NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Eco-friendly management of Meloidogyne incognita in cadmium-contaminated soil by using nematophagous fungus Purpureocillium lavendulum YMF1.683: Efficacy and mechanism.

Root-knot nematodes (RKNs) are distributed globally, including in agricultural fields contaminated by heavy metals (HM), and can cause serious crop damages. Having a method that could control RKNs in HM-contaminated soil while limit HM accumulation in crops could provide significant benefits to both farmers and consumers. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum YMF1.683 exhibited a high nematocidal activity against the RKN Meloidogyne incognita and a high tolerance to CdCl2. Comparing to the P. lavendulum YMF1.838 which showed low tolerance to Cd2+, strain YMF1.683 effectively suppressed M. incognita infection and significantly reduced the Cd2+ uptake in tomato root and fruit in soils contaminated by 100 mg/kg Cd2+. Transcriptome analyses and validation of gene expression by RT-PCR revealed that the mechanisms contributed to high Cd-resistance in YMF1.683 mainly included activating autophagy pathway, increasing exosome secretion of Cd2+, and activating antioxidation systems. The exosomal secretory inhibitor GW4869 reduced the tolerance of YMF1.683 to Cd2+, which firstly demonstrated that fungal exosome was involved in HM tolerance. The up-regulation of glutathione synthesis pathway, increasing enzyme activities of both catalase and superoxide dismutase also played important roles in Cd2+ tolerance of YMF1.683. In Cd2+-contaminated soil, YMF1.683 limited Cd2+-uptake in tomato by up-regulating the genes of ABCC family in favor of HM sequestration in plant, and down-regulating the genes of ZIP, HMA, NRAMP, YSL families associated with HM absorption, transport, and uptake in plant. Our results demonstrated that YMF1.683 could be a promising bio-agent in eco-friendly management of M. incognita in Cd2+ contaminated soils.

In silico secretome analyses of the polyphagous root-knot nematode Meloidogyne javanica: a resource for studying M. javanica secreted proteins.

BACKGROUND: Plant-parasitic nematodes (PPNs) that cause most damage include root-knot nematodes (RKNs) which are a major impediment to crop production. Root-knot nematodes, like other parasites, secrete proteins which are required for parasite proliferation and survival within the host during the infection process. RESULTS: Here, we used various computational tools to predict and identify classically and non-classically secreted proteins encoded in the Meloidogyne javanica genome. Furthermore, functional annotation analysis was performed using various integrated bioinformatic tools to determine the biological significance of the predicted secretome. In total, 7,458 proteins were identified as secreted ones. A large percentage of this secretome is comprised of small proteins of ≤ 300 aa sequence length. Functional analyses showed that M. javanica secretome comprises cell wall degrading enzymes for facilitating nematode invasion, and migration by disintegrating the complex plant cell wall components. In addition, peptidases and peptidase inhibitors are an important category of M. javanica secretome involved in compatible host-nematode interactions. CONCLUSION: This study identifies the putative secretome encoded in the M. javanica genome. Future experimental validation analyses can greatly benefit from this global analysis of M. javanica secretome. Equally, our analyses will advance knowledge of the interaction between plants and nematodes.

Streptomyces hydrogenans strain DH-16 alleviates negative impact of Meloidogyne incognita stress by modifying physio-biochemical attributes in Solanum lycopersicum plants.

The current study assessed the nematicidal and plant growth promoting potential of metabolites produced by Streptomyces hydrogenans strain DH-16 on morphological and physiological activities in 60 days old Solanum lycopersicum plants grown under Meloidogyne incognita stress. M. incognita infestation altered the levels of various photosynthetic pigments, various stress markers, enzymatic and non-enzymatic antioxidants in S. lycopersicum plants grown under in-vivo conditions. However, treatment with culture cells, supernatant and extract produced by S. hydrogenans strain DH-16 significantly reduced the number of galls in M. incognita infested plants when compared with untreated M. incognita infected plants. Moreover, the culture cells/ supernatant/ extract remarkably lowered the levels of stress markers (Hydrogen peroxide and Malondialdehyde) in infected plants and enhanced the activities of non-enzymatic antioxidants (glutathione, tocopherol) and enzymatic antioxidants (Catalase, Superoxide dismutase, Ascorbate peroxidase, Guaiacol peroxidase, Gluatathione-S-transferase and Polyphenol oxidase) in metabolites treated M. incognita infected plants. The enhanced level of different photosynthetic attributes were also evaluated by studying gas exchange parameters and different plant pigments. Moreover, an increment in the content of phenolic compounds such as total phenols, anthocyanin and flavonoids were also reflected in treated and nematode infested plants. The present study also evaluated the microscopic analysis depicting cell viability, nuclear damage and hydrogen peroxide localization in differently treated plants. The outcome of the present study therefore endorses the efficacy of DH-16 as a potential biocontrol agent that help plants in mitigating M. incognita stress.

A novel sugar beet cyst nematode effector 2D01 targets the Arabidopsis HAESA receptor-like kinase.

Plant-parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185-amino-acid secreted protein, was previously shown to be up-regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana-H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface-associated leucine-rich repeat (LRR) receptor-like kinase (RLK) involved in signalling the activation of cell wall-remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA-LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01-AtHAE interaction may play an important role in sugar beet cyst nematode parasitism.

Detection of rare nematode resistance Rhg1 haplotypes in Glycine soja and a novel Rhg1 α-SNAP.

This study pursued the hypothesis that wild plant germplasm accessions carrying alleles of interest can be identified using available single nucleotide polymorphism (SNP) genotypes for particular alleles of other (unlinked) genes that contribute to the trait of interest. The soybean cyst nematode (SCN, Heterodera glycines [HG]) resistance locus Rhg1 is widely used in farmed soybean [Glycine max (L.) Merr.]. The two known resistance-conferring haplotypes, rhg1-a and rhg1-b, typically contain three or seven to 10 tandemly duplicated Rhg1 segments, respectively. Each Rhg1 repeat carries four genes, including Glyma.18G022500, which encodes unusual isoforms of the vesicle-trafficking chaperone α-SNAP. Using SoySNP50K data for NSFRAN07 allele presence, we discovered a new Rhg1 haplotype, rhg1-ds, in six accessions of wild soybean, Glycine soja Siebold & Zucc. (0.5% of the ∼1,100 G. soja accessions in the USDA collection). The α-SNAP encoded by rhg1-ds is unique at an important site of amino acid variation and shares with the rhg1-a and rhg1-b α-SNAP proteins the traits of cytotoxicity and altered N-ethylmaleimide sensitive factor (NSF) protein interaction. Copy number assays indicate three repeats of rhg1-ds. G. soja PI 507613 and PI 507623 exhibit resistance to HG type 2.5.7 SCN populations, in part because of contributions from other loci. In a segregating F2 population, rhg1-b and rhg1-ds made statistically indistinguishable contributions to resistance to a partially virulent HG type 2.5.7 SCN population. Hence, the unusual multigene copy number variation Rhg1 haplotype was present but rare in ancestral G. soja and was present in accessions that offer multiple traits for SCN resistance breeding. The accessions were initially identified for study based on an unlinked SNP.

Signatures of adaptation to a monocot host in the plant-parasitic cyst nematode Heterodera sacchari.

Interactions between plant-parasitic nematodes and their hosts are mediated by effectors, i.e. secreted proteins that manipulate the plant to the benefit of the pathogen. To understand the role of effectors in host adaptation in nematodes, we analysed the transcriptome of Heterodera sacchari, a cyst nematode parasite of rice (Oryza sativa) and sugarcane (Saccharum officinarum). A multi-gene phylogenetic analysis showed that H. sacchari and the cereal cyst nematode Heterodera avenae share a common evolutionary origin and that they evolved to parasitise monocot plants from a common dicot-parasitic ancestor. We compared the effector repertoires of H. sacchari with those of the dicot parasites Heterodera glycines and Globodera rostochiensis to understand the consequences of this transition. While, in general, effector repertoires are similar between the species, comparing effectors and non-effectors of H. sacchari and G. rostochiensis shows that effectors have accumulated more mutations than non-effectors. Although most effectors show conserved spatiotemporal expression profiles and likely function, some H. sacchari effectors are adapted to monocots. This is exemplified by the plant-peptide hormone mimics, the CLAVATA3/EMBRYO SURROUNDING REGION-like (CLE) effectors. Peptide hormones encoded by H. sacchari CLE effectors are more similar to those from rice than those from other plants, or those from other plant-parasitic nematodes. We experimentally validated the functional significance of these observations by demonstrating that CLE peptides encoded by H. sacchari induce a short root phenotype in rice, whereas those from a related dicot parasite do not. These data provide a functional example of effector evolution that co-occurred with the transition from a dicot-parasitic to a monocot-parasitic lifestyle.

MiDaf16-like and MiSkn1-like gene families are reliable targets to develop biotechnological tools for the control and management of Meloidogyne incognita.

Meloidogyne incognita is a plant-parasitic root-knot nematode (RKN, PPN) responsible for causing damage to several crops worldwide. In Caenorhabditis elegans, the DAF-16 and SKN-1 transcription factors (TFs) orchestrate aging, longevity, and defense responses to several stresses. Here, we report that MiDaf16-like1 and MiSkn1-like1, which are orthologous to DAF-16 and SKN-1 in C. elegans, and some of their targets, are modulated in M. incognita J2 during oxidative stress or plant parasitism. We used RNAi technology for the stable production of siRNAs in planta to downregulate the MiDaf16-like1 and MiSkn1-like1 genes of M. incognita during host plant parasitism. Arabidopsis thaliana and Nicotiana tabacum overexpressing a hairpin-derived dsRNA targeting these genes individually (single-gene silencing) or simultaneously (double-gene silencing) were generated. T2 plants were challenged with M. incognita and the number of eggs, galls, and J2, and the nematode reproduction factor (NRF) were evaluated. Our data indicate that MiDaf16-like1, MiSkn1-like1 and some genes from their networks are modulated in M. incognita J2 during oxidative stress or plant parasitism. Transgenic A. thaliana and N. tabacum plants with single- or double-gene silencing showed significant reductions in the numbers of eggs, J2, and galls, and in NRF. Additionally, the double-gene silencing plants had the highest resistance level. Gene expression assays confirmed the downregulation of the MiDaf16-like1 and MiSkn1-like1 TFs and defense genes in their networks during nematode parasitism in the transgenic plants. All these findings demonstrate that these two TFs are potential targets for the development of biotechnological tools for nematode control and management in economically important crops.

Sting nematodes modify metabolomic profiles of host plants.

Plant-parasitic nematodes are devastating pathogens of many important agricultural crops. They have been successful in large part due to their ability to modify host plant metabolomes to their benefit. Both root-knot and cyst nematodes are endoparasites that have co-evolved to modify host plants to create sophisticated feeding cells and suppress plant defenses. In contrast, the ability of migratory ectoparasitic nematodes to modify host plants is unknown. Based on global metabolomic profiling of sting nematodes in African bermudagrass, ectoparasites can modify the global metabolome of host plants. Specifically, sting nematodes suppress amino acids in susceptible cultivars. Upregulation of compounds linked to plant defense have negative impacts on sting nematode population densities. Pipecolic acid, linked to systemic acquired resistance induction, seems to play a large role in protecting tolerant cultivars from sting nematode feeding and could be targeted in breeding programs.

Meloidogyne incognita (Nematoda: Meloidogynidae) sterol-binding protein Mi-SBP-1 as a target for its management.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes.

What determines host specificity in hyperspecialized plant parasitic nematodes?

BACKGROUND: In hyperspecialized parasites, the ability to grow on a particular host relies on specific virulence factors called effectors. These excreted proteins are involved in the molecular mechanisms of parasitism and distinguish virulent pathogens from non-virulent related species. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida are major plant-parasitic nematodes developing on numerous solanaceous species including potato. Their close relatives, G. tabacum and G. mexicana are stimulated by potato root diffusate but unable to establish a feeding site on this plant host. RESULTS: RNA sequencing was used to characterize transcriptomic differences among these four Globodera species and to identify genes associated with host specificity. We identified seven transcripts that were unique to PCN species, including a protein involved in ubiquitination. We also found 545 genes that were differentially expressed between PCN and non-PCN species, including 78 genes coding for effector proteins, which represent more than a 6-fold enrichment compared to the whole transcriptome. Gene polymorphism analysis identified 359 homozygous non-synonymous variants showing a strong evidence for selection in PCN species. CONCLUSIONS: Overall, we demonstrated that the determinant of host specificity resides in the regulation of essential effector gene expression that could be under the control of a single or of very few regulatory genes. Such genes are therefore promising targets for the development of novel and more sustainable resistances against potato cyst nematodes.

The soybean Rhg1 amino acid transporter gene alters glutamate homeostasis and jasmonic acid-induced resistance to soybean cyst nematode.

Rhg1 (resistance to Heterodera glycines 1) is an important locus that contributes to resistance against soybean cyst nematode (SCN; Heterodera glycines Ichinohe), which is the most economically damaging disease of soybean worldwide. Simultaneous overexpression of three genes encoding a predicted amino acid transporter, an α-soluble N-ethylmaleimide-sensitive factor attachment protein (α-SNAP) and a predicted wound-induced protein resulted in resistance to SCN provided by this locus. However, the roles of two of these genes (excluding α-SNAP) remain unknown. Here, we report the functional characterization of Glyma.18G022400, a gene at the Rhg1 locus that encodes the predicted amino acid transporter Rhg1-GmAAT. Although the direct role of Rhg1-GmAAT in glutamate transport was not demonstrated, multiple lines of evidence showed that Rhg1-GmAAT impacts glutamic acid tolerance and glutamate transportation in soybean. Transcriptomic and metabolite profiling indicated that overexpression of Rhg1-GmAAT activated the jasmonic acid (JA) pathway. Treatment with a JA biosynthesis inhibitor reduced the resistance provided by the Rhg1-containing PI88788 to SCN, which suggested that the JA pathway might play a role in Rhg1-mediated resistance to SCN. Our results could be helpful for the clarification of the mechanism of resistance to SCN provided by Rhg1 in soybean.

The Meloidogyne graminicola effector Mg16820 is secreted in the apoplast and cytoplasm to suppress plant host defense responses.

On invasion of roots, plant-parasitic nematodes secrete effectors to manipulate the cellular regulation of the host to promote parasitism. The root-knot nematode Meloidogyne graminicola is one of the most damaging nematodes of rice. Here, we identified a novel effector of this nematode, named Mg16820, expressed in the nematode subventral glands. We localized the Mg16820 effector in the apoplast during the migration phase of the second-stage juvenile in rice roots. In addition, during early development of the feeding site, Mg16820 was localized in giant cells, where it accumulated in the cytoplasm and the nucleus. Using transient expression in Nicotiana benthamiana leaves, we demonstrated that Mg16820 directed to the apoplast was able to suppress flg22-induced reactive oxygen species production. In addition, expression of Mg16820 in the cytoplasm resulted in the suppression of the R2/Avr2- and Mi-1.2-induced hypersensitive response. A potential target protein of Mg16820 identified with the yeast two-hybrid system was the dehydration stress-inducible protein 1 (DIP1). Bimolecular fluorescence complementation resulted in a strong signal in the nucleus. DIP1 has been described as an abscisic acid (ABA)-responsive gene and ABA is involved in the biotic and abiotic stress response. Our results demonstrate that Mg16820 is able to act in two cellular compartments as an immune suppressor and targets a protein involved in the stress response, therefore indicating an important role for this effector in parasitism.

Characterization of bi-domain drosomycin-type antifungal peptides in nematodes: An example of convergent evolution.

Drosomycin-type antifungal peptides (DTAFPs) are natural effectors of the innate immune system, which are restrictedly distributed in plants and ecdysozoans. Mehamycin is a bi-domain DTAFP (abbreviated as bDTAFP) firstly found in the Northern root-knot nematode Meloidogyne hapla. Here, we report its structural and functional features and the evolution of bDTAFPs in nematodes. Different from classical DTAFPs, mehamycin contains an insertion, called single Disulfide Bridge-linked Domain (abbreviated as sDBD), located in a loop region of the drosomycin scaffold. Despite this, recombinant mehamycin likely adopts a similar fold to drosomycin, as revealed by the circular dichroism spectral analysis. Functionally, it showed some weak activity against three species of fungi but relatively stronger activity against seven species of Gram-positive bacteria, indicative of functional diversification between mehamycin and classical DTAFPs. By computational data mining of the nematode databases, we identified polymorphic genes encoding mehamycin and a new multigene family of bDTAFPs (named roremycins) from Rotylenchulus reniformis. A combination of data suggests that the origination of sDBDs from M. hapla and R. reniformis is a consequence of convergent evolution, in which some probably suffered positive selection during evolution. Our study may be valuable in understanding the role of these unique antimicrobial peptides in the innate immunity of nematodes.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation.

A cryotechnique-based method for low abundance protein immunolocalization in tomato (Solanum lycopersicum) roots infected with a nematode, Globodera rostochiensis.

Plant-parasitic cyst forming nematodes induce in host roots a specific feeding site called a syncytium. Modifications induced by the pathogen in cells incorporated into syncytium include their hypertrophy and changes in apoplast caused by over-expression of plant proteins, e.g. cellulases. As a result cell wall openings between syncytial elements are formed. The major aim of our investigation was to immunolocalize cellulases involved in these cell-wall modifications. Experiments were conducted on tomato (Solanum lycopersicum cv. "Money Maker") infected with Globodera rostochiensis. Root segments containing syncytia were processed using two techniques: conventional method of embedding in LR-White resin and cryotechnique of progressive lowering of temperature (PLT). It is believed that the latter is superior to other techniques in keeping in place cell components and preserving antigenicity of macromolecules. It is especially useful when low abundance proteins have to be immunodetected at their place of action. The main principle of the PLT technique is a stepwise lowering of temperature throughout probe dehydration, infiltration and embedding in an appropriate resin. Two-step immunolocalization and visualization using fluorochrome (FITC) at light microscopy level or colloidal gold particles at transmission electron microscopy level was performed in this study. The labeling of cellulase 7 protein at both microscopy levels was more intensive and specific on PLT-treated sections as compared to sections obtained from the classical method. Our results confirm the usefulness of the PLT cryotechnique for plant immunocytochemistry and indicate that in nematode-infected roots cellulase 7 is predominantly present in the syncytia.

Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.).

Relative gene expression analyses by qRT-PCR (quantitative reverse transcription PCR) require an internal control to normalize the expression data of genes of interest and eliminate the unwanted variation introduced by sample preparation. A perfect reference gene should have a constant expression level under all the experimental conditions. However, the same few housekeeping genes selected from the literature or successfully used in previous unrelated experiments are often routinely used in new conditions without proper validation of their stability across treatments. The advent of RNA-Seq and the availability of public datasets for numerous organisms are opening the way to finding better reference genes for expression studies. Globodera rostochiensis is a plant-parasitic nematode that is particularly yield-limiting for potato. The aim of our study was to identify a reliable set of reference genes to study G. rostochiensis gene expression. Gene expression levels from an RNA-Seq database were used to identify putative reference genes and were validated with qRT-PCR analysis. Three genes, GR, PMP-3, and aaRS, were found to be very stable within the experimental conditions of this study and are proposed as reference genes for future work.

Novel global effector mining from the transcriptome of early life stages of the soybean cyst nematode Heterodera glycines.

Soybean cyst nematode (SCN) Heterodera glycines is an obligate parasite that relies on the secretion of effector proteins to manipulate host cellular processes that favor the formation of a feeding site within host roots to ensure its survival. The sequence complexity and co-evolutionary forces acting upon these effectors remain unknown. Here we generated a de novo transcriptome assembly representing the early life stages of SCN in both a compatible and an incompatible host interaction to facilitate global effector mining efforts in the absence of an available annotated SCN genome. We then employed a dual effector prediction strategy coupling a newly developed nematode effector prediction tool, N-Preffector, with a traditional secreted protein prediction pipeline to uncover a suite of novel effector candidates. Our analysis distinguished between effectors that co-evolve with the host genotype and those conserved by the pathogen to maintain a core function in parasitism and demonstrated that alternative splicing is one mechanism used to diversify the effector pool. In addition, we confirmed the presence of viral and microbial inhabitants with molecular sequence information. This transcriptome represents the most comprehensive whole-nematode sequence currently available for SCN and can be used as a tool for annotation of expected genome assemblies.

Progressive metabolic impairment underlies the novel nematicidal action of fluensulfone on the potato cyst nematode Globodera pallida.

BACKGROUND: Fluensulfone is a new nematicide with an excellent profile of selective toxicity against plant parasitic nematodes. Here, its effects on the physiology and biochemistry of the potato cyst nematode Globodera pallida have been investigated and comparisons made with its effect on the life-span of the free-living nematode Caenorhabditis elegans to provide insight into its mode of action and its selective toxicity. RESULTS: Fluensulfone exerts acute effects (≤1h; ≥100µM) on stylet thrusting and motility of hatched second stage G. pallida juveniles (J2s). Chronic exposure to lower concentrations of fluensulfone (≥3days; ≤30µM), reveals a slowly developing metabolic insult in which G. pallida J2s sequentially exhibit a reduction in motility, loss of a metabolic marker for cell viability, high lipid content and tissue degeneration prior to death. These effects are absent in adults and dauers of the model genetic nematode Caenorhabditis elegans. CONCLUSION: The nematicidal action of fluensulfone follows a time-course which progresses from an early impact on motility through to an accumulating metabolic impairment, an inability to access lipid stores and death.

In silico modeling and characterization of phytoparasitic nematodes translationally-controlled tumor proteins.

Plant parasitic nematodes infect a wide range of hosts representing the largest source of biotic stress experienced by plants. Meloidogyne genus comprises the most important parasitic nematodes, also known as root-knot nematodes. These parasitic organisms obtain nutrients to support their development through complex interactions with their hosts. The translationally-controlled tumor protein (TCTP) is widely expressed in eukaryotic organisms, and is related to a great diversity of biological processes such as calcium binding, cell proliferation and growth, pluripotency, regulation of apoptosis, microtubules stabilization, and histamine release. TCTP has been identified in the secretions of plant-parasitic nematodes, and may play a role in suppressing the plant immunity and programmed cell, hence promoting nematode parasitism. Our results revealed a high conservation of the evaluated protein sequences and little variation in their physico-chemical characteristics, such as isoelectric points and hidropathicity. Phylogenetic analysis also revealed the presence of three main groups of TCTPs, corresponding to plant parasitic, animal parasitic and free-living nematodes. Six plant parasitic TCTPs tertiary structure models were generated by homology modeling. The constructed models were highly similar and most of the structural variations occurred outside the characterized functional domains. To our knowledge, these are the first theoretical models of plant parasitic nematodes TCTPs and these results may provide a theoretical basis for future studies of host plant resistance to nematode infection.