Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation

Authors: Thekke Veetil, Thanuja; McCoppin, Nancy; DOMIER, LESLIE - Retired ARS Employee; HAJIMORAD, M - University Of Tennessee; LAMBERT, KRIS - University Of Illinois; LIM, HYOUN-SUB - Chungnam National University; Hartman, Glen

Submitted to: Molecular and Biochemical Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2022
Publication Date: 5/28/2022
Citation: Thekke Veetil, T., McCoppin, N.K., Domier, L.L., Hajimorad, M.R., Lambert, K.N., Lim, H., Hartman, G.L. 2022. Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation. Molecular and Biochemical Parasitology. 250. Article 111489. https://doi.org/10.1016/j.molbiopara.2022.111489.
DOI: https://doi.org/10.1016/j.molbiopara.2022.111489

Interpretive Summary: Plant parasitic nematodes cause significant losses in crop production each year. Yet little is known about the molecular basis for the interactions between plant and nematodes that lead to disease. It has been difficult to manipulate the expression of genes involved in these interactions because many plant parasitic nematodes are obligate parasites and reproduce sexually. This study describes the development of methods to express foreign genes in plant parasitic nematodes that utilizes brief high voltage electrical pulses to permeabilize nematode membranes and allow the uptake of messenger RNAs encoding a reporter gene. The technique successfully applied to soybean cyst nematode, rootknot nematode and the free-living model nematode, Caenorhabditis elegans and was less laborious than microinjection, which is commonly used with C. elegans. The ability to transiently express messenger RNA constructs in economically important plant parasitic nematodes provides a rapid means to evaluate nematode and/or foreign genes for their potential roles in nematode management. This technique will be of interest to researchers studying plant-nematode interactions and mechanisms of gene expression in nematodes.

Technical Abstract: Despite their economic significance in agricultural cropping systems, a lack of suitable molecular tools for manipulating gene expression has hindered progress in the functional genomics of plant parasitic nematodes (PPN). Obligate sexual reproduction and the obligate nature of PPN-host interactions further complicate the development of in vivo gene delivery and expression systems in these pests. Methods such as microinjection and microprojectile bombardment have been developed for introducing gene constructs into the free-living nematode, Caenorhabditis elegans. However, these procedures can be laborious and inefficient. Electroporation has been used extensively to introduce macromolecules, including single-stranded RNAs, into eukaryotic and prokaryotic cells. The technique has also been used for the delivery of DNA and double-stranded RNA constructs into nematodes by whole-animal electroporation. Here, we report the successful expression of a nematode-optimized NanoLuc luciferase mRNA in the form of in vitro transcripts following whole-animal electroporation of Heterodera glycines, Meloidogyne hapla, and C. elegans. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their potential roles in nematode management.