Toward genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles.

Affiliation

Kranse O(1), Beasley H(1), Adams S(2), Pires-daSilva A(2), Bell C(3), Lilley CJ(3), Urwin PE(3), Bird D(4), Miska E(5), Smant G(6), Gheysen G(7), Jones J(8)(9), Viney M(10), Abad P(11), Maier TR(12), Baum TJ(12), Siddique S(13), Williamson V(14), Akay A(15), Eves-van den Akker S(1).
Author information:
(1)Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, UK.
(2)School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK.
(3)Centre for Plant Sciences, School of Biology, University of Leeds, Leeds, LS2 9JT, UK.
(4)Entomology and Plant Pathology, NC State University, Raleigh, NC 27695-7613, USA.
(5)Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Genetics, University of Cambridge, Cambridge CB2 1QN, UK.
(6)Laboratory of Nematology, Department of Plant Sciences, Wageningen University & Research, 6708 PB Wageningen, The Netherlands.
(7)Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, 9000 Ghent, Belgium.
(8)Cell & Molecular Sciences Department, The James Hutton Institute, Dundee, DD2 5DA, UK.
(9)School of Biology, Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK.
(10)Department of Evolution, Ecology and Behaviour, University of Liverpool, Liverpool L69 7ZB, UK.
(11)INRAE, Université Côte d'Azur, CNRS, ISA, F-06903 Sophia Antipolis, France.
(12)Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011, USA.
(13)Department of Entomology and Nematology, University of California, Davis, Davis, CA 95616, USA.
(14)Department of Plant Pathology, University of California, Davis, Davis, CA 95616, USA.
(15)Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK.

Abstract

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.