Biology subjects

Eves-van den Akker, S.

Publications and source records attributed to Eves-van den Akker, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A cross-kingdom interactome predicted by AlphaFold3 reveals a DNF2-centered interface required for symbiotic accommodation

Legumes convert atmospheric nitrogen into ammonium through symbiotic bacteria housed in root nodules, yet the molecular interactions between rhizobial and host proteins inside nodules remain poorly understood. Here we employed AlphaFold3 to construct a cross-kingdom interactome between Medicago truncatula and its symbiont Sinorhizobium meliloti. Screening more than 217,000 protein pairs yielded 7,137 putative interactions, providing a valuable resource for the broader symbiosis community. Within this network, we focused on DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), a host protein required for rhizobial persistence within nodules. We showed that DNF2 localizes to the peribacteroid space and associates with previously uncharacterized secreted rhizobial proteins (SRPs), suggesting it may function as a hub for host-symbiont communication. Notably, knockout of two DNF2-interacting proteins, SRP86 and SRP485, results in white, nitrogen-fixation-deficient nodules with abnormal symbiosomes and elevated expression of senescence-associated genes, closely phenocopying the dnf2 loss-of-function mutant. Together, our findings define a DNF2-SRP molecular framework underlying symbiotic accommodation, and illustrate the potential of AI-guided interactome mapping to uncover molecular mechanisms of plant-microbe interactions with relevance to sustainable agriculture.

plant biology↗

bioRxiv2026

3D printing and deep learning enable holistic and dynamic analyses of tens of thousands of parasites infecting hundreds of genotypes

Host-parasite interactions are dynamic systems, where parasites usually outnumber hosts by one or more orders of magnitude. However, our understanding is often limited by the assessment of parts of the host, at arbitrary time points, and/or aggregate parasite responses. Here we combined custom-built 3D-printed hardware and deep-learning-based algorithms to enable holistic (i.e. all infecting individuals on the whole plant), spatio-temporal, and parasite-centric analyses of plant-parasitism by nematodes from timelapse videos of infection over months. In so doing, we tracked the dynamic growth and development of all individual parasites, at the organismal level, for thousands of hosts across hundreds of genotypes of Arabidopsis thaliana. Categorising traits into the static (i.e. in an acquired image at a given time point) and dynamic (i.e. phenotypic changes over time), we revealed a greater extent of host-genetic control of parasite traits, and new physiological limits of the species under these conditions. Using this capability, we identify Quantitative Trait Loci (QTL) in the host plant associated with 18 phenotypic traits in the parasite as a resource for the community. Finally, we leverage the large and diverse dataset to understand fundamental features of the parasite, independent of host genotype, revealing aspects of the life cycle which are pseudo-deterministic as well as local, deleterious interactions between co-infecting parasites. Given that plant-parasitic nematodes cause an estimated $100 billion in agricultural damages per year, these insights are contextualised in a global challenge driven by plant-parasitic nematodes.

pathology↗

bioRxiv2026

Diversity at the HYP1 locus in potato cyst nematodes does not result from developmentally-programmed somatic mutations

Most genetic diversity stems from spontaneous mutations, that is, errors in DNA repair or replication. But for dozens of organisms across the tree of life, mutations at specific loci are not spontaneous but developmentally programmed: effectively, some organisms edit their own DNA sequences. This is perhaps most common among pathogens and parasites, many of which use editing to diversify genes that produce important antigens. Plant-parasitic potato cyst nematodes are damaging agricultural pests that establish a lifelong feeding site inside the root of their host plant. We previously observed extensive diversity of rare alleles at HYP1, the most highly expressed gene that encodes a protein secreted by potato cyst nematodes during parasitism. Importantly, HYP1 alleles differ from each other by complex, in-frame rearrangements of short repeated sequence motifs within a single exon. Combining several lines of evidence, we previously hypothesized that potato cyst nematodes use developmentally-programmed mutations, or editing, to diversify HYP1 alleles in the soma. In the current work, we now test this hypothesis. We employ highly accurate long-read DNA sequencing of a simplified genetic system to identify potential rare edited alleles, we use a transgenic yeast system to describe large de novo mutations at HYP1, and we interpret our findings in light of key population genetic parameters as well as the genetic diversity surrounding HYP1 and across the genome. Significance StatementIn parasites, genetic diversity is valuable fuel for the coevolutionary arms race with their hosts. However, in parasitic nematode worms, low genetic diversity is often observed. Several other parasites and pathogens (e.g., single-celled eukaryotes) generate genetic diversity in an unusual way: they edit the DNA sequence of an important gene in their own genome, instead of waiting for rare spontaneous mutations to occur. This has never been observed in a plant parasite, but we previously described a major parasitism gene (HYP1) in plant-parasitic nematodes with such diverse DNA sequences that it looks like it could perhaps be edited. Now, we generate better data for a simplified genetic system and show that much of the observed HYP1 diversity was actually cryptic DNA sequencing errors. Although there is real genetic diversity at HYP1, it is best explained not by editing but by fundamental evolutionary forces that operate across the genome.

genetics↗

bioRxiv2026

A transcription factor-pair work in concert to regulate gene expression across the life cycle of the pinewood nematode, Bursaphelenchus xylophilus

The migratory endoparasitic pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, causing significant economic and ecological losses in conifer forest ecosystems in Europe and Asia. Understanding the molecular mechanisms regulating PWN parasitism-related genes may lead to new sustainable solutions for control. Based on previous PWN transcriptomic datasets from the pre-parasitic and parasitic stages and from the pharyngeal gland cells (GC), an in silico analysis was performed to identify transcription factors (TF) highly expressed in the GC. Seven candidates TF genes were selected, and their spatial expression validated by in situ hybridisation. From those, two GC-expressed TFs, BXY_079 and BXY_022, each encoding zinc finger domains, were successfully knocked down by RNA interference. Transcriptomic data from silenced BXY_079 and BXY_022 TFs, analysed with existing life cycle specific transcriptomic data, showed that both TFs control genes expressed at similar times, by repressing male-related genes while activating genes expressed during the J3 and D3 stages, yet each represents the extreme of the others minor function. In addition to these common roles, BXY_079 also activates parasitism-related genes in the J2 stage. These BXY_079-activated parasitism-related genes predominantly encode proteins with lytic functions, including secreted peptidases and glycoside hydrolases. Consistent with their proposed role in parasitism, these genes are highly expressed during the parasitic juvenile stages and are likely involved in nematode feeding, tissue penetration, and migration within the host. In contrast, BXY_022 also represses the expression of several genes related to the reproduction system, such as major sperm proteins and cytosolic motility proteins, particularly in the adult male stage. Taken together, both dual-functional TFs work together, non-redundantly, to regulate gene expression across the life cycle, while each is additionally specialised to regulate diverse and distinct gene sets: ranging from genes implicated in lytic parasitic functions to sexual dimorphism.

pathology↗

bioRxiv2026

Antagonistic action of a nuclear hormone receptor pair coordinates a switch from lytic to biotrophic effector production in a plant-parasitic nematode

Pathogens secrete overlapping and sequential waves of effectors to manipulate their host, and yet the regulators that conduct the ensemble are poorly understood. Here, we identify the Dorsal Gland Regulator DGR-1 in the beet cyst nematode Heterodera schachtii. DGR-1 controls the expression of 131 putative effectors, acting as a dual-functional switch that "switches off" early stage effectors involved in plant invasion, and "switches on" later stage effectors associated with biotrophic establishment in the host. Interestingly, DGR-1 works antagonistically with the only other known transcriptional regulator of effectors in plant-parasitic nematodes, the Subventral Gland Regulator-1 (SUGR-1), to coordinate this apparent switch from lytic to biotrophic effector production. Together, DGR-1 and SUGR-1 control the expression of nearly one half of all H. schachtii early-stage effectors, and over one fifth of effectors of any kind. The requirement to activate lytic effector functions in the cortex, and biotrophic effector functions in the vascular cylinder, suggests that this transcription factor pair must differentially respond to signals from the host. Consistent with this, we find that diffusates from the roots of A. thaliana Casparian strip mutant myb36/sgn3, which are enriched in molecules ordinarily restricted to the vascular cylinder, upregulate dgr-1, but not sugr-1, compared to Col-0. Taken together, these data indicate that H. schachtii responds to compartmentalised host-derived signals to appropriately regulate spatiotemporal effector expression during infection. Given that misregulating DGR-1 results in delayed development of parasitic nematodes in Arabidopsis and Mustard, strategies which impair effector regulation as a whole may hold promise for crop protection against these agriculturally important pathogens.

pathology↗

bioRxiv2026

Better late than never: the impact of hatching time on Heterodera schachtii parasitism

Plant-parasitic nematodes are a diverse, polyphyletic group of plant pathogens which can infect most plant tissues and all major crops. Amongst the most damaging clades are the cyst nematodes, which can remain dormant in the soil for decades as infection-competent, developmentally arrested, second-stage juveniles in eggs. Hatching is stimulated by a variety of factors. However, the impact of hatching factor responsiveness on nematode morphology, physiology, gene expression, and infection biology has not been explored. We examined the impact of hatching time on the beet cyst nematode, Heterodera schachtii. We found that late hatchers invaded host roots and established feeding sites in greater numbers than early hatchers. We demonstrate variation in baseline parasitism gene expression and in responsiveness of genes to effectostimulins, small, plant-derived molecules which upregulate parasitism genes. Three quarters of effectostimulin-induced transcriptional changes were also modulated, either positively or negatively, by hatching time. While there were no observable morphological differences between early and late hatching nematodes on the day of their emergence from the egg, the late hatchers displayed signs of faster utilisation of internal energy reserves after 7 days at 4{degrees}C, as evidenced by less body area attributed to fat, than early hatchers. Finally, we found no evidence of substantive genetic differences between early and late hatchers, they were representative of a single population, despite the observed differences in infection, gene expression, and physiology. Taken together, non-genetic differences likely drive late hatchers to more rapidly utilise their internal energy reserves, to be more responsive to host-derived signals, and to be ultimately more infective than their early hatching counterparts.

molecular biology↗

bioRxiv2026

The sugar-beet cyst nematode effector Hs2B11 targets the Arabidopsis serine protease inhibitor AtPR-6 to favor parasitism

Cyst nematodes secrete effector proteins to manipulate host cell biology and suppress immunity, yet the mechanisms underlying these interactions remain largely unexplored. In this study, we characterize the function of Hs2B11, a Heterodera schachtii effector that was previously shown to be expressed in the dorsal gland of sugar-beet cyst nematodes (BCN). Here, we report that in Arabidopsis thaliana Hs2B11 functions as an immune regulator that modulates the production of elicitor-induced oxidative species, likely to favor parasitism. To elucidate the molecular basis of this immune suppression, we performed a yeast-two-hybrid screen and identified the host serine protease inhibitor AtPR-6 as a direct interactor of Hs2B11. We show that AtPR-6 acts as a positive regulator of plant immunity; its expression is induced upon nematode infection and knock-out of AtPR-6 compromises oxidative species production leading to higher susceptibility to H. schachtii infection. Conversely, AtPR-6 overexpression enhances immune responses resulting in increased resistance to BCN infection. Detailed analysis of this interaction demonstrated that Hs2B11 interacts with AtPR-6 using its carboxyl-terminal domain. AlphaFold2 predicts that the C-terminal domain forms a beta-solenoid-like structure with a ladder of serine residues organized across one of its surfaces. We propose that using this interface, Hs2B11 targets AtPR-6 via molecular titration, preventing the inhibitor from regulating host proteases that control immune signaling. These findings highlight a counter-defense strategy where a nematode effector neutralizes a specific host protease inhibitor to subvert plant immunity.

plant biology↗

bioRxiv2025

The host range paradox of Meloidogyne incognita: a physiological and transcriptomic analysis of nine susceptible interactions across six plant orders

The plant-parasitic nematode Meloidogyne incognita is the pathogen with the broadest host range among all known biotrophic interactions. This species is also the single most damaging of a group of agriculturally important plant-parasites, which together are estimated to contribute to losses in excess of $170 billion/year to world agriculture. Understanding how M. incognita is able to infect representatives from most orders of flowering plants, covering more than 3000 species, addresses a fundamentally important question of how pathogens adapt to their host-environment, and may inform control of a pathogen which threatens global food security. Here, we analyse the plant-nematode infection phenotype, and cross-kingdom transcriptome, of nine interactions across six orders of flowering plants at 25 days post infection. At this stage, majority of nematodes found within roots were immature and mature females (49.2% - 91.8%). Our data show that the phylogenetic distribution of hosts does not explain the phenotypic distribution of parasitism. Interestingly, though, M. incognita do have distinct transcriptional responses to different groups of hosts, but in a pattern which is independent of host phylogenetic. Three distinct nematode "transcriptional programmes" - Group 1, 2, and 3 - are evident, and we find that effectors are neither uniformly deployed across hosts, nor across groups of hosts. Importantly, we show that this differential deployment of effectors can have profound consequences for host specificity. Finally, we show that there is essentially no widespread core gall transcriptome at 25 days post infection, prompting the proposal of a model best described as "all roads lead to Rome".

plant biology↗

bioRxiv2025

DNA-protein interactions in Meloidogyne incognita

BackgroundThe root-knot nematode Meloidogyne incognita, is a highly destructive parasite that manipulates host plant processes through effector proteins, affecting agriculture globally. Despite advances in genomic and transcriptomic studies, the regulatory mechanisms controlling effector gene expression, especially at the chromatin level, are still poorly understood. Gene regulation studies in plant-parasitic nematodes (PPN) face several challenges, including the absence of transformation systems and technical barriers in chromatin preparation, particularly for transcription factors (TFs) expressed in secretory gland cells. Conventional methods like Chromatin Immunoprecipitation (ChIP) are limited in PPN due to low chromatin yields, the impermeability of nematode cuticles, and difficulties in producing antibodies for low-abundance TFs. These issues call for alternative approaches, such as dCas9-based CAPTURE (CRISPR Affinity Purification in siTU of Regulatory Elements) that allows studying chromatin interactions by using a catalytically inactive dCas9 protein to target specific genomic loci without relying on antibodies. ResultsThis study presents an optimized in vitro dCas9-based CAPTURE for M. incognita that addresses key challenges in chromatin extraction and stability. The protocol focuses on the promoter region of the effector gene 6F06, a critical gene for parasitism. Several optimizations were made, including improvements in nematode disruption, chromatin extraction, and protein-DNA complex stability. This method successfully isolated chromatin-protein complexes and identified four putative chromatin-associated proteins, including BANF1, linked to chromatin remodelling complexes like SWI/SNF. ConclusionThe optimized in vitro dCas9-based CAPTURE protocol offers a new tool for investigating chromatin dynamics and regulatory proteins in non-transformable nematodes. This method expands the scope of effector gene regulation research and provides new insights into parasitism in M. incognita. Future research will aim to validate these regulatory proteins and extend the method to other effector loci, potentially guiding the development of novel nematode control strategies.

molecular biology↗

bioRxiv2024

The SUbventral-Gland master Regulator (SUGR) of nematode virulence

All pathogens must tailor their gene expression to their environment. Therefore, targeting host:parasite biology that regulates these changes in gene expression could open up routes to pathogen control. Here, we show that in the plant-parasitic nematode Heterodera schachtii, host signals (termed effectostimulins) within plant roots activate the master regulator sugr1. SUGR1, then, directly binds effector promoters, and orchestrates their production. Effector production, in turn, facilitates host entry, releasing more effectostimulins. These data show that gene expression during the very earliest stages of parasitism is defined by a feed forward loop for host entry. Importantly, we demonstrate that blocking SUGR1 blocks parasitism, underlining the SUGR1 signalling cascade as a valuable target for crop protection. Given that nematodes also parasitise humans and other animals, the potential impact is broad: disrupting effector production could, in principle, be applied to any pathogen that secrets effectors. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/576598v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@cebedaorg.highwire.dtl.DTLVardef@153fe87org.highwire.dtl.DTLVardef@16b6957org.highwire.dtl.DTLVardef@d0e920_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

The origin, deployment, and evolution of a plant-parasitic nematode effectorome

Plant-parasitic nematodes constrain global food security. During parasitism, they secrete effectors into the host plant from two types of pharyngeal gland cells. These effectors elicit profound changes in host biology to suppress immunity and establish a unique feeding organ from which the nematode draws nutrition. Despite the importance of effectors in nematode parasitism, there has been no comprehensive identification and characterisation of the effector repertoire of any plant-parasitic nematode. To address this, we advance techniques for gland cell isolation and transcriptional analysis to define a stringent annotation of putative effectors for the cyst nematode Heterodera schachtii at three key life-stages. We define 659 effector gene loci: 293 "known" high-confidence homologs of plant-parasitic nematode effectors, and 366 "novel" effectors with high gland cell expression. In doing so we define a comprehensive "effectorome" of a plant-parasitic nematode. Using this effector definition, we provide the first systems-level understanding of the origin, deployment and evolution of a plant-parasitic nematode effectorome. The robust identification of the comprehensive effector repertoire of a plant-parasitic nematode will underpin our understanding of nematode pathology, and hence, inform strategies for crop protection.

plant biology↗

bioRxiv2023

A gene with a thousand alleles: the HYPer-variable effectors of plant-parasitic nematodes

Pathogens are engaged in a fierce evolutionary arms race with their host. The genes at the forefront of the engagement between kingdoms, including effectors and immune receptors, are part of diverse and highly mutable gene families. Even in this context, we discovered unprecedented variation in the HYPer-variable (HYP) effectors of plant-parasitic nematodes. We discovered single effector gene loci that can harbour potentially thousands of allelic variants. These alleles vary in the number, but principally in the organisation, of motifs within a central Hyper Variable Domain (HVD). Using targeted long-read sequencing, we dramatically expand the HYP repertoire of two plant-parasitic nematodes, Globodera pallida and G. rostochiensis, such that we can define distinct sets of species-specific "rules" underlying the apparently flawless genetic rearrangements. Finally, by analysing the HYP complement of 68 individual nematodes we made the unexpected finding that despite the huge number of alleles, most individuals are homozygous. Taken together, these data point to a novel mechanism of programmed genetic variation, termed HVD-editing, where alterations are locus-specific, strictly governed by rules, and can theoretically produce thousands of variants without errors.

genetics↗

bioRxiv2023

Whole mount multiplexed visualization of DNA, mRNA, and protein in plant-parasitic nematodes

BackgroundPlant-parasitic nematodes compromise the agriculture of a wide variety of the most common crops worldwide. Obtaining information on the fundamental biology of these organisms and how they infect the plant has been restricted by the ability to visualize intact nematodes using small molecule stains, antibodies, or in situ hybridization. Consequently, there is limited information available about the internal composition of the nematodes or the biology of the effector molecules they use to reprogram their host plant. ResultsWe present the Sperling prep -a whole mount method for nematode preparation that enables staining with small molecules, antibodies, or in situ hybridization chain reaction. This method does not require specialized apparatus and utilizes typical laboratory equipment and materials. By dissociating the strong cuticle and interior muscle layers, we enabled entry of the small molecule stains into the tissue. After permeabilization, small molecule stains can be used to visualize the nuclei with the DNA stain DAPI and the internal structures of the digestive tract and longitudinal musculature with the filamentous actin stain phalloidin. The permeabilization even allows entry of larger antibodies, albeit with lower efficiency. Finally, this method works exceptionally well with in situ HCR. Using this method, we have visualized effector transcripts specific to the dorsal gland and the subventral grand of the sugar beet cyst nematode, Heterodera schachtii, multiplexed in the same nematode. ConclusionWe were able to visualize the internal structures of the nematode as well as key effector transcripts that are used during plant infection and parasitism. Therefore, this method provides an important toolkit for studying the biology of plant-parasitic nematodes.

plant biology↗

bioRxiv2022

A low-cost and open-source solution to automate imaging and analysis of cyst nematode infection assays for Arabidopsis thaliana

BackgroundCyst nematodes are one of the major groups of plant-parasitic nematode, responsible for considerable crop losses worldwide. Improving genetic resources, and therefore resistant cultivars, is an ongoing focus of many pest management strategies. One of the major bottlenecks in identifying the plant genes that impact the infection, and thus the yield, is phenotyping. The current available screening method is slow, has unidimensional quantification of infection limiting the range of scorable parameters, and does not account for phenotypic variation of the host. The ever-evolving field of computer vision may be the solution for both the above-mentioned issues. To utilise these tools, a specialised imaging platform is required to take consistent images of nematode infection in quick succession. ResultsHere, we describe an open-source, easy to adopt, imaging hardware and trait analysis software method based on a pre-existing nematode infection screening method in axenic culture. A cost-effective, easy-to-build and -use, 3D-printed imaging device was developed to acquire images of the root system of Arabidopsis thaliana infected with the cyst nematode Heterodera schachtii, replacing costly microscopy equipment. Coupling the output of this device to simple analysis scripts allowed the measurement of some key traits such as nematode number and size from collected images, in a semi-automated manner. Additionally, we used this combined solution to quantify an additional trait, root area before infection, and showed both the confounding relationship of this trait on nematode infection and a method to account for it. ConclusionTaken together, this manuscript provides a low-cost and open-source method for nematode phenotyping that includes the biologically relevant nematode size as a scorable parameter, and a method to account for phenotypic variation of the host. Together these tools highlight great potential in aiding our understanding of nematode parasitism.

plant biology↗

bioRxiv2021

The genome and lifestage-specific transcriptomes of a plant-parasitic nematode and its host reveal susceptibility genes involved in trans-kingdom synthesis of vitamin B.

Plant-parasitic nematodes are a major, and in some cases a dominant, threat to crop production in all agricultural systems. The relative scarcity of classical resistance genes highlights a pressing need to identify new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality genome of the model cyst nematode Heterodera schachtii to provide a platform for the first system-wide dual analysis of host and parasite gene expression over time, covering all major stages of the interaction. This novel approach enabled the analysis of the hologenome of the infection site, to identify metabolic pathways that were incomplete in the parasite but complemented by the host. Using a combination of bioinformatic, genetic, and biochemical approaches, we show that the highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is critically important for parasitism. Knockout of either the plant-encoded or the now nematode-encoded steps in the pathway blocks parasitism. Our experiments establish a reference for cyst nematodes, use this platform to further our fundamental understanding of the evolution of plant-parasitism by nematodes, and show that understanding congruent differential expression of metabolic pathways represents a new way to find nematode susceptibility genes, and thereby, targets for future genome editing-mediated generation of nematode-resistant crops.

genetics↗

bioRxiv2021

Genome expression dynamics reveals parasitism regulatory landscape of the root-knot nematode Meloidogyne incognita and a promoter motif associated with effector genes

Root-knot nematodes are the major contributor to the crop losses caused by nematodes. Root-knot nematodes secrete effectors into the plant, derived from two sets of pharyngeal gland cells, to manipulate host physiology and immunity. Successful completion of the life cycle, involving successive molts from egg to adult, covers morphologically and functionally distinct stages and will require precise control of gene expression, including effectors. The details of how root-knot nematodes regulate transcription remain sparse. Here, we report a life stage-specific transcriptome of Meloidogyne incognita. Combined with an available annotated genome, we explore the spatio-temporal regulation of gene expression. We reveal gene expression clusters and predicted functions that accompany the major developmental transitions. Focusing on effectors, we identify a putative cis-regulatory motif associated with expression in the dorsal glands: providing an insight into effector regulation. We combine the presence of this motif with several other criteria to predict a novel set of putative dorsal gland effectors. Finally, we show this motif, and thereby its utility, is broadly conserved across the Meloidogyne genus and termed it Mel-DOG. Taken together, we provide the first genome-wide analysis of spatio-temporal gene expression in a root-knot nematode, and identify a new set of candidate effector genes that will guide future functional analyses.

genomics↗

bioRxiv2020

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

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.

genetics↗

bioRxiv2018

The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes

Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock. Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7Mb), and horizontal gene transfer events (151 genes). Using previously published effector sequences, the newly generated H. glycines genome, and comparisons to other nematode genomes, we investigate the evolutionary mechanisms responsible for the emergence and diversification of effector genes.

genomics↗