Root-knot nematodes produce functional mimics of tyrosine-sulfated plant peptides.

Root-knot nematodes (Meloidogyne spp.) are highly evolved obligate parasites threatening global food security. These parasites have a remarkable ability to establish elaborate feeding sites in roots, which are their only source of nutrients throughout their life cycle. A wide range of nematode effectors have been implicated in modulation of host pathways for defense suppression and/or feeding site development. Plants produce a diverse array of peptide hormones including PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides, which promote root growth via cell expansion and proliferation. A sulfated PSY-like peptide RaxX (required for activation of XA21 mediated immunity X) produced by the biotrophic bacterial pathogen (Xanthomonas oryzae pv. oryzae) has been previously shown to contribute to bacterial virulence. Here, we report the identification of genes from root-knot nematodes predicted to encode PSY-like peptides (MigPSYs) with high sequence similarity to both bacterial RaxX and plant PSYs. Synthetic sulfated peptides corresponding to predicted MigPSYs stimulate root growth in Arabidopsis. MigPSY transcript levels are highest early in the infection cycle. Downregulation of MigPSY gene expression reduces root galling and egg production, suggesting that the MigPSYs serve as nematode virulence factors. Together, these results indicate that nematodes and bacteria exploit similar sulfated peptides to hijack plant developmental signaling pathways to facilitate parasitism.

Immunolocalization and Ultrastructure Show Ingestion of Cry Protein Expressed in Glycine max by Heterodera glycines and Its Mode of Action.

Great interest exists in developing a transgenic trait that controls the economically important soybean (Glycine max) pest, soybean cyst nematode (SCN, Heterodera glycines), due to its adaptation to native resistance. Soybean plants expressing the Bacillus thuringiensis delta-endotoxin, Cry14Ab, were recently demonstrated to control SCN in both growth chamber and field testing. In that communication, ingestion of the Cry14Ab toxin by SCN second stage juveniles (J2s) was demonstrated using fluorescently labeled Cry14Ab in an in vitro assay. Here, we show that consistent with expectations for a Cry toxin, Cry14Ab has a mode of action unique from the native resistance sources Peking and PI 88788. Further, we demonstrate in planta the ingestion and localization of the Cry14Ab toxin in the midgut of nematodes feeding on roots expressing Cry14Ab using immunogold labeling and transmission electron microscopy. We observed immunolocalization of the toxin and resulting intestinal damage primarily in the microvillus-like structure (MvL)-containing region of the midgut intestine but not in nematodes feeding on roots lacking toxin. This demonstrated that Cry14Ab was taken up by the J2 SCN, presumably through the feeding tube within the plant root cell that serves as its feeding site. This suggests that relatively large proteins can be taken up through the feeding tube. Electron microscopy showed that Cry14Ab caused lysis of the midgut MvL membrane and eventual degradation of the MvL and the lysate, forming particulate aggregates. The accumulated electron-dense aggregate in the posterior midgut intestine was not observed in SCN in nonCry14Ab-expressing plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Host-induced RNA interference targeting the neuromotor gene FMRFamide-like peptide-14 (Mi-flp14) perturbs Meloidogyne incognita parasitic success in eggplant.

KEY MESSAGE: The study demonstrates the successful management of Meloidogyne incognita in eggplant using Mi-flp14 RNA interference, showing reduced nematode penetration and reproduction without off-target effects across multiple generations. Root-knot nematode, Meloidogyne incognita, causes huge yield losses worldwide. Neuromotor function in M. incognita governed by 19 neuropeptides is vital for parasitism and parasite biology. The present study establishes the utility of Mi-flp14 for managing M. incognita in eggplant in continuation of our earlier proof of concept in tobacco (US patent US2015/0361445A1). Mi-flp14 hairpin RNA construct was used for generating 19 independent transgenic eggplant events. PCR and Southern hybridization analysis confirmed transgene integration and its orientation, while RT-qPCR and Northern hybridization established the generation of dsRNA and siRNA of Mi-flp14. In vitro and in vivo bio-efficacy analysis of single-copy events against M. incognita showed reduced nematode penetration and development at various intervals that negatively impacted reproduction. Interestingly, M. incognita preferred wild-type plants over the transgenics even when unbiased equal opportunity was provided for the infection. A significant reduction in disease parameters was observed in transgenic plants viz., galls (40-48%), females (40-50%), egg masses (35-40%), eggs/egg mass (50-55%), and derived multiplication factor (60-65%) compared to wild type. A unique demonstration of perturbed expression of Mi-flp14 in partially penetrated juveniles and female nematodes established successful host-mediated RNAi both at the time of penetration even before the nematodes started withdrawing plant nutrients and later stage, respectively. The absence of off-target effects in transgenic plants was supported by the normal growth phenotype of the plants and T-DNA integration loci. Stability in the bio-efficacy against M. incognita across T1- to T4-generation transgenic plants established the utility of silencing Mi-flp14 for nematode management. This study demonstrates the significance of targeting Mi-flp14 in eggplant for nematode management, particularly to address global agricultural challenges posed by M. incognita.

Plant-Parasitic Nematode Genera Associated with Date Palm in Central Iraq.

A survey of plant-parasitic nematode genera associated with roots of mature date palm trees was conducted in central Iraq to study the occurrence, population density and geographical distribution. This study in Baghdad, Babil, Diyala, Karbela, Najaf, and Wasit governorates was conducted from 2019 to 2023, during the months from October to April. A total of 150 soil and root samples were gathered from 46 fields, and plant parasitic nematodes were extracted and morphologically identified to genus level. Eleven genera of plant parasitic nematodes were detected in the declining order of frequency: Tylenchorhynchus spp. (20%) in Baghdad, Babil, Diyala, Karbala, and Najaf; Meloidogyne spp., (17.3%) in Baghdad, Babil, Diyala, Najaf, and Wasit; Paratylenchus spp. (16%) in Diyala and Najaf; Helicotylenchus spp. (12%) in Baghdad, Diyala, Najaf, and Wasit; Tylenchus spp. (12%) in Baghdad, Babil, Diyala, Najaf, and Wasit; Aphelenchoides spp. (6.7%) in Babil, Diyala, Karbala, and Wasit; Pratylenchus spp. (4%) in Baghdad, Diyala, Najaf, and Wasit; Ditylenchus spp. (3.3%) in Babil and Najaf; Hoplolaimus spp. (1.3%) in Diyala; and Heterodera spp. and Scutellonema spp. (0.7% each) in Diyala. Population density was low in Baghdad. High population density was recorded for Meloidogyne spp., (37625 J2 /1000 cm3 soil) and lowest population density occurred with Paratylenchus spp. (20 nematodes /1000 cm3 soil) in Diyala (Al-Jadidah). This appears to be the first report of occurrences of plant-parasitic nematodes of date palm in central Iraq.

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.

The role of AtPP2-A3 and AtPP2-A8 genes encoding Nictaba-related lectin domains in the defense response of Arabidopsis thaliana to Heterodera schachtii.

MAIN CONCLUSION: Expression levels of AtPP2-A3 and AtPP2-A8 are reduced in syncytia induced by Heterodera schachtii and decline of their expression levels decreases host susceptibility, whereas their overexpression promotes susceptibility to parasite. Plant-parasitic nematodes cause huge crop losses worldwide. Heterodera schachtii is a sedentary cyst-forming nematode that induces a feeding site called a syncytium via the delivery of secreted chemical substances (effectors) to host cells, which modulate host genes expression and phytohormone regulation patterns. Genes encoding the Nictaba-related lectin domain have been found among the plant genes with downregulated expression during the development of syncytia induced by H. schachtii in Arabidopsis thaliana roots. To investigate the role of two selected Nictaba-related genes in the plant response to beet cyst nematode parasitism, mutants and plants overexpressing AtPP2-A3 or AtPP2-A8 were infected, and promoter activity and protein localization were analyzed. In wild-type plants, AtPP2-A3 and AtPP2-A8 were expressed only in roots, especially in the cortex and rhizodermis. After nematode infection, their expression was switched off in regions surrounding a developing syncytium. Astonishingly, plants overexpressing AtPP2-A3 or AtPP2-A8 were more susceptible to nematode infection than wild-type plants, whereas mutants were less susceptible. Based on these results and changes in AtPP2-A3 and AtPP2-A8 expression patterns after treatments with different stress phytohormones, we postulate that the AtPP2-A3 and AtPP2-A8 genes play important roles in the defense response to beet cyst nematode infection.

Characterization of microRNAs in the cyst nematode Heterodera glycines identifies possible candidates involved in cross-kingdom interactions with its host Glycine max.

The soybean cyst nematode (SCN - Heterodera glycines) is one of the most damaging pests to the cultivated soybean worldwide. Using a wide array of stylet-secreted effector proteins, this nematode can restructure its host cells into a complex and highly active feeding structure called the syncytium. Tight regulation of these proteins is thought to be essential to the successful formation of this syncytium. To date, multiple mechanisms have been proposed to regulate the expression of these proteins including through post-transcriptional regulation. MicroRNAs (miRNAs) are a class of small, roughly 22-nucleotide-long, non-coding RNA shown to regulate gene expression through its interaction with the 3' untranslated region of genes. These same small RNAs have also been hypothesized to be able to cross over kingdom barriers and regulate genes in other species in a process called cross-kingdom interactions. In this study, we characterized the miRNome of the SCN via sequencing of small-RNAs isolated from whole nematodes and exosomes representing all developmental stages. We identified 121 miRNA loci encoding 96 distinct miRNA families including multiple lineage- and species-specific candidates. Using a combination of plant- and animal-specific miRNA target predictors, we generated a unique repertoire of miRNA:mRNA interacting partners in the nematode and its host plant leading to the identification of a set of nine probable cross-kingdom miRNA candidates.

A Real-Time PCR Assay to Detect and Quantify Root-Knot Nematodes from Soil Extracts.

Root-knot nematodes cause forking and stubbing of the growing carrot root tip, decreasing market value and reducing yield by up to 45%. Since crop damage by these nematodes depends on their initial population densities at planting, preplant detection of potentially low nematode numbers is critical for predicting future yield loss. The aim of this study was to overcome some of the drawbacks of the labor- and time-intensive process of root-knot nematode identification and quantification by developing and field testing a real-time PCR (qPCR) assay. Primers were designed targeting the root-knot nematode Meloidogyne incognita species complex, which includes M. incognita as well as the closely related Meloidogyne javanica and Meloidogyne arenaria. The qPCR assay successfully detected each species and showed little amplification for nontarget nematode groups except for the sister group Meloidogyne enterolobii, which is not known to occur in California. Predicted nematode densities related well with microscopic counts of nematodes from prepared solutions, as well as from solutions extracted from field soil. In a greenhouse experiment, the qPCR assay distinguished between low, medium, and high levels of M. incognita infection and qPCR predicted densities at planting were negatively related in linear models with final carrot fresh weight, length, and diameter. These results suggest that qPCR assays could be a valuable diagnostic tool to predict nematode infections and prevent crop losses.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Plant Parasitic Nematodes Database: A Comprehensive Genomic Data Platform for Plant Parasitic Nematode Research.

Plant parasitic nematodes are important phytopathogens that greatly affect the growth of agricultural and forestry plants. Scientists have conducted several studies to prevent and treat the diseases they cause. With the advent of the genomics era, the genome sequencing of plant parasitic nematodes has been considerably accelerated, and a large amount of data has been generated. This study developed the Plant Parasitic Nematodes Database (PPND), a platform to combine these data. The PPND contains genomic, transcriptomic, protein, and functional annotation data, allowing users to conduct BLAST searches and genome browser analyses and download bioinformatics data for in-depth research. PPND will be continuously updated, and new data will be integrated. PPND is anticipated to become a comprehensive genomics data platform for plant parasitic nematode research.

Isolation, Identification and Molecular Mechanism Analysis of the Nematicidal Compound Spectinabilin from Newly Isolated Streptomyces sp. DT10.

Plant parasitic nematodes (PPNs) are highly destructive and difficult to control, while conventional chemical nematicides are highly toxic and cause serious environmental pollution. Additionally, resistance to existing pesticides is becoming increasingly common. Biological control is the most promising method for the controlling of PPNs. Therefore, the screening of nematicidal microbial resources and the identification of natural products are of great significance and urgency for the environmentally friendly control of PPNs. In this study, the DT10 strain was isolated from wild moss samples and identified as Streptomyces sp. by morphological and molecular analysis. Using Caenorhabditis elegans as a model, the extract of DT10 was screened for nematicidal activity, which elicited 100% lethality. The active compound was isolated from the extracts of strain DT10 using silica gel column chromatography and semipreparative high-performance liquid chromatography (HPLC). The compound was identified as spectinabilin (chemical formula C28H31O6N) using liquid chromatography mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Spectinabilin exhibited a good nematicidal activity on C. elegans L1 worms, with a half-maximal inhibitory concentration (IC50) of 2.948 µg/mL at 24 h. The locomotive ability of C. elegans L4 worms was significantly reduced when treated with 40 µg/mL spectinabilin. Further analysis of spectinabilin against known nematicidal drug target genes in C. elegans showed that it acts via target(s) different from those of some currently used nematicidal drugs such as avermectin and phosphine thiazole. This is the first report on the nematicidal activity of spectinabilin on C. elegans and the southern root-knot nematode Meloidogyne incognita. These findings may pave the way for further research and application of spectinabilin as a potential biological nematicide.

Distribution of Plant-Parasitic Nematodes in Michigan Corn Fields.

Plant-parasitic nematodes (PPNs) can cause substantial economic yield losses to many agronomic crops in the United States. A regional-scale survey was completed across 20 counties to determine PPNs prevalence in Michigan corn and how factors such as soil type, tillage, irrigation, and cropping systems influence their distribution. Ten different major genera of PPNs were identified in Michigan corn fields: Longidorus (needle), Helicotylenchus (spiral), Pratylenchus (lesion), Meloidogyne (root-knot), Heterodera (cyst), Hoplolaimus (lance), Tylenchorhynchus or Merlinius (stunt), Paratylenchus (pin), Criconemella (ring), and Xiphinema (dagger). No significant differences among different categories of tillage for lesion, stunt, or needle nematode prevalence was detected. Lesion nematodes were most prevalent in muck soil, while stunt nematode prevalence was significantly affected by the soil type. Needle nematodes were least abundant in irrigated soils and in contrast, stunt nematodes were higher in non-irrigated soils. Spiral nematodes were the most common PPNs in Michigan corn in all cropping systems. These findings will be helpful in planning future nematode studies in Michigan and in developing and evaluating corn nematode management strategies.

GABA Immunoreactivity and Pharmacological Effects vary Among Stylet-Bearing Nematodes.

Plant-parasitic nematodes conduct a series of sophisticated behaviors to complete their life cycles. Among these, locomotion behaviors, including finding the host and migrating to the feeding site, directly affect the success of parasitism. Thus, disrupting locomotion behaviors has the potential to control these parasites. γ-Aminobutyric acid (GABA) is the prominent inhibitory neurotransmitter in nematodes. GABA-immunoreactive neurons are mostly found in motor neurons, where they regulate behaviors in the model nematode C. elegans. However, the GABA system in most stylet-bearing nematodes has received little attention. Using immunohistochemistry, we found variation in the pattern of GABA-immunoreactivity among two major plant-parasites and a fungal feeder. Some of these GABA-immunoreactive neurons lack clear homologs to C. elegans. Pharmaceutical assays showed that applying GABA, its agonist, and its antagonist, can disrupt the locomotion behaviors of these nematodes, although sensitivity to a given compound varied between species. Our data suggest that the GABA system is a potential target for the control of plant-parasitic nematodes.

Characterization of Hoplolaimus seinhorsti and Hoplolaimus pararobustus (Tylenchina: Hoplolaimidae) from banana, with phylogeny and species delineation in the genus Hoplolaimus.

The morphological and molecular characterisations of two lance nematode species isolated from the rhizosphere of banana, Hoplolaimus seinhorsti and H. pararobustus, are provided based on an integrative study that includes light and scanning electron microscopy, phylogenetic analysis and two tree-based molecular species delimitation methods (GMYC and bPTP). Nineteen new sequences were obtained, including 5 partial 18S rRNA, 6 D2-D3 of 28S rRNA, 1 ITS rRNA and 7 COI mtDNA (the first COI sequences of H. seinhorsti and H. pararobustus), and an updated morphological character comparison of 37 Hoplolaimus species is presented. The tree-based molecular species-delimitation approaches employed gave markedly differing results, and also showed remarkable discrepancies among the investigated genes, although the bPTP output was found to agree well with established morphological species delimitations. Both species-delimitation approaches did, however, provide the same output for the COI mtDNA sequences, and the COI mtDNA gene sequence was also found to correspond better to established morphological species. It is therefore recommended by this paper as representing the most suitable barcode marker for Hoplolaimus species identification. This integrative study also resulted in the corrective reassignment of 17 gene sequences that were previously unidentified or incorrectly classified, as well as concluding that H. pararobustus consists of two cryptic species.

Host-delivered RNAi-mediated silencing of the root-knot nematode (Meloidogyne incognita) effector genes, Mi-msp10 and Mi-msp23, confers resistance in Arabidopsis and impairs reproductive ability of the root-knot nematode.

MAIN CONCLUSION: Mi-msp10 and Mi-msp23 effector genes play a significant role during Meloidogyne incognita parasitism on Arabidopsis roots. The role of these genes was confirmed by demonstrating the decrease of the level of susceptibility of Arabidopsis by the  silencing of Mi-msp10 and Mi-msp23 genes using HD-RNAi technology. Root-knot nematodes (RKNs) are the most damaging pathogens severely affecting global food production. The sustainable options to minimize menace of nematode populations through economically feasible measures are limited. Thus, the development of innovative and target-specific strategies that aid in their management is imperative. RNAi technology has emerged as a sustainable and target-specific alternative to control phytonematodes. Here, we characterized two novel subventral gland and dorsal gland-specific effectors, Mi-msp10 and Mi-msp23, to determine their potential effectiveness in controlling M. incognita. Comparative developmental profiling using qRT-PCR revealed higher expression of both effectors in the adult nematode female. Furthermore, functional evaluation of Mi-msp10 and Mi-msp23 dsRNA cassettes was performed using host-delivered RNAi (HD-RNAi) in Arabidopsis. The transgenic lines were examined against M. incognita, and the phenotypic effect of HD-RNAi was evident with a 61% and 51% reduction in gall formation in the Mi-msp10 and Mi-msp23 RNAi lines, respectively. A significant drop in the nematode adult females by 59% for Mi-msp10 and 49% for Mi-msp23-RNAi lines was observed. Similarly, production in egg masses decreased significantly by 76% (Mi-msp10) and 60% (Mi-msp23) for the RNAi lines, which eventually decreased the reproductive factor by 92% and 75%, respectively. The gene expression analysis showed a significant decrease in the transcript level by up to 72% (Mi-msp10) and 66% (Mi-msp23) in M. incognita females feeding on RNAi lines, providing further evidence of effective gene silencing. Overall, our findings provide useful information and support further development of RNAi-based strategies to control M. incognita.

The heterologous expression of conserved Glycine max (soybean) mitogen activated protein kinase 3 (MAPK3) paralogs suppresses Meloidogyne incognita parasitism in Gossypium hirsutum (upland cotton).

Two conserved Glycine max (soybean) mitogen activated protein kinase 3 (MAPK3) paralogs function in defense to the parasitic soybean cyst nematode Heterodera glycines. Gene Ontology analyses of RNA seq data obtained from MAPK3-1-overexpressing (OE) and MAPK3-2-OE roots compared to their control, as well as MAPK3-1-RNA interference (RNAi) and MAPK3-2-RNAi compared to their control, hierarchically orders the induced and suppressed genes, strengthening the hypothesis that their heterologous expression in Gossypium hirsutum (upland cotton) would impair parasitism by the root knot nematode (RKN) Meloidogyne incognita. MAPK3-1 expression (E) in G. hirsutum suppresses the production of M. incognita root galls, egg masses, and second stage juveniles (J2s) by 80.32%, 82.37%, and 88.21%, respectfully. Unexpectedly, egg number increases by 28.99% but J2s are inviable. MAPK3-2-E effects are identical, statistically. MAPK3-1-E and MAPK3-2-E decreases root mass 1.49-fold and 1.55-fold, respectively, as compared to the pRAP15-ccdB-E control. The reproductive factor (RF) of M. incognita for G. hirsutum roots expressing MAPK3-1-E or MAPK3-2-E decreases 60.39% and 50.46%, respectively, compared to controls. The results are consistent with upstream pathogen activated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI) functioning in defense to H. glycines. The experiments showcase the feasibility of employing MAPK3, through heterologous expression, to combat M. incognita parasitism, possibly overcoming impediments otherwise making G. hirsutum's defense platform deficient. MAPK homologs are identified in other important crop species for future functional analyses.

Observations on the Population Genetic Structure of the Leaf Galling Nematode, Ditylenchus gallaeformans.

Ditylenchus gallaeformans is a plant parasitic nematode that induces galls on aboveground parts of Melastomataceae plants. It differs from most gall-inducing nematodes in that it is not an endoparasite and has been considered as a possible biological control agent against invasive species of Miconia. Little is known about D. gallaeformans biology, genetic differences among populations, and host preferences. This study examined the genetic differences among D. gallaeformans populations from different locations and host species and the phylogenetic relationships among them. Nematodes were collected from galls in plants from Costa Rica, Dominica, and Trinidad. The Cytochrome c oxidase 1 (cox1) region was sequenced from a total of 33 individual nematodes isolated from 33 different plant individuals, representing 21 species of Melastomataceae. Phylogenetic reconstructions, haplotype networks, and analysis of molecular variance showed that the species is monophyletic and has three major clades, which were mostly consistent with geographic location but not with host species. The first clade was composed by two subclades, one with individuals from Costa Rica and one with individuals from Dominica. The second and third clades comprised nematodes only from Trinidad. Overall, there is no evidence of host-species specialization in D. gallaeformans. Biocontrol efforts using the nematode against invasive Miconia could focus on geographical location matching but likely will not need to match host species.

Co-Silencing of the Voltage-Gated Calcium Channel ß Subunit and High-Voltage Activated α1 Subunit by dsRNA Soaking Resulted in Enhanced Defects in Locomotion, Stylet Thrusting, Chemotaxis, Protein Secretion, and Reproduction in Ditylenchus destructor.

The voltage-gated calcium channel (VGCC) ß subunit (Cavß) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Cavß subunit gene of D. destructor, namely, DdCavß, was subjected to molecular characterization. In situ hybridization assays showed that DdCavß was expressed in all nematode tissues. Transcriptional analyses showed that DdCavß was expressed during each developmental stage of D. destructor, and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCavß was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCavß or HVA Cavα1 alone and co-silencing of the DdCavß and HVA Cavα1 genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor. Co-silencing of the HVA Cavα1 and Cavß subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.

Tomato Sterol 22-desaturase Gene CYP710A11: Its Roles in Meloidogyne incognita Infection and Plant Stigmasterol Alteration.

Sterols are isoprenoid-derived lipids that play essential structural and functional roles in eukaryotic cells. Plants produce a complex mixture of sterols, and changes in plant sterol profiles have been linked to plant-pathogen interactions. ß-Sitosterol and stigmasterol, in particular, have been associated with plant defense. As nematodes have lost the ability to synthesize sterols de novo, they require sterols from the host. Tomato (Solanum lycopersicum) plants infected by the plant parasitic nematode Meloidogyne incognita show a reduced level of stigmasterol and a repression of the gene CYP710A11, encoding the sterol C-22 desaturase that is responsible for the conversion of ß-sitosterol to stigmasterol. In this study, we investigated the role of the tomato sterol C-22 desaturase gene CYP710A11 in the response to infection by M. incognita. We explored the plant-nematode interaction over time by analyzing the plant sterol composition and CYP710A11 gene regulation in S. lycopersicum after M. incognita infection. The temporal gene expression analysis showed that 3 days after inoculation with M. incognita, the CYP710A11 expression was significantly suppressed in the tomato roots, while a significant decrease in the stigmasterol content was observed after 14 days. A cyp710a11 knockout mutant tomato line lacking stigmasterol was analyzed to better understand the role of CYP710A11 in nematode development. M. incognita grown in the mutant line showed reduced egg mass counts, presumably due to the impaired growth of the mutant. However, the nematodes developed as well as they did in the wild-type line. Thus, while the suppression of CYP710A11 expression during nematode development may be a defense response of the plant against the nematode, the lack of stigmasterol did not seem to affect the nematode. This study contributes to the understanding of the role of stigmasterol in the interaction between M. incognita and tomato plants and shows that the sterol C-22 desaturase is not essential for the success of M. incognita.

Roles of Species-Specific Legumains in Pathogenicity of the Pinewood Nematode Bursaphelenchus xylophilus.

Peptidases are very important to parasites, which have central roles in parasite biology and pathogenesis. In this study, by comparative genome analysis, genome-wide peptidase diversities among plant-parasitic nematodes are estimated. We find that genes encoding cysteine peptidases in family C13 (legumain) are significantly abundant in pine wood nematodes Bursaphelenchus genomes, compared to those in other plant-parasitic nematodes. By phylogenetic analysis, a clade of B. xylophilus-specific legumain is identified. RT-qPCR detection shows that these genes are highly expressed at early stage during the nematode infection process. Utilizing transgene technology, cDNAs of three species-specific legumain were introduced into the Arabidopsis γvpe mutant. Functional complementation assay shows that these B. xylophilus legumains can fully complement the activity of Arabidopsis γVPE to mediate plant cell death triggered by the fungal toxin FB1. Secretory activities of these legumains are experimentally validated. By comparative transcriptome analysis, genes involved in plant cell death mediated by legumains are identified, which enrich in GO terms related to ubiquitin protein transferase activity in category molecular function, and response to stimuli in category biological process. Our results suggest that B. xylophilu-specific legumains have potential as effectors to be involved in nematode-plant interaction and can be related to host cell death.

Description of Rotylenchus zhongshanensis sp. nov. (Tylenchomorpha: Hoplolaimidae) and discovery of its endosymbiont Cardinium.

A new bisexual species of Rotylenchus is described and illustrated based on morphological, morphometric and molecular characterizations. Rotylenchus zhongshanensis sp. nov. is characterized by having a conoid lip region complying with the basic pattern for Hoplolaimidae, but with pharyngeal glands slightly overlapping intestine dorsally and cuticle thickened abnormally in female tail terminus. Females have robust stylet (30.1-33.8 µm). The pharyngeal gland has short dorsal (11.2-16.8 µm) overlap on the intestine. The vulva is located at 48.0-56.5% of body length, and phasmids are pore-like, 4-6 annuli posterior to the anus. For males, phasmids are pore-like, 11-17 annuli posterior to cloaca. The spicules are ventrally arcuate (21.0-28.5 µm) with gubernaculum in 5-8 µm length. The rRNA and mitochondrial COI genes were successfully sequenced from the assembled whole-genome sequences of the new species, and were used for reconstructing the phylogenetic relationships of the new species. A new strain of cyto-endosymbiont Cardinium was also discovered from the genome sequences of R. zhongshanensis sp. nov. The 16S rRNA phylogeny analyses revealed that this new bacterial strain is closed to that from cyst and root-lesion nematodes.