As the use of RNAi in crop sciences gains momentum, the US Department of Agriculture has continued focusing on the gene-silencing technology, with agency researchers kicking off at least five different RNAi-related research projects in recent months.
Two of the projects, both of which began this month, are centered on identifying gene targets for RNAi-based control of the whitefly, or Bemisia tabaci.
B. tabaci has become an important agricultural pest in recent decades as it developed resistance to conventional insecticides, not only feeding on crop plants but also acting as a vector for viruses including tomato yellow leaf curl virus, a begomovirus, and tomato chlorosis virus, a begomovirus.
With an eye toward these two viruses, USDA investigator William Wintermantle aims to run a global transcriptome analysis of two species of whiteflies that have been exposed to either plants infected with the viruses or healthy plants.
Specifically, B. tabaci colonies raised on Brassica oleracea will be introduced to virus-infected tomato plants, while a Trialeurodes vaporariorum whitefly colony raised on Physalis wrightii will be introduced to normal tomato plants, as well as blackberry and Nicotiana benthamiana plants.
Wintermantle's group will then conduct RNA sequencing on the insects to identify differentially expressed genes.
"Once whitefly functional genes or their predicted precursor sequences are identified, dsRNAs will be designed and synthesized," according to the USDA. These oligos will be fed to B. tabaci in varying concentrations, either via an artificial diet or by introduction into tomato plants by a nutrient solution, and the effects on whitefly mortality, virus acquisition, and virus transmission evaluated.
Through the work, Wintermantle aims to determine whether viral infection in the tomato plant can alter gene expression in the whitefly and whether the virus affects the same pathways in different whitefly species. Additionally, the work is expected to yield RNAi approaches for altering key biological functions in the insects.
Conducting similar work is USDA plant pathologist Kaishu Ling, who will study B. tabaci colonies raised on B. oleracea and introduced to healthy or tomato yellow leaf curl virus-infected tomato plants.
As in Wintermantle's studies, Ling will design dsRNA against target genes identified through sequencing studies, and administer them to whiteflies in order to determine their effects on mortality and virus transmission.
Wintermantle's project begins on Sept.16 and runs until March 15, 2015, while Ling's effort started on Sept. 1 and runs until the end of Sept. 2014.
Also aiming to use RNAi to combat plant pathogens are scientists from the USDA's food and feed safety unit, who are interested in using the technology to provide corn and cotton plants with resistance to the fungus Aspergillus flavus, which produces the carcinogen aflatoxin.
Part of the group's research will focus on determining if the antifungal peptide D4E1 can be produced by transgenic plants in order to create A. flavus resistance. However, the scientists will also try to develop RNAi vectors targeting genes essential to the pathogen and introduce them into corn and cotton plants. RNAi-altered plants that show increased resistance to A. flavus infection will be further studied under greenhouse and field conditions during abiotic stress conditions such as drought.
This project began on July 30 and runs for five years.
The production of aflatoxin by Aspergillus is at the center of another USDA research program, this one being run by investigators who hope to use RNAi to prevent the accumulation of the toxin in peanuts, in addition to studying the genetic components of peanut pathogens and developing high-yield varieties of the plant.
The team, lead by Renee Arias, aims to develop RNAi constructs against key Aspergillus genes. The most effective ones will be introduced into peanut plants, which will then be challenged with aflatoxigenic strains of the pathogen.
The program began on July 30 and runs for five years.
Meanwhile, a group within the USDA's crop bioprotection research unit recently began work on RNAi-based approaches to control the production of mycotoxins by Fusarium fungi in corn.
This effort will include an evaluation of known resistance-associated corn genes to determine their effects on insects and mycotoxic fungi, and to see whether they can be introduced into plants. However, it will also test whether RNAi can be used to inhibit mycotoxin production in fungi Fusarium as an alternative approach.
The RNAi constructs that prove most effective in blocking mycotoxin production in culture will be used to produce transgenic corn, the ears of which will be inoculated with fungi to see if the effect can be replicated in vivo.
This effort started on May 31 and runs until the end of May 2018.