The <i>Pratylenchus penetrans</i> transcriptome as a source for the development of alternative control strategies:mining for putative genes involved in parasitism and evaluation of <i>in planta</i> RNAi

The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for selection and use to design effective control strategies against root lesion nematodes

Myoinhibitory peptide signaling as a modulator of aversive gustatory learning in C. elegans

Learning and memory processes are crucial for animals to adapt their choices and behavioral responses according to previous experiences to increase their chances of survival. Neuropeptides are essential signaling molecules in the brain and are emerging as important modulators of learned behaviors. For many neuropeptides, however, the central function in the nervous system remains largely unknown and their precise role is not well understood. In this study, we used the genetically tractable neurobiological model Caenorhabditis elegans to investigate the neuropeptidergic modulation of learning and memory. We hypothesized that the best candidate neuropeptides to study in learning and memory are those that are conserved across the Animal Kingdom and of which the cellular expression is largely observed in brain areas known to be involved in learning and memory. Such a neuropeptide system is the evolutionarily conserved myoinhibitory peptide (MIP) system, initially discovered in locusts and mainly studied in insects. Although MIP neuropeptides and their receptors have not been directly implicated in learning, we hypothesized that they may modulate learning circuits based on their expression in the insect mushroom body, an area of the central nervous system that is crucial for olfactory and gustatory conditioning in insects. C. elegans has three predicted MIP receptor orthologs: SPRR-1, SPRR-2, and SPRR-3 (sex peptide receptor related), belonging to the rhodopsin class of G protein coupled receptors (GPCRs) and named after their sequence similarity with the Drosophila MIP receptor which is also known as Sex Peptide Receptor. Previous studies in our lab showed that the SPRR-1 and SPRR-2 receptors can be activated by peptides encoded by the NLP-42 and NLP-38 precursors in vitro, respectively. In addition to activating the MIP-related receptors in vitro, these peptides exhibit the characteristic protostomian C-terminal MIP motif, which is why we suggest to rename the NLP-38 precursor as MIP-1 and NLP-42 as MIP-2. Here, we attempted to find the ligand(s) of the C. elegans SPRR-3 receptor as well as the putative human MIPRs GPR139 and GPR142. However, none of the ~350 currently identified and predicted C. elegans neuropeptides could activate these receptors in our in vitro GPCR activation assay. We next analyzed the in vivo expression patterns of SPRR-1, SPRR-2, SPRR-3, NLP-38, and NLP-42 using fluorescent reporter genes. Expression was revealed in the C. elegans nervous system including in chemosensory and interneurons that function in gustatory and olfactory learning, thereby pointing to a role for this system in gustatory and/or olfactory learning. Guided by our in vivo expression analysis and the previously reported MIP expression analysis in insects we compared the behavior of mutants with impaired MIP signaling with wild-type behavior in gustatory associative learning assays in C. elegans. We found that MIP-1 signaling through activation of the SPRR-2 receptor modulates two types of aversive, but not appetitive, gustatory associative learning. On the one hand, MIP-1/SPRR-2 signaling modulates short-term gustatory plasticity, wherein worms learn to avoid normally attractive NaCl concentrations when salt is paired with short-term food withdrawal. On the other hand, MIP-1/SPRR-2 as well as SPRR-3 signaling modulates aversion towards normally attractive NaCl concentrations when paired with a long, 6 hours, starvation period. We additionally showed that the memory formed upon this salt avoidance learning shows hallmarks of long-term memory. By contrast, an appetitive association between the presence of food and NaCl is not modulated by MIP signaling and does not display hallmarks of long-term memory. Taken together, our findings expand our current knowledge on the neuropeptidergic modulation of learning and memory. Our results suggest that MIP signaling promotes learning and memory and appears to specifically modulate aversive gustatory learning in C. elegans. Moreover, MIP signaling regulates both short-term memory and memory displaying characteristics of long-term memory. Given that the putative deuterostomian MIPRs are expressed in brain regions related to learning and memory but otherwise remain largely unexplored, these findings lay a foundation for further research into the involvement of MIP receptor signaling in avoidance learning in other organisms, including humans. With the recent increased interest in avoidance learning from psychology literature, this study contributes to our understanding of how aversive events in life become anchored in memory and contribute to normal as well as disordered psychological functioning.nrpages: 223status: publishe