Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and ...establish specialized feeding structures called ‘giant-cells’, from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism.
Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization.
Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity.
The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.
Adaptation to changing environmental conditions represents a challenge to parthenogenetic organisms, and until now, how phenotypic variants are generated in clones in response to the selection ...pressure of their environment remains poorly known. The obligatory parthenogenetic root‐knot nematode species Meloidogyne incognita has a worldwide distribution and is the most devastating plant‐parasitic nematode. Despite its asexual reproduction, this species exhibits an unexpected capacity of adaptation to environmental constraints, for example, resistant hosts. Here, we used a genomewide comparative hybridization strategy to evaluate variations in gene copy numbers between genotypes of M. incognita resulting from two parallel experimental evolution assays on a susceptible vs. resistant host plant. We detected gene copy number variations (CNVs) associated with the ability of the nematodes to overcome resistance of the host plant, and this genetic variation may reflect an adaptive response to host resistance in this parthenogenetic species. The CNV distribution throughout the nematode genome is not random and suggests the occurrence of genomic regions more prone to undergo duplications and losses in response to the selection pressure of the host resistance. Furthermore, our analysis revealed an outstanding level of gene loss events in nematode genotypes that have overcome the resistance. Overall, our results support the view that gene loss could be a common class of adaptive genetic mechanism in response to a challenging new biotic environment in clonal animals.
Summary
Because plant wilting has been described as a consequence of cadmium (Cd2+) toxicity, we investigate Cd2+ effects on plant water losses, gas exchanges and stomatal behaviour in Arabidopsis ...thaliana L. Effects of 1‐week Cd2+ application in hydroponic condition (CdCl2 10–100 µm) were analyzed. A 10‐µm Cd2+ concentration had no significant effect on the plant–water relationship and carbon assimilation. At higher Cd2+ concentrations, a Cd2+ ‐dependent decrease in leaf conductance and CO2 uptake was observed despite the photosynthetic apparatus appeared not to be affected as probed by fluorescence measurements. In epidermal strip bioassays, nanomolar Cd2+ concentrations reduced stomatal opening under light in A. thaliana, Vicia faba and Commelina communis. Application of 5 µm ABA limited the root‐to‐shoot translocation of cadmium. However, the Cd2+‐induced stomatal closure was likely ABA‐independent, since a 5‐day treatment with 50 µm Cd2+ did not affect the plant relative water content. Additionally, a similar Cd2+‐induced stomatal closure was observed in the ABA insensitive mutant abi1‐1. Interestingly, this mutant displayed a higher transpiration rate than the wild type but did not accumulate more Cd2+, arguing that Cd2+ uptake is not dependent only on the transpiration flow. Application of putative calcium channels inhibitors suppressed the inhibitory effect of Cd2+ in epidermal strip experiments, suggesting that Cd2+ could enter the guard cell through calcium channels. Patch‐clamp studies with V. faba guard cell protoplasts showed that plasma membrane K+ channels were insensitive to external Cd2+ application whereas Ca2+ channels were found permeable to Cd2+. In conclusion, we propose that Cd2+ affects guard cell regulation in an ABA‐independent manner by entering the cytosol via Ca2+ channels.
• Root‐knot nematodes (RKNs) are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into ...specialized feeding cells. Nematode effectors synthesized in the oesophageal glands and injected into the plant tissue through the syringe‐like stylet certainly play a central role in these processes. • In a search for nematode effectors, we used comparative genomics on expressed sequence tag (EST) datasets to identify Meloidogyne incognita genes encoding proteins potentially secreted upon the early steps of infection. • We identified three genes specifically expressed in the oesophageal glands of parasitic juveniles that encode predicted secreted proteins. One of these genes, Mi‐EFF1 is a pioneer gene that has no similarity in databases and a predicted nuclear localization signal. We demonstrate that RKNs secrete Mi‐EFF1 within the feeding site and show Mi‐EFF1 targeting to the nuclei of the feeding cells. • RKNs were previously shown to secrete proteins in the apoplasm of infected tissues. Our results show that nematodes sedentarily established at the feeding site also deliver proteins within plant cells through their stylet. The protein Mi‐EFF1 injected within the feeding cells is targeted at the nuclei where it may manipulate nuclear functions of the host cell.
Secreted peptides and their specific receptors frequently orchestrate cell‐to‐cell communication in plants. Phytosulfokines (PSKs) are secreted tyrosine‐sulphated peptide hormones, which trigger ...cellular dedifferentiation and redifferentiation upon binding to their membrane receptor. Biotrophic plant pathogens frequently trigger the differentiation of host cells into specialized feeding structures, which are essential for successful infection. We found that oomycete and nematode infections were characterized by the tissue‐specific transcriptional regulation of genes encoding Arabidopsis PSKs and the PSK receptor 1 (PSKR1). Subcellular analysis of PSKR1 distribution showed that the plasma membrane‐bound receptor internalizes after binding of PSK‐α. Arabidopsis pskr1 knockout mutants were impaired in their susceptibility to downy mildew infection. Impaired disease susceptibility depends on functional salicylic acid (SA) signalling, but not on the massive up‐regulation of SA‐associated defence‐related genes. Knockout pskr1 mutants also displayed a major impairment of root‐knot nematode reproduction. In the absence of functional PSKR1, giant cells arrested their development and failed to fully differentiate. Our findings indicate that the observed restriction of PSK signalling to cells surrounding giant cells contributes to the isotropic growth and maturation of nematode feeding sites. Taken together, our data suggest that PSK signalling in Arabidopsis promotes the differentiation of host cells into specialized feeding cells.
Summary Statement
Secreted peptides frequently determine plant development and the interaction with the environment. We analysed the role of tyrosine‐sulphated phytosulfokines (PSKs) for the interaction of Arabidopsis thaliana with leaf and root pathogens, including an oomycete, a bacterium and a root‐knot nematode. Functional analyses of loss‐of‐function and gain‐of‐function mutant and transgenic lines revealed that the different pathogens require Arabidopsis PSK signalling for cellular reprogramming events that are associated with infection. Our findings indicate that PSK signalling represents a central ‘hub’ in the host, which is targeted by unrelated pathogens for the establishment of disease.
Root-knot nematodes (genus Meloidogyne) are plant parasites causing huge economic loss in the agricultural industry and affecting severely numerous developing countries. Control methods against these ...plant pests are sparse, the preferred one being the deployment of plant cultivars bearing resistance genes against Meloidogyne species. However, M. enterolobii is not controlled by the resistance genes deployed in the crop plants cultivated in Europe. The recent identification of this species in Europe is thus a major concern. Here, we sequenced the genome of M. enterolobii using short and long-read technologies. The genome assembly spans 240 Mbp with contig N50 size of 143 kbp, enabling high-quality annotations of 59,773 coding genes, 4,068 non-coding genes, and 10,944 transposable elements (spanning 8.7% of the genome). We validated the genome size by flow cytometry and the structure, quality and completeness by bioinformatics metrics. This ensemble of resources will fuel future projects aiming at pinpointing the genome singularities, the origin, diversity, and adaptive potential of this emerging plant pest.
Discovered in the 1960s, Meloidogyne graminicola is a root‐knot nematode species considered as a major threat to rice production. Yet, its origin, genomic structure, and intraspecific diversity are ...poorly understood. So far, such studies have been limited by the unavailability of a sufficiently complete and well‐assembled genome. In this study, using a combination of Oxford Nanopore Technologies and Illumina sequencing data, we generated a highly contiguous reference genome (283 scaffolds with an N50 length of 294 kb, totaling 41.5 Mb). The completeness scores of our assembly are among the highest currently published for Meloidogyne genomes. We predicted 10,284 protein‐coding genes spanning 75.5% of the genome. Among them, 67 are identified as possibly originating from horizontal gene transfers (mostly from bacteria), which supposedly contribute to nematode infection, nutrient processing, and plant defense manipulation. Besides, we detected 575 canonical transposable elements (TEs) belonging to seven orders and spanning 2.61% of the genome. These TEs might promote genomic plasticity putatively related to the evolution of M. graminicola parasitism. This high‐quality genome assembly constitutes a major improvement regarding previously available versions and represents a valuable molecular resource for future phylogenomic studies of Meloidogyne species. In particular, this will foster comparative genomic studies to trace back the evolutionary history of M. graminicola and its closest relatives.
The genome of the rice root‐knot nematode (Meloidogyne graminicola) was assembled by combining short and long sequencing reads. Genes and transposable elements were annotated and then analyzed, revealing that lateral gene transfers from bacteria may have greatly contributed to the evolution of parasitism. This genome draft will foster comparative genomic studies to trace back the evolutionary history of M. graminicola and its closest relatives.
Root-knot nematodes (genus Meloidogyne) exhibit a diversity of reproductive modes ranging from obligatory sexual to fully asexual reproduction. Intriguingly, the most widespread and devastating ...species to global agriculture are those that reproduce asexually, without meiosis. To disentangle this surprising parasitic success despite the absence of sex and genetic exchanges, we have sequenced and assembled the genomes of three obligatory ameiotic and asexual Meloidogyne. We have compared them to those of relatives able to perform meiosis and sexual reproduction. We show that the genomes of ameiotic asexual Meloidogyne are large, polyploid and made of duplicated regions with a high within-species average nucleotide divergence of ~8%. Phylogenomic analysis of the genes present in these duplicated regions suggests that they originated from multiple hybridization events and are thus homoeologs. We found that up to 22% of homoeologous gene pairs were under positive selection and these genes covered a wide spectrum of predicted functional categories. To biologically assess functional divergence, we compared expression patterns of homoeologous gene pairs across developmental life stages using an RNAseq approach in the most economically important asexually-reproducing nematode. We showed that >60% of homoeologous gene pairs display diverged expression patterns. These results suggest a substantial functional impact of the genome structure. Contrasting with high within-species nuclear genome divergence, mitochondrial genome divergence between the three ameiotic asexuals was very low, signifying that these putative hybrids share a recent common maternal ancestor. Transposable elements (TE) cover a ~1.7 times higher proportion of the genomes of the ameiotic asexual Meloidogyne compared to the sexual relative and might also participate in their plasticity. The intriguing parasitic success of asexually-reproducing Meloidogyne species could be partly explained by their TE-rich composite genomes, resulting from allopolyploidization events, and promoting plasticity and functional divergence between gene copies in the absence of sex and meiosis.
Root-knot nematodes (RKNs) (Meloidogyne spp.) are obligate endoparasites of major worldwide economic importance. They exhibit a wide continuum of variation in their reproductive strategies, ranging ...from amphimixis to obligatory mitotic parthenogenesis. Molecular phylogenetic studies have highlighted divergence between mitotic and meiotic parthenogenetic RKN species and probable interspecific hybridization as critical steps in their speciation and diversification process. The recent completion of the genomes of two RKNs, Meloidogyne hapla and Meloidogyne incognita, that exhibit striking differences in their mode of reproduction (with and without sex, respectively), their geographic distribution, and their host range has opened the way for deciphering the evolutionary significance of (a)sexual reproduction in these parasites. Accumulating evidence suggests that whole-genome duplication (in M. incognita) and horizontal gene transfers (HGTs) represent major forces that have shaped the genome of current RKN species and may account for the extreme adaptive capacities and parasitic success of these nematodes.
Root-knot nematodes (RKNs; genus Meloidogyne) are a class of plant parasites that infect the roots of many plant species. It is believed that RKNs target certain signaling molecules derived from ...plants to locate their hosts; however, currently, no plant compound has been unambiguously identified as a universal RKN attractant. To address this question, we screened a chemical library of synthetic compounds for Meloidogyne incognita attractants. The breakdown product of aminopropylamino-anthraquinone, 1,3-diaminopropane, as well as its related compounds, putrescine and cadaverine, were found to attract M. incognita. After examining various polyamines, M. incognita were found to be attracted specifically by natural compounds that possess three to five methylene groups between two terminal amino groups. Using cryo-TOF-SIMS/SEM, cadaverine was indeed detected in soybean root cortex cells and the surrounding rhizosphere, establishing a chemical gradient. In addition to cadaverine, putrescine and 1,3-diaminopropane were also detected in root exudate by HPLC-MS/MS. Furthermore, exogenously applied cadaverine is sufficient to enhance M. incognita infection of Arabidopsis seedlings. These results suggest that M. incognita is likely attracted by polyamines to locate the appropriate host plants, and the naturally occurring polyamines have potential applications in agriculture in developing protection strategies for crops from RKN infection.
This study identified the naturally occurring polyamines including cadaverine, putrescine, and 1,3-diaminopropane as the root-knot nematode Meloidogyne incognita attractants. Using cryo-TOF-SIMS/SEM, cadaverine was indeed detected in soybean root cortex cells and the surrounding rhizosphere, establishing a chemical gradient.
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