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

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 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, 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.

Genome Expression Dynamics Reveal the Parasitism Regulatory Landscape of the Root-Knot Nematode Meloidogyne incognita and a Promoter Motif Associated with Effector Genes

“…With respect to insights, one ongoing hypothesis is that a small number of regulators act in concert for effector expression, and thus, the gene disruption of these few regulators can simultaneously disrupt the function of hundreds of putative effectors and have a major effect on parasitism. Progress in understanding effector biogenesis and biology is limited by a lack of tools for functional genetics on plant-parasitic nematodes, mostly due to their biology and gonad accessibility, but although this is challenging, recent improvements in mRNA delivery appear realistic and promising [ 80 ]. The current approaches for their study are mostly based on the analyses of a single effector at a time through its overexpression in plants, target gene silencing via RNAi, and the identification of target host proteins [ 9 ].…”

Genome expression dynamics reveals parasitism regulatory landscape of the root-knot nematodeMeloidogyne incognitaand a promoter motif associated with effector genes

“…With respect to insights, one ongoing hypothesis is that a small number of regulators act in concert for effector expression, and thus, the gene disruption of these few regulators can simultaneously disrupt the function of hundreds of putative effectors and have a major effect on parasitism. Progress in understanding in effector biogenesis and biology is limited by a lack of tools for functional genetics on plant-parasitic nematodes, mostly due to their biology and gonad accessibility, but although this is hitherto challenging recent improvements on mRNA delivery appear realistic and promising [80]. The current approaches for their study are mostly based on the analyses of a single effector at a time through its overexpression in plants, target gene silencing via RNAi and the identification of target host proteins [9].…”

Recent applications of biotechnological approaches to elucidate the biology of plant–nematode interactions

“…Generally, plant-parasitic nematode biology is not conducive to genetic modification (long life cycles, obligate biotrophy, inaccessible immature germlines, etc.) and as a result, genetic modification techniques that are readily deployed in other nematode species are prohibitively difficult [13]. As a result, reverse genetics in PPN is almost exclusively reliant on gene silencing by RNA interference (RNAi).…”

“…A recent advance provides the first example of reporter gene expression in any plantparasitic nematode [13]. This was achieved by bathing Heterodera schachtii juveniles in a solution containing octopamine (to stimulate uptake) and mRNA encoding a reporter protein encapsulated in a lipid bilayer (termed a liposome).…”