standard Ribonucleoprotein Particles Improve Oral RNAi Delivery to Crop Pests

Boll weevil.jpg

Anthonomus grandis, the boll weevil. USDA ARS image ID K2742-6

The efficacy of RNAi in insects depends in part on efficient delivery and uptake of dsRNA.  Gillet et al. (2017) report in Frontiers of Physiology on their use of protein-complexed dsRNA to silence genes in Anthonomous grandis following oral delivery, and make a number of interesting observations.

Oral delivery of dsRNA for the purposes of silencing insect genes is often convenient but equally important it is a mode of delivery that makes the creation of insect resistant plants feasible through the use of transgenic plant technologies.  Transgenic cotton expressing insect specific dsRNAs, according to the authors, is something of considerable interest in Brazil where Anthonomous grandis remains a major pest.

Gillet et al.  provide a nice introduction to previous efforts to use dsRNA in insects and highlight two aspects of this technology that are problematic in many insects:  degradation of dsRNA in the gut and hemolymph, and poor uptake of dsRNA by cells.

These are problems that are also central to developing RNAi-based therapeutics for humans, as Gillet et al. explain, and consequently there is a rich selection of potential solutions that might be adapted for use in insects because of the very active research and development efforts that have been ongoing for many years in the human therapeutics space.

Gillet et al.  have chosen a strategy involving the association of  dsRNA with proteins that mitigate the problems of degradation and uptake, and, interestingly, report some success from their efforts.

Cell penetrating peptides are being used to enhance the uptake of a variety of cargoes. PTD is a derivative of HIV Tat. Image credit. http://www.bio-itworld.com/archive/051904/firstbase.html

The authors use a chimeric protein consisting of domains with dsRNA binding activity and a domain that promote cell uptake.

Cell penetrating peptides are a broad class of proteins characterized by their property of being readily taken up by cells by any one of a number of processes.   The Tat protein from the HIV is a well-studied cell penetrating peptide that has been developed into a tool for moving various cargoes into cells. Gillet et al. used an enhanced version of Tat referred to as PTD (peptide transduction domain).

diagram of PKR

The basic structure and function of human protein kinase R. Image credit: https://medicine.ekmd.huji.ac.il/En/Publications/ResearchersPages/Pages/pages_raymondkProjects/project07.aspx

Attached to PTD are the two dsRNA binding domains  of the human RNA dependent protein kinase (DRBD), a mediator of antiviral and inflammatory responses.

This is a PTD-DRBD fusion protein. Credit: Dowdy Lab/UC San Diego

So, the dsRNA binding domains associate with the RNA and the cell penetrating peptide ‘carries’ the ribonucleoprotein particle into the cell.  You can read more about PTD-DRBD in Eguchi et al. (2009).

Gillet et al. report evidence that the rate of dsRNA degradation by benzonase, a bacterial nuclease with protein family members in A. grandis, was reduced.  It is not exactly clear why but perhaps simply by reduced access due to binding of PKR RNA binding domains.  They also present some evidence that the PTD-DSRB ribonucleoprotein enhanced uptake into gut epithelial cells of A. grandis attributable to PTD.  And, finally, the authors fed the ribonucleoprotein complex to A. grandis and reported reduced gene expression of a chitin synthase gene – not enough to kill the insects but a significant reduction nonetheless.

There are many approaches to delivering dsRNA to cells and this one seems to have promise and, importantly, is adaptable to being used in transgenic plants.

 

 

Gillet, F-X, Garcia, RA, Macedo, LLP, Albuquerque, EVS, Silva, MCM, Grossi-de-Sa, MF. Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests. Frontiers in Physiology 2017; 8. doi.org: 10.3389/fphys.2017.00256

Eguchi, A, Meade, BR, Chang, Y-C, Fredrickson, CT, Willert, K, Puri, N, Dowdy, SF. Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat Biotech 2009; 27:567-571. doi:10.1038/nbt.1541

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