Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities

Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.     

Characterization of Microsporidia-Induced Developmental Arrest and a Transmembrane Leucine-Rich Repeat Protein in Caenorhabditis elegans

Microsporidia comprise a highly diverged phylum of intracellular, eukaryotic pathogens, with some species able to cause life-threatening illnesses in immunocompromised patients. To better understand microsporidian infection in animals, we study infection of the genetic model organism Caenorhabditis elegans and a species of microsporidia, Nematocida parisii, which infects Caenorhabditis nematodes in the wild. We conducted a targeted RNAi screen for host C. elegans genes important for infection and growth of N. parisii, using nematode larval arrest as an assay for infection. Here, we present the results of this RNAi screen, and our analyses on one of the RNAi hits from the screen that was ultimately not corroborated by loss of function mutants. This hit was an RNAi clone against F56A8.3, a conserved gene that encodes a transmembrane protein containing leucine-rich repeats (LRRs), a domain found in numerous pathogen receptors from other systems. This RNAi clone caused C. elegans to be resistant to infection by N. parisii, leading to reduced larval arrest and lower pathogen load. Characterization of the endogenous F56A8.3 protein revealed that it is expressed in the intestine, localized to the membrane around lysosome-related organelles (LROs), and exists in two different protein isoforms in C. elegans. We used the CRISPR-Cas9 system to edit the F56A8.3 locus and created both a frameshift mutant resulting in a truncated protein and a complete knockout mutant. Neither of these mutants was able to recapitulate the infection phenotypes of the RNAi clone, indicating that the RNAi-mediated phenotypes are due to an off-target effect of the RNAi clone. Nevertheless, this study describes microsporidia-induced developmental arrest in C. elegans, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved F56A8.3 gene and its protein product.     

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.     

Insecticidal features displayed by the beneficial rhizobacterium Pseudomonas chlororaphis PCL1606

The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial the ability to repel and to fight against eukaryotic organisms, such as nematodes and insect larvae, thus protecting the plant host and itself. Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium isolated from avocado roots and show efficient biocontrol against fungal soil-borne disease. The main antimicrobial compound produced by P. chlororaphis PCL606 is 2-hexyl-5-propyl resorcinol (HPR), which plays a crucial role in effective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN), and homologous fit genes. To test the insecticidal activity and to determine the bases for such activity, single and double mutants on the biosynthetic genes of these four compounds were tested in a Galleria mellonella larval model using inoculation by injection. The results revealed that Fit toxin and HPR in combination are involved in the insecticide phenotype of P. chlororaphis PCL1606, and additional compounds such as HCN and PRN could be considered supporting compounds.

     

Identification of MicroRNAs in Meloidogyne incognita Using Deep Sequencing

MicroRNAs play important regulatory roles in eukaryotic lineages. In this paper, we employed deep sequencing technology to sequence and identify microRNAs in M. incognita genome, which is one of the important plant parasitic nematodes. We identified 102 M. incognita microRNA genes, which can be grouped into 71 nonredundant miRNAs based on mature sequences. Among the 71 miRANs, 27 are known miRNAs and 44 are novel miRNAs. We identified seven miRNA clusters in M. incognita genome. Four of the seven clusters, miR-100/let-7, miR-71-1/miR-2a-1, miR-71-2/miR-2a-2 and miR-279/miR-2b are conserved in other species. We validated the expressions of 5 M. incognita microRNAs, including 3 known microRNAs (miR-71, miR-100b and let-7) and 2 novel microRNAs (NOVEL-1 and NOVEL-2), using RT-PCR. We can detect all 5 microRNAs. The expression levels of four microRNAs obtained using RT-PCR were consistent with those obtained by high-throughput sequencing except for those of let-7. We also examined how M. incognita miRNAs are conserved in four other nematodes species: C. elegans, A. suum, B. malayi and P. pacificus. We found that four microRNAs, miR-100, miR-92, miR-279 and miR-137, exist only in genomes of parasitic nematodes, but do not exist in the genomes of the free living nematode C. elegans. Our research created a unique resource for the research of plant parasitic nematodes. The candidate microRNAs could help elucidate the genomic structure, gene regulation, evolutionary processes, and developmental features of plant parasitic nematodes and nematode-plant interaction.     

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gα_o-coupled 5-HT₁-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl^- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.     

Disruption of the C. elegans Intestinal Brush Border by the Fungal Lectin CCL2 Phenocopies Dietary Lectin Toxicity in Mammals

Lectins are non-immunoglobulin carbohydrate-binding proteins without enzymatic activity towards the bound carbohydrates. Many lectins of e.g. plants or fungi have been suggested to act as toxins to defend the host against predators and parasites. We have previously shown that the Coprinopsis cinerea lectin 2 (CCL2), which binds to α1,3-fucosylated N-glycan cores, is toxic to Caenorhabditis elegans and results in developmental delay and premature death. In this study, we investigated the underlying toxicity phenotype at the cellular level by electron and confocal microscopy. We found that CCL2 directly binds to the intestinal apical surface and leads to a highly damaged brush border with loss of microvilli, actin filament depolymerization, and invaginations of the intestinal apical plasma membrane through gaps in the terminal web. We excluded several possible toxicity mechanisms such as internalization and pore-formation, suggesting that CCL2 acts directly on intestinal apical plasma membrane or glycocalyx proteins. A genetic screen for C. elegans mutants resistant to CCL2 generated over a dozen new alleles in bre 1, ger 1, and fut 1, three genes required for the synthesis of the sugar moiety recognized by CCL2. CCL2-induced intestinal brush border defects in C. elegans are similar to the damage observed previously in rats after feeding the dietary lectins wheat germ agglutinin or concanavalin A. The evolutionary conserved reaction of the brush border between mammals and nematodes might allow C. elegans to be exploited as model organism for the study of dietary lectin-induced intestinal pathology in mammals.     

Identification of Pseudomonas aeruginosa Phenazines that Kill Caenorhabditis elegans

Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.     

Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.     

Molecular characterization of two homologs of the Caenorhabditis elegans cadmium-responsive gene cdr-1: cdr-4 and cdr-6

A novel cadmium-inducible gene, cdr-1, was previously identified and characterized in the nematode Caenorhabditis elegans and found to mediate resistance to cadmium toxicity. Subsequently, six homologs of cdr-1 were identified in C. elegans. Here, we describe two homologs: cdr-4, which is metal inducible, and cdr-6, which is noninducible. Both cdr-4 and cdr-6 mRNAs contain open reading frames of 831 nt and encode predicted 32-kDa integral membrane proteins, which are similar to CDR-1. cdr-4 expression is induced by arsenic, cadmium, mercury, and zinc exposure as well as by hypotonic stress. In contrast, cdr-6 is constitutively expressed at a high level in C. elegans, and expression is not affected by these stressors. Both cdr-4 and cdr-6 are transcribed in postembryonic pharyngeal and intestinal cells in C. elegans. In addition, cdr-4 is transcribed in developing embryos. Like CDR-1, CDR-4 is targeted to intestinal cell lysosomes in vivo. Inhibition of CDR-4 and/or CDR-6 expression does not render C. elegans more susceptible to cadmium toxicity; however, there is a significant decrease in their lifespan in the absence of metal. Although nematodes in which CDR-4 and/or CDR-6 expression is knocked down accumulate fluid in the pseudocoelomic space, exposure to hypertonic conditions did not significantly affect growth or reproduction in these nematodes. These results suggest that CDR expression is required for optimal viability but does not function in osmoregulation.     

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes,Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function¹. These proteins are found at the presynaptic and postsynaptic membranes ². Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission ¹. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders ³. Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegansnrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon ⁴. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength ⁵. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response ⁶.To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring ⁷. The behavioral phenotypes were confirmed using specific RNAi clones ⁸. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding ⁹. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.     

Feeding on Host Plants with Different Concentrations and Structures of Pyrrolizidine Alkaloids Impacts the Chemical-Defense Effectiveness of a Specialist Herbivore

Sequestration of chemical defenses from host plants is a strategy widely used by herbivorous insects to avoid predation. Larvae of the arctiine moth Utetheisa ornatrix feeding on unripe seeds and leaves of many species of Crotalaria (Leguminosae) sequester N-oxides of pyrrolizidine alkaloids (PAs) from these host plants, and transfer them to adults through the pupal stage. PAs confer protection against predation on all life stages of U. ornatrix. As U. ornatrix also uses other Crotalaria species as host plants, we evaluated whether the PA chemical defense against predation is independent of host plant use. We fed larvae from hatching to pupation with either leaves or seeds of one of eight Crotalaria species (C. incana, C. juncea, C. micans, C. ochroleuca, C. pallida, C. paulina, C. spectabilis, and C. vitellina), and tested if adults were preyed upon or released by the orb-weaving spider Nephila clavipes. We found that the protection against the spider was more effective in adults whose larvae fed on seeds, which had a higher PA concentration than leaves. The exceptions were adults from larvae fed on C. paulina, C. spectabilis and C. vitellina leaves, which showed high PA concentrations. With respect to the PA profile, we describe for the first time insect-PAs in U. ornatrix. These PAs, biosynthesized from the necine base retronecine of plant origin, or monocrotaline- and senecionine-type PAs sequestered from host plants, were equally active in moth chemical defense, in a dose-dependent manner. These results are also partially explained by host plant phylogeny, since PAs of the host plants do have a phylogenetic signal (clades with high and low PA concentrations in leaves) which is reflected in the adult defense.     

Nematicidal effect of volatiles produced by Trichoderma sp.

Trichoderma is an important biocontrol agent that produces metabolites harmful to nematodes. We investigated the volatile organic compounds (VOCs) of Trichoderma sp. YMF 1.00416 and examined their abilities to kill nematodes. Chemical investigations of the VOCs from this strain led to the isolation and identification of three metabolites: a new compound, 1β-vinylcyclopentane-1α,3α-diol (1) and two known metabolites, 6-pentyl-2H-pyran-2-one (2) and 4-(2-hydroxyethyl)phenol (3). Nematicidal activity assays showed that compound 2 was nematicidal, and killed > 85% of Panagrellus redivivus, Caenorhabditis elegans, and Bursaphelenchus xylophilus in 48 h at 200 mg/L in a 2 mL vial. Our results will help identify new nematicides.     

Pseudomonas fluorescens NZI7 repels grazing by C. elegans,a natural predator

The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, ‘EDB'' (‘edible''). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.     

On-Demand Isolation and Manipulation of C. elegans by In Vitro Maskless Photopatterning

Caenorhabditis elegans (C. elegans) is a model organism for understanding aging and studying animal behavior. Microfluidic assay techniques have brought widespread advances in C. elegans research; however, traditional microfluidic assays such as those based on soft lithography require time-consuming design and fabrication cycles and offer limited flexibility in changing the geometric environment during experimentation. We present a technique for maskless photopatterning of a biocompatible hydrogel on an NGM (Agar) substrate, enabling dynamic manipulation of the C. elegans culture environment in vitro. Maskless photopatterning is performed using a projector-based microscope system largely built from off-the-shelf components. We demonstrate the capabilities of this technique by building micropillar arrays during C. elegans observation, by fabricating free-floating mechanisms that can be actuated by C. elegans motion, by using freehand drawing to isolate individual C. elegans in real time, and by patterning arrays of mazes for isolation and fitness testing of C. elegans populations. In vitro photopatterning enables rapid and flexible design of experiment geometry as well as real-time interaction between the researcher and the assay such as by sequential isolation of individual organisms. Future adoption of image analysis and machine learning techniques could be used to acquire large datasets and automatically adapt the assay geometry.     

Material Properties of Caenorhabditis elegans Swimming at Low Reynolds Number

Undulatory locomotion, as seen in the nematode Caenorhabditis elegans, is a common swimming gait of organisms in the low Reynolds number regime, where viscous forces are dominant. Although the nematode's motility is expected to be a strong function of its material properties, measurements remain scarce. Here, the swimming behavior of C. elegans is investigated in experiments and in a simple model. Experiments reveal that nematodes swim in a periodic fashion and generate traveling waves that decay from head to tail. The model is able to capture the experiments' main features and is used to estimate the nematode's Young's modulus E and tissue viscosity η. For wild-type C. elegans, we find E ≈ 3.77 kPa and η ≈ −860 Pa·s; values of η for live C. elegans are negative because the tissue is generating rather than dissipating energy. Results show that material properties are sensitive to changes in muscle functional properties, and are useful quantitative tools with which to more accurately describe new and existing muscle mutants.