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.

Functional analysis of 3 genes in xenobiotic detoxification pathway of Bursaphelenchus xylophilus against matrine.

Bursaphelenchus xylophilus is the causative agent of pine wilt disease. It has caused devastating damage to ecosystems worldwide, owing to the characteristic of being widely spread and uncontrollable. However, the current methods of control are mainly based on pesticides, which can cause irreversible damage to the ecosystem. Therefore, the search for new drug targets and the development of environmentally friendly nematicides is especially valuable. In this study, three key genes of the xenobiotic detoxification pathways were cloned from B. xylophilus, which were subsequently subjected to bioinformatic analysis. The bioassay experiment was carried out to determine the concentration of matrine required for further tests. Subsequently, enzyme activity detection and three gene expression pattern analysis were performed on matrine treated nematodes. Finally, RNA interference was conducted to verify the functions carried out by the three genes in combating matrine. The results indicated that cytochrome P450 and glutathione S-transferase of B. xylophilus were activated by matrine, which induced high expression of BxCYP33C4, BxGST1, and BxGST3. After RNA interference of three genes of B. xylophilus, the sensitivity of B. xylophilus to matrine was increased and the survival rate of nematodes was reduced to various degrees in comparison to the control group. Overall, the results fully demonstrated that BxCYP33C4, BxGST1, and BxGST3 are valuable drug targets for B. xylophilus. Furthermore, the results suggested that matrine has value for development and exploitation in the prevention and treatment of B. xylophilus.

Toxicity of Bacillus thuringiensis Strains Derived from the Novel Crystal Protein Cry31Aa with High Nematicidal Activity against Rice Parasitic Nematode Aphelenchoides besseyi.

The plant parasitic nematode, Aphelenchoides besseyi, is a serious pest causing severe damage to various crop plants and vegetables. The Bacillus thuringiensis (Bt) strains, GBAC46 and NMTD81, and the biological strain, FZB42, showed higher nematicidal activity against A. besseyi, by up to 88.80, 82.65, and 75.87%, respectively, in a 96-well plate experiment. We screened the whole genomes of the selected strains by protein-nucleic acid alignment. It was found that the Bt strain GBAC46 showed three novel crystal proteins, namely, Cry31Aa, Cry73Aa, and Cry40ORF, which likely provide for the safe control of nematodes. The Cry31Aa protein was composed of 802 amino acids with a molecular weight of 90.257 kDa and contained a conserved delta-endotoxin insecticidal domain. The Cry31Aa exhibited significant nematicidal activity against A. besseyi with a lethal concentration (LC50) value of 131.80 µg/mL. Furthermore, the results of in vitro experiments (i.e., rhodamine and propidium iodide (PI) experiments) revealed that the Cry31Aa protein was taken up by A. besseyi, which caused damage to the nematode's intestinal cell membrane, indicating that the Cry31Aa produced a pore-formation toxin. In pot experiments, the selected strains GBAC46, NMTD81, and FZB42 significantly reduced the lesions on leaves by up to 33.56%, 45.66, and 30.34% and also enhanced physiological growth parameters such as root length (65.10, 50.65, and 55.60%), shoot length (68.10, 55.60, and 59.45%), and plant fresh weight (60.71, 56.45, and 55.65%), respectively. The number of nematodes obtained from the plants treated with the selected strains (i.e., GBAC46, NMTD81, and FZB42) and A. besseyi was significantly reduced, with 0.56, 0.83., 1.11, and 5.04 seedling mL-1 nematodes were achieved, respectively. Moreover, the qRT-PCR analysis showed that the defense-related genes were upregulated, and the activity of hydrogen peroxide (H2O2) increased while malondialdehyde (MDA) decreased in rice leaves compared to the control. Therefore, it was concluded that the Bt strains GBAC46 and NMTD81 can promote rice growth, induce high expression of rice defense-related genes, and activate systemic resistance in rice. More importantly, the application of the novel Cry31Aa protein has high potential for the efficient and safe prevention and green control of plant parasitic nematodes.

Nematicidal activity of furanoeremophilenes against Meloidogyne incognita and Nacobbus aberrans.

BACKGROUND: While searching for novel small molecules for new organic pesticide agents against plant-parasitic nematodes, we found that the hexane extract from the roots of Senecio sinuatos and its main secondary metabolite, 3ß-angeloyloxy-6ß-hydroxyfuranoeremophil-1(10)-ene (1), possess nematicidal activity against the second stage juvenile (J2) of Meloidogyne incognita and Nacobbus aberrans. Both species reduce yield of various vegetable crops. These results encouraged us to synthesize esters 3-9 formed by diol 2, obtained by alkaline hydrolysis of 1 and acetic anhydride, benzoic acid, 2-nitrobenzoic acid, 2-bromobenzoic acid, 4-nitrobenzoic acid, 4-bromobenzoic acid, and 4-methoxybenzoic acid, respectively. The nematicidal activity of these esters was evaluated and compared with that of the free benzoic acids. RESULTS: Natural product 1 and derivatives 2-9 were obtained and characterized by their physical and spectroscopic properties, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) experiments; X-ray diffraction analysis established their absolute configuration. The nematicidal activity of compounds 1-9 was assessed in vitro against M. incognita and N. aberrans J2 and was compared to activity shown by benzoic acid, 2-nitrobenzoic acid, 2-bromobenzoic acid, 4-nitrobenzoic acid, 4-bromobenzoic acid, and 4-methoxybenzoic acid. The esters suppressed nematodes more than free benzoic acid. Nacobbus aberrans J2 were suppressed, with compounds 5, 6, and 8 being the most active. CONCLUSION: Esters formed by 3ß,6ß-dihydroxyfuranoeremophil-1(10)-ene and ortho- or para-substituted benzoic acids containing electron acceptor groups had nematicidal activity against N. aberrans. These compound can potentially serve as a model for the development of new organic nematicidal agents. © 2022 Society of Chemical Industry.

Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode Aphelenchoides besseyi.

RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and in planta methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes (Ab-far-1, Ab-far-4, and combinatorial Ab-far-1 and Ab-far-4) of the rice white tip nematode (RWTN), Aphelenchoides besseyi, were used as target genes to construct a fungal RNAi vector, and the Ab-far-n dsRNA transgenic Botrytis cinerea (ARTBn) were generated using Agrobacterium-mediated transformation technology. After RWTN feeding on ARTBn, the expression of Ab-far-1 and Ab-far-4 in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The Ab-far-1 and Ab-far-4 co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB1 were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs.Abbreviations: FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, Aphelenchoides besseyi); Ab-far-n (Fatty acid and retinol binding protein gene of A. besseyi); ARTB1 (Ab-far-1 hpRNA transgenic Botrytis cinerea); ARTB4 (Ab-far-4 hpRNA transgenic Botrytis cinerea); ARTB1/4 (combinatorial Ab-far-1 and Ab-far-4 hpRNA transgenic B. cinerea); EVTB (Empty vector transgenic B. cinerea); GRTB (eGFP hpRNA transgenic B. cinerea); WTB (Wild-type B. cinerea).

Potential Molecular Mimicry Proteins Responsive to α-pinene in Bursaphelenchus xylophilus.

: Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic analysis of B. xylophilus. The expression levels of three genes encoding secreted B. xylophilus proteins were influenced by α-pinene. We cloned one gene encoding a thaumatin-like protein, Bx-tlp-2 (accession number MK000287), and another gene encoding a cysteine proteinase inhibitor, Bx-cpi (accession number MK000288). Additionally, α-pinene appeared to induce Bx-tlp-1 expression, but had the opposite effect on Bx-cpi expression. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees revealed that the α-pinene content was consistent with the expression levels of Bx-tlp-1 (Bx-cpi) and Pm-tlp (Pm-cpi) over time. Thus, these genes likely have important roles contributing to the infection of pine species by B. xylophilus. The results of this study may be relevant for future investigations of the functions of Bx-tlp-1, Bx-tlp-2 and Bx-cpi, which may provide a point to explore the relationship between B. xylophilus and host pines.

Development of a Redox Dye-Based Rapid Colorimetric Assay for the Quantitation of Viability/Mortality of Pine Wilt Nematode.

Control of pine wilt disease, which is caused by pine wilt nematode, Bursaphelenchus xylophilus, is heavily dependent on the use of chemicals such as abamectin. Although such chemicals are highly effective, demands for alternatives that are derived preferentially from natural sources, are increasing out of environmental concerns. One of the challenges to discovery of alternative control agents is lack of fast and efficient screening method that can be used in high-throughput manner. Here we described the development of colorimetric assay for the rapid and accurate screening of candidate nematicidal compounds/biologics targeting B. xylophilus. Contrary to the conventional method, which relies on laborious visual inspection and counting of nematode population under microscope, our method utilizes a redox dye that changes its color in response to metabolic activity of nematode population in a given sample. In this work, we optimized parameters of our colorimetric assay including number of nematodes and amount of redox dye, and tested applicability of our assay for screening of chemicals and biologics. We demonstrated that our colorimetric assay can applied to rapid and accurate quantification of nematode viability/mortality in a nematode population treated with candidate chemicals/biologics. Application of our method would facilitate high-throughput endeavors aiming at finding environment-friendly control agents for deadly disease of pine trees.

STATAWAARS: a promoter motif associated with spatial expression in the major effector-producing tissues of the plant-parasitic nematode Bursaphelenchus xylophilus.

BACKGROUND: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. RESULTS: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel "pioneer" effectors. CONCLUSIONS: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda.

Assessment of the behaviour and survival of nematodes under low oxygen concentrations.

Oxygen is required for the completion of almost all known metazoan lifecycles, but many metazoans harbour abilities to withstand varying degrees and periods of hypoxia. Caenorhabditis elegans, one of the most popular model organism is extensively used as a model for the study of hypoxia and anoxia biology and it has been found that this nematode is capable of tolerance to varying degrees of hypoxia. Considering the extremely high diversity of nematodes, the effects of low oxygen concentration and mechanisms of adaptation to oxygen depletion differ among species. In this study, we used a simple assay to examine anoxia tolerance in four nematode species, including three free-living and one plant parasitic nematode. We found that the plant parasitic nematode Bursaphelenchus xylophilus can survive more than 14 days under anoxic conditions. Comparisons of behaviour during anoxia induction and the repertoire of oxygen sensation genes among the tested species suggested the existence of different oxygen sensation systems between B. xylophilus and C. elegans, which quickly introduce suspended animation in response to oxygen depletion to survive long-term anoxia.

Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

Immune response of insect vectors to transmitted pathogens or insect hosts against parasites are well studied, whereas the mechanism of tripartite interactions remains elusive. In this study, we investigated the immune interactions of the vector beetle Monochamus alternatus ( Ma) to the devastating plant parasitic nematode Bursaphelenchus xylophilus ( Bx) and the insect parasitic nematode Howardula phyllotretae ( Hp). We report the unique immune mechanism by which the vector beetle tolerates many devastating Bx in its trachea, yet that immune tolerance is compromised by the parasitic nematode Hp. Contact with either nematode species triggers epithelial reactive oxygen species (ROS) production in Ma. Only the entry of Bx, not Hp, infection, induces increased expression of antioxidative genes, through which the ROS levels are balanced in the trachea of beetles. Furthermore, we found that up-regulation of antioxidative genes was induced by the interaction of Toll receptors. In contrast, beetles infected by Hp retain high levels of oxidative stress and melanization in trachea, and as a result, decrease Bx loading. This study highlights the role of Toll receptors in mediating the activation of antioxidative genes in immune tolerance to plant parasitic nematodes, and suggests the use of insect parasites as a biologic control.-Zhou, J., Zhao, L.-L., Yu, H.-Y., Wang, Y.-H., Zhang, W., Hu, S.-N., Zou, Z., Sun, J.-H. Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

Trehalose metabolism genes render rice white tip nematode Aphelenchoides besseyi (Nematoda: Aphelenchoididae) resistant to an anaerobic environment.

After experiencing anaerobic environments, Aphelenchoides besseyi will enter a state of suspended animation known as anoxybiosis, during which it may use trehalose as an energy supply to survive. To explore the function of trehalose metabolism, two trehalose-6-phosphate synthase (TPS) genes (Ab-tps1 and Ab-tps2) encoding enzymes catalysing trehalose synthesis, and three trehalase (TRE) genes (Ab-ntre1, Ab-ntre2 and Ab-atre) encoding enzymes catalysing the hydrolysis of trehalose, were identified and investigated. Ab-tps1 and Ab-tps2 were active during certain periods of anoxybiosis for A. besseyi, and Ab-tps2, Ab-ntre1, Ab-ntre2 and Ab-atre were active during certain periods of recovery. The results of RNA interference experiments suggested that TRE genes regulated each other and both TPS genes, while a single TPS gene only regulated the other TPS gene. However, two TPS genes together could regulate TRE genes, which indicated a feedback mechanism between these genes. All these genes also positively regulated the survival and resumption of active metabolism of the nematode. Genes functioning at re-aeration have a greater impact on nematode survival, suggesting that these genes could play roles in anoxybiosis regulation, but may function within restricted time frames. Changes in trehalose levels matched changes in TRE activity during the anoxybiosis-re-aeration process, suggesting that trehalose may act as an energy supply source. The observation of up-regulation of TPS genes during anoxybiosis suggested a possible signal role of trehalose. Trehalose metabolism genes could also work together to control trehalose levels at a certain level when the nematode is under anaerobic conditions.

Effects of the plant volatile trans­2-hexenal on the dispersal ability, nutrient metabolism and enzymatic activities of Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus causes pine wilt disease (PWD), which severely damages pine species. The plant volatile trans­2-hexenal has strong activity against nematodes, although the precise mechanism of this inhibitory action remains unclear. In this paper, the fumigant effects of the LC10 and LC30 of trans­2-hexenal on B. xylophilus were demonstrated. The trans­2-hexenal treatments significantly inhibited the dispersal ability of nematodes. The results also indicated that trans­2-hexenal affects the metabolism of nutrients and the activity of digestive enzymes. Among detoxifying enzymes, after treatment with trans­2-hexenal, glutathione S-transferase activity increased significantly and general esterase activity decreased significantly. Based on these results, trans­2-hexenal disturbs the normal physiological and biochemical activities of this nematode. These results provide valuable insight into the nematicidal mechanisms of trans­2-hexenal.

Effects of trans-2-hexenal on reproduction, growth and behaviour and efficacy against the pinewood nematode, Bursaphelenchus xylophilus.

BACKGROUND: Bursaphelenchus xylophilus is a serious quarantined pest that causes severe damage and major economic losses to pine forests. Because of the adverse effects of some traditional nematicides on humans and the environment, the search for new plant toxicants against these nematodes has intensified. Nematicidal activity of trans-2-hexenal, which is a six-carbon aldehyde present in many plants, was tested against the nematode. RESULTS: trans-2-Hexenal showed significant efficacy against B. xylophilus in a dose range of 349.5-699 g m-3 by fumigation of pinewood logs. Additionally, it had significant nematicidal activity against different life stages of B. xylophilus in an in vitro test, with second-stage larvae (L2s) being the most sensitive, with an LC50 value of 9.87 µg mL-1 at 48 h. Egg hatch was also significantly inhibited. Further studies revealed that trans-2-hexenal inhibited the reproductive activity of B. xylophilus, with negative effects on reproduction rate and egg numbers. Moreover, trans-2-hexenal reduced the body length of B. xylophilus. Respiratory rate and thrashing behaviour of B. xylophilus also decreased following treatment with this compound. CONCLUSION: trans-2-Hexenal had significant nematicidal activity against B. xylophilus, providing a basis for elucidation of the mode of action of trans-2-hexenal against plant-parasitic nematodes in future studies. © 2016 Society of Chemical Industry.

Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus.

Bursaphelenchus mucronatus (B. mucronatus) isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while ß-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus' pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future.

Molecular identification and functional characterization of the fatty acid- and retinoid-binding protein gene Rs-far-1 in the burrowing nematode Radopholus similis (Tylenchida: Pratylenchidae).

Fatty acid- and retinoid-binding protein (FAR) is a nematode-specific protein expressed in the nematode hypodermis. It is involved in nematode development, reproduction, and infection and can disrupt the plant defense reaction. In this study, we obtained the full-length sequence of the far gene from Radopholus similis (Rs-far-1), which is 828 bp long and includes a 558 bp ORF encoding 186 amino acids. A protein homology analysis revealed that Rs-FAR-1 is 75% similar to Mj-FAR-1 from Meloidogyne javanica. A neighbor-joining phylogenetic tree was inferred and showed that Rs-FAR-1 is most similar to Pv-FAR-1 from Pratylenchus vulnus. A fluorescence-based ligand-binding analysis confirmed that Rs-FAR-1 can combine with fatty acids and retinol. qPCR was used to assess Rs-far-1 expression levels at different developmental stages in different R. similis populations, and its expression was 2.5 times greater in the highly pathogenic Rs-C population than in the less pathogenic Rs-P population. The highest expression was found in females, followed by eggs, juveniles and males. When R. similis was treated with Rs-far-1 dsRNA for 36 h, the reproduction and pathogenicity decreased significantly. In situ hybridization revealed Rs-far-1 transcripts in the R. similis hypodermis. Additionally, R. similis treated with Rs-far-1 dsRNA or water were inoculated into Arabidopsis thaliana. Allene oxide synthase (AOS) expression in A. thaliana was upregulated during early infection in both treatments and then returned to the expression levels of the control plant. Compared with the control plant, AOS expression significantly decreased in A. thaliana inoculated with water-treated R. similis but significantly increased in A. thaliana inoculated with Rs-far-1 dsRNA-treated R. similis. This finding indicates that Rs-far-1 regulates AOS expression in A. thaliana. Rs-FAR-1 plays a critical role in R. similis development, reproduction, and infection and can disturb the plant defense reaction. Therefore, Rs-far-1 is an important target gene to control R. similis.

Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea).

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

Screening and functional analysis of the peroxiredoxin specifically expressed in Bursaphelenchus xylophilus--the causative agent of pine wilt disease.

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode.

Molecular characterization and functions of fatty acid and retinoid binding protein gene (Ab-far-1) in Aphelenchoides besseyi.

Rice white tip nematode, Aphelenchoides besseyi, is a kind of plant parasitic nematodes that cause serious losses in rice and many other crops. Fatty acid and retinoid binding protein (FAR) is a specific protein in nematodes and is related to development, reproduction, infection to the host, and disruption of plant defense reactions, so the inhibition of FAR function is the potential approach to control A. besseyi. The full-length of Ab-far-1 cDNA is 805 bp, including 546 bp of ORF that encodes 181 amino acids. Software analysis revealed that the Ab-FAR-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a hydrophobic secretory signal peptide, but did not have glycosylation sites. The Ab-FAR-1 had 52% homology to Gp-FAR-1 protein. The Ab-FAR-1 and Gp-FAR-1 were grouped in the same branch according to the phylogenetic tree. Fluorescence-based ligand binding analysis confirmed that the recombinant Ab-FAR-1 (rAb-FAR-1) bound with the fluorescent analogues 11-((5-dimethylaminonaphthalene-1-sulfonyl) amino) undecannoic acid (DAUDA), cis-parinaric acid and retinol, but the oleic acid would compete with the binding site. Quantitative PCR (qPCR) was used to assess the expression level of Ab-far-1 at different development stages of A. besseyi, the highest expression was found in the females, followed by eggs, juveniles and males. Using in situ hybridization technique, Ab-far-1 mRNA was present in the hypodermis of juveniles and adults, the ovaries of females and the testes of males. When A. besseyi was treated with Ab-far-1 dsRNA for 48 h, the silencing efficiency of Ab-far-1 was the best and the number of nematodes on the carrot was the least. Thus FAR plays important roles in the development and reproduction of nematodes. This study is useful and helpful to figure out a new way to control the plant parasitic nematodes.

Making headway in understanding pine wilt disease: what do we perceive in the postgenomic era?

The advent of next generation sequencing has revolutionized research approaches to biology by making entire genome sequences available and marking a new age in biology that has the potential to open innovative research avenues in various fields. Genome sequencing is now being applied in the fields of forest ecology and forest pathology, which previously had limited access to molecular techniques. One of the most advanced areas of progress is the study of "pine wilt disease", which is caused by the parasitic nematode, Bursaphelenchus xylophilus. The entire genome sequence of B. xylophilus was determined in 2011, and since then, proteomic studies have been conducted to understand the molecular basis of the parasitism and pathogenicity of B. xylophilus. These postgenomic studies have provided numerous molecular insights and greatly changed our understanding of the pathogenesis of pine wilt disease. Here, we review the recent advances in genomic and proteomic approaches that address some of the longstanding questions behind the pathogenesis of pine wilt disease and have identified future questions and directions in this regard.