Plant-parasitic nematodes are a current and future 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 progress in plant-parasitic nematology, 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 characterise the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimise 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, and, taken together, will expedite the development of genetic modification protocols for sedentary endoparasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

Gene sequences of H. schachtii