In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index

Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family

Parasitic Nematodes Modulate PIN-Mediated Auxin Transport to Facilitate Infection

Plant-parasitic nematodes are destructive plant pathogens that cause significant yield losses. They induce highly specialized feeding sites (NFS) in infected plant roots from which they withdraw nutrients. In order to establish these NFS, it is thought that the nematodes manipulate the molecular and physiological pathways of their hosts. Evidence is accumulating that the plant signalling molecule auxin is involved in the initiation and development of the feeding sites of sedentary plant-parasitic nematodes. Intercellular transport of auxin is essential for various aspects of plant growth and development. Here, we analysed the spatial and temporal expression of PIN auxin transporters during the early events of NFS establishment using promoter-GUS/GFP fusion lines. Additionally, single and double pin mutants were used in infection studies to analyse the role of the different PIN proteins during cyst nematode infection. Based on our results, we postulate a model in which PIN1-mediated auxin transport is needed to deliver auxin to the initial syncytial cell, whereas PIN3 and PIN4 distribute the accumulated auxin laterally and are involved in the radial expansion of the NFS. Our data demonstrate that cyst nematodes are able to hijack the auxin distribution network in order to facilitate the infection process

(Homo)glutathione Deficiency Impairs Root-knot Nematode Development in Medicago truncatula

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes

A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network

Imbalanced Lignin Biosynthesis Promotes the Sexual Reproduction of Homothallic Oomycete Pathogens

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-l-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-l-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment

NEMATIC A simple and versatile tool for the in silico analysis of plant-nematode interactions

Novel approaches for the control of agriculturally damaging nematodes are sorely needed. Endoparasitic nematodes complete their life cycle within the root vascular cylinder, inducing specialized feeding cells giant cells for root-knot nematodes and syncytia for cyst nematodes. Both nematodes hijack parts of the transduction cascades involved in developmental processes, or partially mimic the plant responses to other interactions with microorganisms, but molecular evidence of their differences and commonalities is still under investigation. Transcriptomics has been used to describe global expression profiles of their interaction with Arabidopsis, generating vast lists of differentially expressed genes. Although these results are available in public databases and publications, the information is scattered and difficult to handle. Here, we present a rapid, visual, user-friendly and easy to handle spreadsheet tool, called NEMATIC (NEMatode-Arabidopsis Transcriptomic Interaction Compendium; http//www.uclm.es/grupo/gbbmp/english/nematic.asp). It combines existing transcriptomic data for the interaction between Arabidopsis and plant-endoparasitic nematodes with data from different transcriptomic analyses regarding hormone and cell cycle regulation, development, different plant tissues, cell types and various biotic stresses. NEMATIC facilitates efficient insilico studies on plant-nematode biology, allowing rapid cross-comparisons with complex datasets and obtaining customized gene selections through sequential comparative and filtering steps. It includes gene functional classification and links to utilities from several databases. This data-mining spreadsheet will be valuable for the understanding of the molecular bases subjacent to feeding site formation by comparison with other plant systems, and for the selection of genes as potential tools for biotechnological control of nematodes, as demonstrated in the experimentally confirmed examples provided. © 2013 BSPP AND JOHN WILEY & SONS LTD

Evaluation of the ability of lectin from snowdrop (Galanthus nivalis) to protect plants against root-knot nematodes

International audienceArabidopsis thaliana transgenic plants expressing the mannose binding lectin GNA from snowdrop (Galanthus nivalis), under the control of the constitutive 35S promoter of the Cauliflower Mosaic Virus (CaMV) with an enhancer sequence were produced. They were used to investigate the potential of this lectin to control the root-knot nematode Meloidogyne incognita. Homozygous lines expressing GNA in roots at an estimated range of 0.1–1.3% of total soluble proteins were selected. Protection against root-knot nematodes was investigated in an in vitro test. Some lines had a significant decrease (20–50%) in root gall number. However, one line showed a higher gall number compared to the control line. This effect, which is not correlated to GNA protein root level, suggests a complex action mechanism of lectin against nematodes. Nevertheless, GNA seems to have a strong potential against M. incognita and could be used as a component of integrated pest management program

Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode

Root-knot nematodes (RKNs) are sedentary biotrophic parasites that induce the differentiation of root cells into feeding cells that provide the nematodes with the nutrients necessary for their development. The development of new control methods against RKNs relies greatly on the functional analysis of genes that are crucial for the development of the pathogen or the success of parasitism. In the absence of genetic transformation, RNA interference (RNAi) allows for phenotype analysis of nematode development and nematode establishment in its host after sequence-specific knock-down of the targeted genes. Strategies used to induce RNAi in RKNs are so far restricted to small-scale analyses. In the search for a new RNAi strategy amenable to large-scale screenings the possibility of using RNA viruses to produce the RNAi triggers in plants was tested. Tobacco rattle virus (TRV) was tested as a means to introduce double-stranded RNA (dsRNA) triggers into the feeding cells and to mediate RKN gene silencing. It was demonstrated that virus-inoculated plants can produce dsRNA and siRNA silencing triggers for delivery to the feeding nematodes. Interestingly, the knock-down of the targeted genes was observed in the progeny of the feeding nematodes, suggesting that continuous ingestion of dsRNA triggers could be used for the functional analysis of genes involved in early development. However, the heterogeneity in RNAi efficiency between TRV-inoculated plants appears as a limitation to the use of TRV-mediated silencing for the high-throughput functional analysis of the targeted nematode gene

Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode

Root-knot nematodes (RKNs) are sedentary biotrophic parasites that induce the differentiation of root cells into feeding cells that provide the nematodes with the nutrients necessary for their development. The development of new control methods against RKNs relies greatly on the functional analysis of genes that are crucial for the development of the pathogen or the success of parasitism. In the absence of genetic transformation, RNA interference (RNAi) allows for phenotype analysis of nematode development and nematode establishment in its host after sequence-specific knock-down of the targeted genes. Strategies used to induce RNAi in RKNs are so far restricted to small-scale analyses. In the search for a new RNAi strategy amenable to large-scale screenings the possibility of using RNA viruses to produce the RNAi triggers in plants was tested. Tobacco rattle virus (TRV) was tested as a means to introduce double-stranded RNA (dsRNA) triggers into the feeding cells and to mediate RKN gene silencing. It was demonstrated that virus-inoculated plants can produce dsRNA and siRNA silencing triggers for delivery to the feeding nematodes. Interestingly, the knock-down of the targeted genes was observed in the progeny of the feeding nematodes, suggesting that continuous ingestion of dsRNA triggers could be used for the functional analysis of genes involved in early development. However, the heterogeneity in RNAi efficiency between TRV-inoculated plants appears as a limitation to the use of TRV-mediated silencing for the high-throughput functional analysis of the targeted nematode gene