A heritable antiviral RNAi response limits Orsay virus infection in Caenorhabditis elegans N2

Orsay virus (OrV) is the first virus known to be able to complete a full infection cycle in the model nematode species Caenorhabditis elegans. OrV is transmitted horizontally and its infection is limited by antiviral RNA interference (RNAi). However, we have no insight into the kinetics of OrV replication in C. elegans. We developed an assay that infects worms in liquid, allowing precise monitoring of the infection. The assay revealed a dual role for the RNAi response in limiting Orsay virus infection in C. elegans. Firstly, it limits the progression of the initial infection at the step of recognition of dsRNA. Secondly, it provides an inherited protection against infection in the offspring. This establishes the heritable RNAi response as anti-viral mechanism during OrV infections in C. elegans. Our results further illustrate that the inheritance of the anti-viral response is important in controlling the infection in the canonical wild type Bristol N2. The OrV replication kinetics were established throughout the worm life-cycle, setting a standard for further quantitative assays with the OrV-C. elegans infection model

Loss-of-function of b-catenin bar-1 slows development and activates the Wnt pathway in Caenorhabditis elegans

C. elegans is extensively used to study the Wnt-pathway and most of the core-signalling components are known. Four beta-catenins are important gene expression regulators in Wnt-signalling. One of these, bar-1, is part of the canonical Wnt-pathway. Together with Wnt effector pop-1, bar-1 forms a transcription activation complex which regulates the transcription of downstream genes. The effects of bar-1 loss-of-function mutations on many phenotypes have been studied well. However, the effects on global gene expression are unknown. Here we report the effects of a loss-of-function mutation bar-1(ga80). By analysing the transcriptome and developmental phenotyping we show that bar-1(ga80) impairs developmental timing. This developmental difference confounds the comparison of the gene expression profile between the mutant and the reference strain. When corrected for this difference it was possible to identify genes that were directly affected by the bar-1 mutation. We show that the Wnt-pathway itself is activated, as well as transcription factors elt-3, pqm-1, mdl-1 and pha-4 and their associated genes. The outcomes imply that this response compensates for the loss of functional bar-1. Altogether we show that bar-1 loss-of function leads to delayed development possibly caused by an induction of a stress response, reflected by daf-16 activated genes

Gene-environment and protein degradation signatures characterize genomic and phenotypic diversity in wild Caenorhabditis elegans populations

Background: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research on the nematode Caenorhabditis elegans has been instrumental for unraveling genotype-phenotype relations, and has important implications for understanding the biology of mammals, but almost all studies, including forward and reverse genetic screens, are limited by investigations in only one canonical genotype. This hampers the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. It is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild C. elegans populations. Results: We used multiple wild C. elegans populations freshly isolated from local sites to investigate gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness, and behavioral traits. The genotype, transcriptome, and a number of fitness traits showed a direct link with the original site of the strains. The separation between the isolation sites was prevalent on all chromosomes, but chromosome V was the largest contributor to this variation. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Comparing polymorphic genes between the populations with a set of genes extracted from 19 different studies on gene expression in C. elegans exposed to biotic and abiotic factors, such as bacteria, osmotic pressure, and temperature, revealed a significant enrichment for genes involved in gene-environment interactions and protein degradation. Conclusions: We found that wild C. elegans populations are characterized by gene-environment signatures, and we have unlocked a wealth of genotype-phenotype relations for the first time. Studying natural isolates provides a treasure trove of evidence compared with that unearthed by the current research in C. elegans, which covers only a diminutive part of the myriad of genotype-phenotype relations that are present in the wild

A rapid and massive gene expression shift marking adolescent transition in C. elegans

Organismal development is the most dynamic period of the life cycle, yet we have only a rough understanding of the dynamics of gene expression during adolescent transition. Here we show that adolescence in Caenorhabditis elegans is characterized by a spectacular expression shift of conserved and highly polymorphic genes. Using a high resolution time series we found that in adolescent worms over 10,000 genes changed their expression. These genes were clustered according to their expression patterns. One cluster involved in chromatin remodelling showed a brief up-regulation around 50 h post-hatch. At the same time a spectacular shift in expression was observed. Sequence comparisons for this cluster across many genotypes revealed diversifying selection. Strongly up-regulated genes showed signs of purifying selection in non-coding regions, indicating that adolescence-active genes are constrained on their regulatory properties. Our findings improve our understanding of adolescent transition and help to eliminate experimental artefacts due to incorrect developmental timing

Microarray experiment comparing gene expression in the bar-1 mutant EW15 with wild-type strain N2 in Caenorhabditis elegans

The Wnt-pathway is a key-signalling pathway in metazoans. The beta-catenins, which include bar-1, have a function in gene expression regulation in Wnt-signalling. In this study, the effect of a loss-of-function mutation (bar-1(ga80) in the strain EW15) on gene-expression was tested. To this end age-synchronized populations of both N2 (wild-type) and EW15 were grown for 48 hours at 20 degrees Celsius, after which populations were collected and total RNA was isolated. For each genotype six replicates were collected in three batches of two replicates

Transcriptome profiling by microarray of Caenorhabditis elegans MT2124 x CB4856 RILs used for eQTL mapping

We crossed the MT2124 strain carrying the let-60 (n1046) gain of function mutation with the wild-type strain CB4856. From this cross a panel of mutation introgression recombinant inbred lines (miRILs) was generated. Using this population, the influence of genetic variation on the the let-60 (gf) mutation can be investigated. This analysis can pinpoint genetic loci A panel of 33 miRILs, the parental strains (4x), and transgenic lines amx-2(lf);Si[amx-2(Bristol)];let-60(gf) and amx-2(lf);Si[amx-2(Hawaii)];let-60(gf) containing the N2- or CB4856-allele of amx-2 in a amx-2(lf);let-60(gf) background were sampled. All the strains were grown on nematode growth medium dishes seeded with E. coli OP50. The temperature at which the strains were kept was 20 degrees Celsius and the strains were grown for 72 hours after synchronization by bleaching. Thereafter RNA was isolated, labelled, and hybridized on microarray. The gene expression profiles of the miRILs were used for eQTL mapping. The gene expression profiles of the transgenic strains were used for trans-band confirmation

Microarray on Caenorhabditis elegans N2 and CB4856 infected with Orsay virus

This experiment investigates the gene expression differences upon Orsay virus infection in the Caenorhabdits elegans strains N2 and CB4856. Assays measuring viral load found that the N2 strain displays higher viral loads upon infection than the CB4856 strain. The goal of the experiment was to identify gene-expression differences that could explain the differences in viral load. We (mock-)infected 26h-old C. elegans populations with Orsay virus and took samples after 30h of infection. For each treatment-strain combination 8 samples were collected. Thereafter RNA was isolated, labelled, and hybridized on microarray

Gene expression profiling in control, heat-shock, and recovery treatment in an introgression line population of Caenorhabditis elegans L4 larvae

This experiment investigates the genetic architecture of gene expression (eQTL) in three different treatments in an N2xCB4856 introgression line population of Caenorhabditis elegans. The goal is to compare the effect of small CB4856 introgressions in an N2 genetic background versus a previously conducted experiment using recombinant inbred lines (RILs) derived from the same parental strains. We exposed ILs to a control (56 ILs), heat-stress (56 ILs), and recovery treatment (55 ILs). Additionally, also both parental strains, N2 and CB4856 were exposed to these three treatments (in at least 3 replicas per treatment). More specifically, these three conditions can be characterized as: (i) the control treatment was grown for 48 hours at 20C, (ii) the heat-stress treatment was grown for 46 hours at 20C followed by 2 hours at 35C, and (iii) the recovery treatment was grown for 46 hours at 20C, followed by 2 hours at 35C and thereafter 2 hours at 20C. Thereafter RNA was isolated, labelled and hybridized on microarray. The gene expression profiles were used for comparison versus findings in the RIL population (E-MTAB-5779)

Microarray from roots of Arabidopsis Col-0 and knock-down UPL3, infected and non-infected with Heterodera schachtii

In this experiment we evaluate the transcriptional response to beet cyst nematode infection in wild-type (Col-0) and a upl3 knocked-down Arabidopsis thaliana. The data indicates that the upl3 knock-down alone does not induce a strong transcriptional regulation in the plant. However, the interaction of the mutation and infection by H. schachtii results in a strong transcriptional differential regulation, likely involving the plant’s immune responses. Samples were taken from whole root systems of 14 day-old seedlings (0 days post-infection) and mock or H. schahtii-infected 21 day-old seedlings (7 days post-infection). Each sample contained at least 18 plants and 4 biological replicates of each sample were collected

Gene expression effects in Caenorhabditis elegans strains of feeding on Erwinia rhapontici or Escherichia coli

Diet can have a strong impact on development, to test this effect the strains N2, CB4856, three strains from Orsay (JU1581, JU1944, and JU1949), and three strains from Santeuil (JU1921, JU1930, and JU1932) were grown on two different food-sources. These sources were the standard lab food, Escherichia coli OP50, and the Gram-negative plant pathogen Erwinia rhapontici. The effect of these bacteria on development could also be measured by the start of reproduction, which was delayed while feeding on E. coli. To measure the transcriptional effects, age-synchronized strains were grown for 48 hours at 20 degrees Celsius. The impact of food-source on gene expression was determined by a transcriptional ruler based on the strain N2 (E-MTAB-7019)