RNAi Effector Diversity in Nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes

<em>flp-32</em> Ligand/Receptor Silencing Phenocopy Faster Plant Pathogenic Nematodes

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role offlp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii)Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R-silenced worms also display an increase in migration rate. This work demonstrates that Gp-flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR

A novel <i>Gp-flp</i>-32 receptor (<i>Gp-flp</i>-32R) is expressed in <i>Globodera pallida</i> J2s and is conserved across the nematode phylum.

<p>A novel <i>Gp-flp</i>-32R which shows close homology to the <i>C. elegans</i> VRFa receptor 1 (C26F1.6) is expressed in <i>G. pallida</i> (A). The 389 amino acid protein has seven transmembrane helices; outside to inside helices are boxed in red; inside to outside helices are boxed in blue (A). 11 conserved residues common to rhodopsin family GPCRs are denoted by asterisks (A). Interrogation of available nematode EST, genomic and transcriptomic datasets show that at least 12 of the 16 nematode species from clades IV and V which express <i>flp</i>-32 also express a homologue of the <i>Gp-flp</i>-32R (B), demonstrating inter-clade conservation of the <i>flp</i>-32 receptor. <i>B. xylophilis: Bursaphelenchus xylophilus; G. pallida: Globodera pallida; G rostochiensis: Globodera rostochiensis; M. hapla</i>: <i>Meloidogyne hapla</i>; <i>M. incognita</i>: <i>Meloidogyne incognita</i>; <i>M. paranaensis: Meloidogyne paranaensis; R. similis: Radopholus similis; S. ratti: Strongyloides ratti; A. caninum: Ancylostoma caninum; A. cantonensis: Ancylostoma cantonensis; A. ceylanicum; Ancylostoma ceylanicum; C. elegans: Caenorhabditis elegans; H. polygyrus: Heligmosomoides polygyrus; N. americanus</i>: <i>Necator americanus; N. brasiliensis: Nippostrongylus brasiliensis; T. circumcincta: Teladorsagia circumcincta.</i></p

<i>Meloidogyne incognita flp</i>-32 (<i>Mi-flp</i>-32) silenced nematodes exhibit accelerated migration rates.

<p>Post-RNAi <i>Mi-flp</i>-32 silenced worms displayed an increased speed of migration down a vertical sand column when compared to control worms. After 2 h migration 62% of <i>Mi-flp</i>-32 silenced worms had migrated compared to 35% and 13% of control siRNA and untreated worms, respectively (<i>P</i><0.01; <i>P</i><0.001); after 4 h 99% of <i>Mi-flp</i>-32 silenced worms had migrated compared to 73% and 58% of control siRNA and untreated worms respectively (<i>P</i><0.01; <i>P</i><0.001); after 6 h all <i>Mi-flp</i>-32 silenced worms had completed migration. There was no significant difference in the migratory ability of control siRNA and untreated worms over the 6 h migration period.</p

The rate of potato plant root infection is enhanced in <i>Gp-flp</i>-32-silenced <i>Globodera pallida</i> J2s.

<p>Infection rates of <i>Gp-flp</i>-32, <i>Gp-ace</i> (positive control siRNA), control siRNA (non-native negative control) and untreated worms identified by acid fuschin staining in the roots of potato plants challenged with nematodes post-RNAi. <i>Gp-flp</i>-32 siRNA treated worms show a significantly higher mean infection rate of 74% compared to control siRNA (31%) and untreated (35%) worms (<i>P</i><0.001) as shown by the graph and representative light microscopy images of pink acid fuschin stained nematodes in root segments. There was no significant difference in the mean infection rate of <i>Gp-ace</i> siRNA, control siRNA and untreated worms.</p

<i>Globodera pallida</i> FLP-32 (AMRNALVRFa) is localised in the nervous system of pre-parasitic juveniles.

<p>Confocal microscopy images of <i>G. pallida</i> FLP-32 (AMRNALVRFa) immunoreactivity (IR; green; A) demonstrate staining in the circumpharyngeal nerve ring (CNR) posterior to the pharyngeal bulb (PHB), and in nerve processes (*) running anteriorly towards the stylet protractor muscles (SPM). IR is also evident in the ventral nerve cord (VNC; A) which emanates from the CNR and runs parallel to the body wall muscle (BWM; red) into the tail where a group of AMRNALVRFa-positive cell bodies are located in close proximity to the lumbar ganglia (LG; B). Scale bars = 75 µm.</p

<i>Globodera pallida flp</i>-32 (<i>Gp-flp</i>-32) encodes a single peptide (AMRNALVRFG) and is expressed in at least 16 nematode species.

<p><i>Gp-flp</i>-32 encodes one FMRFamide-like peptide, AMRNALVRFG (highlighted in yellow; A), flanked by dibasic cleavage sites (KK/KR; highlighted in green; A), and preceded by a predicted 28 amino acid N-terminal signal peptide (underlined; A). At least 16 nematode species from clades IV (species boxed in blue) and V (species boxed in red) express <i>flp-32</i>, encoding the single highly conserved AMRNA/SLVRFG FLP-32 peptide (highlighted in yellow; B), flanked by conserved dibasic cleavage sites (KK/KR; highlighted in green; B). <i>B. xylophilis: Bursaphelenchus xylophilus; G. pallida: Globodera pallida; G. rostochiensis: Globodera rostochiensis; M. hapla</i>: <i>Meloidogyne hapla</i>; <i>M. incognita</i>: <i>Meloidogyne incognita</i>; <i>M. paranaensis: Meloidogyne paranaensis; R. similis: Radopholus similis; S. ratti: Strongyloides ratti; A. caninum: Ancylostoma caninum; A. cantonensis: Ancylostoma cantonensis; A. ceylanicum; Ancylostoma ceylanicum; C. elegans: Caenorhabditis elegans; H. polygyrus: Heligmosomoides polygyrus; N. americanus</i>: <i>Necator americanus; N. brasiliensis: Nippostrongylus brasiliensis; T. circumcincta: Teladorsagia circumcincta</i>.</p

<i>Globodera pallida flp</i>-32 (<i>Gp-flp</i>-32) and FLP-32 receptor (<i>Gp-flp</i>-32R) silenced nematodes exhibit accelerated migration rates.

<p>siRNA soaking reduces <i>Gp-flp</i>-32 and <i>Gp-flp</i>-32R transcript levels by 55% and 75% respectively (A) compared to controls (control siRNA and untreated worms; <i>P</i><0.001). Post-RNAi <i>Gp-flp</i>-32 and <i>Gp-flp</i>-32R silenced worms displayed an increased speed of migration down a vertical sand column when compared to control worms (B and C). After 4 h migration 84% of <i>Gp-flp</i>-32 and 91% of <i>Gp-flp</i>-32R worms had migrated compared to 59% and 64% of control siRNA and untreated worms respectively (P<0.01; B and C), after 6 h all <i>Gp-flp</i>-32 and <i>Gp-flp</i>-32R silenced worms had completed migration (B and C). There was no significant difference in the migratory ability of <i>Gp-flp</i>-32 and <i>Gp-flp</i>-32R silenced worms over the 6 h migration period (C).</p