and peanut, and the western flower thrip, a common agricultural pest that not only feeds on a variety of plants but acts as a vector for the virus.
Interested in uncovering how the thrips transmit the virus while avoiding any deleterious effects, Badillo-Vargas and colleagues at KSU previously conducted a proteomic analysis to identify differentially expressed proteins between infected and non-infected insects.
Hypothesizing that thrip larvae mount a response that protects against the virus as it spreads from midgut epithelial cells where it replicates to salivary glands, the site where the virus is transmitted from insect to plant, the investigators generated a partial thrip transcriptome and uncovered “a suite of candidate proteins that respond to virus infection,” according to a paper they published last summer in the Journal of Virology.
Among these were proteins that play roles in the infection cycle of different plant- and animal-infecting viruses and antiviral defense responses, they wrote.
“Once I had a catalog of candidate proteins that interact with the virus, the questions [became], 'What do we know about these proteins and would it be possible to silence some of these genes to see what happens to the virus?” Badillo-Vargas told Gene Silencing News. Following gene silencing, would the insects still become infected with tomato spotted wilt virus, and, if so, would the virus be able to replicate and spread to other tissues?
With the help of the USDA grant, Badillo-Vargas began measuring the expression levels of transcripts representing a subset of the proteins previously identified and their “relative abundance in relation to viral titer.” He then worked to identify “gut proteins from thrips that directly interact with [the virus] during attachment and entry of the virus into the midgut epithelial cells.”
Among those, Badillo-Vargas specifically focused on ones that are highly expressed in the insect gut since any RNAi intervention would be done by feeding the thrips dsRNA, he noted.
Badillo-Vargas said he has identified a number of target genes involved in the acquisition of nutrients from food by the insects, as well as ones that appear to help the virus enter cells. He has generated dsRNAs against these and is now in the process of establishing proof of concept for an RNAi-based thrip-control approach.
Because RNAi has not been established in thrips, he is first attempting to deliver the dsRNA through direct injection simply to show that gene silencing can occur, buoyed by data indicating that the insects harbor the necessary cellular machinery including Dicer and argonaute proteins.
Once that has been done, “we can apply this as a tool to study particular genes and their interaction with the virus,” he said. Ultimately, however, he hopes the approach can be used to control thrip populations and tomato spotted wilt virus transmission, either through the development of an RNAi-based pesticide that can be used to treat plants or the creation of shRNA-expressing transgenic plants.