Dong-Wei Wang

Cathepsin B Cysteine Proteinase is Essential for the Development and Pathogenesis of the Plant Parasitic Nematode Radopholus similis

Radopholus similis is an important plant parasitic nematode which severely harms many crops. Cathepsin B is present in a wide variety of organisms, and plays an important role in many parasites. Understanding cathepsin B of R. similis would allow us to find new targets and approaches for its control. In this study, we found that Rs-cb-1 mRNA was expressed in esophageal glands, intestines and gonads of females, testes of males, juveniles and eggs in R. similis. Rs-cb-1 expression was the highest in females, followed by juveniles and eggs, and was the lowest in males. The maximal enzyme activity of Rs-CB-1 was detected at pH 6.0 and 40 °C. Silencing of Rs-cb-1 using in vitro RNAi (Soaking with dsRNA in vitro) not only significantly inhibited the development and hatching of R. similis, but also greatly reduced its pathogenicity. Using in planta RNAi, we confirmed that Rs-cb-1 expression in nematodes were significantly suppressed and the resistance to R. similis was significantly improved in T2 generation transgenic tobacco plants expressing Rs-cb-1 dsRNA. The genetic effects of in planta RNAi-induced gene silencing could be maintained in the absence of dsRNA for at least two generations before being lost, which was not the case for the effects induced by in vitro RNAi. Overall, our results first indicate that Rs-cb-1 plays key roles in the development, hatching and pathogenesis of R. similis, and that in planta RNAi is an effective tool in studying gene function and genetic engineering of plant resistance to migratory plant parasitic nematodes.

A Nematode Calreticulin, Rs-CRT, Is a Key Effector in Reproduction and Pathogenicity of Radopholus similis

Radopholus similis is a migratory plant-parasitic nematode that causes severe damage to many agricultural and horticultural crops. Calreticulin (CRT) is a Ca2+-binding multifunctional protein that plays key roles in the parasitism, immune evasion, reproduction and pathogenesis of many animal parasites and plant nematodes. Therefore, CRT is a promising target for controlling R. similis. In this study, we obtained the full-length sequence of the CRT gene from R. similis (Rs-crt), which is 1,527-bp long and includes a 1,206-bp ORF that encodes 401 amino acids. Rs-CRT and Mi-CRT from Meloidogyne incognita showed the highest similarity and were grouped on the same branch of the phylogenetic tree. Rs-crt is a multi-copy gene that is expressed in the oesophageal glands and gonads of females, the gonads of males, the intestines of juveniles and the eggs of R. similis. The highest Rs-crt expression was detected in females, followed by juveniles, eggs and males. The reproductive capability and pathogenicity of R. similis were significantly reduced after treatment with Rs-crt dsRNA for 36 h. Using plant-mediated RNAi, we confirmed that Rs-crt expression was significantly inhibited in the nematodes, and resistance to R. similis was significantly improved in transgenic tomato plants. Plant-mediated RNAi-induced silencing of Rs-crt could be effectively transmitted to the F2 generation of R. similis; however, the silencing effect of Rs-crt induced by in vitro RNAi was no longer detectable in F1 and F2 nematodes. Thus, Rs-crt is essential for the reproduction and pathogenicity of R. similis.

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.

The cathepsin S cysteine proteinase of the burrowing nematode Radopholus similis is essential for the reproduction and invasion

BackgroundThe nematode Radopholus similis is an important migratory endoparasite of plants. Cysteine proteinases such as cathepsin S (CPS) play key roles during embryonic development, invasion, and pathogenesis in nematodes and many other animal parasites. This study was designed to investigate the molecular characterization and functions of a cathepsin S protease in R. similis and to find new targets for its control.ResultsRs-CPS of R. similis, Hg-CPS of Heterodera glycines and Ha-CPS of H. avenae are closely genetically related and share the same branch of the phylogenetic tree. Rs-cps is a multi-copy gene that is expressed in the esophageal glands, ovaries, testes, vas deferens, and eggs of R. similis. Rs-cps mRNA transcripts are expressed at varying levels during all developmental stages of R. similis. Rs-cps expression was highest in females. The neurostimulant octopamine did not significantly enhance the ingestion of the dsRNA soaking solution by R. similis but instead had a detrimental effect on nematode activity. The dsRNA soaking solution diffused into the body of R. similis not only through the esophageal lumen but also through the amphids, excretory duct, vagina, anus and cloacal orifice. We confirmed that RNAi significantly suppressed the expression level of Rs-cps and reproductive capability and pathogenicity of R. similis.ConclusionsOur results demonstrate that Rs-cps plays important roles in the reproduction, parasitism and pathogenesis of R. similis and could be used as a new potential target for controlling plant parasitic nematodes.

Transcriptome Analysis of the Chrysanthemum Foliar Nematode, Aphelenchoides ritzemabosi (Aphelenchida: Aphelenchoididae)

The chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi, is a plant parasitic nematode that attacks many plants. In this study, a transcriptomes of mixed-stage population of CFN was sequenced on the Illumina HiSeq 2000 platform. 68.10 million Illumina high quality paired end reads were obtained which generated 26,817 transcripts with a mean length of 1,032 bp and an N50 of 1,672 bp, of which 16,467 transcripts were annotated against six databases. In total, 20,311 coding region sequences (CDS), 495 simple sequence repeats (SSRs) and 8,353 single-nucleotide polymorphisms (SNPs) were predicted, respectively. The CFN with the most shared sequences was B. xylophilus with 16,846 (62.82%) common transcripts and 10,543 (39.31%) CFN transcripts matched sequences of all of four plant parasitic nematodes compared. A total of 111 CFN transcripts were predicted as homologues of 7 types of carbohydrate-active enzymes (CAZymes) with plant/fungal cell wall-degrading activities, fewer transcripts were predicted as homologues of plant cell wall-degrading enzymes than fungal cell wall-degrading enzymes. The phylogenetic analysis of GH5, GH16, GH43 and GH45 proteins between CFN and other organisms showed CFN and other nematodes have a closer phylogenetic relationship. In the CFN transcriptome, sixteen types of genes orthologues with seven classes of protein families involved in the RNAi pathway in C. elegans were predicted. This research provides comprehensive gene expression information at the transcriptional level, which will facilitate the elucidation of the molecular mechanisms of CFN and the distribution of gene functions at the macro level, potentially revealing improved methods for controlling CFN.

Parasitism and pathogenicity of Radopholus similis to Ipomoea aquatica, Basella rubra and Cucurbita moschata and genetic diversity of different populations

Abstract Ten populations of Radopholus similis from different ornamental hosts were tested for their parasitism and pathogenicity to water spinach (Ipomoea aquatic), malabar spinach (Basella rubra), and squash (Cucurbita moschata) in pots. The results showed all three plants were new hosts of R. similis. Growth parameters of plants inoculated with nematodes were significantly lower than those of healthy control plants. All R. similis populations were pathogenic to the three plants, but pathogenicity differed among populations from different hosts. The same R. similis populations also showed different pathogenic effects in the three different plants. RadN5 population from Anthurium andraeanum had the highest pathogenicity to the three studied plants. RadN1 from A. andraeanum had the lowest pathogenicity to squash and RadN7 from Chrysalidocarpus lutesens had the lowest pathogenicity to water spinach and malabar spinach. R. similis is usually associated with root tissues, but here we report that it could be found to move and feed in the stem bases of all three studied plants. Sequence and phylogenetic analyses of DNA markers of the 18S rRNA, 28S rRNA, ITS rRNA, and mitochondrial DNA gene sequences of ten R. similis populations revealed significant genetic diversity. RadN5 and RadN6 populations from anthurium showed a close genetic relationship and could be distinguished from other populations by PCR-RFLP. At the same time, RadN5 and RadN6 populations were the most pathogenic to three studied plants. These results confirm the existence of large biological variability and molecular diversity among R. similis populations from the same or different hosts, and these characteristics are related to pathogenic variability.

Description and molecular analysis of Tylencholaimus helanensis sp. n. from China (Dorylaimida, Tylencholaimidea)

Abstract A new species, Tylencholaimushelanensissp. n., extracted from the rhizosphere soil of unidentified grasses from Helan Mountain, Inner Mongolia, China was identified. The new species is characterized by having a body length of 0.93–1.07 mm with the lip region approximately one-quarter of the body diameter at the posterior end of the neck region wide; female didelphic-amphidelphic; pars proximalis vaginae violin-shaped. Males were not found. SEM observations of the new species were made and a phylogenetic analysis of both the 18S rDNA and the D2-D3 region of 28S rDNA is presented.

Molecular identification and functional characterization of the cathepsin B gene (Ab-cb-1) in the plant parasitic nematode Aphelenchoides besseyi

The rice white tip nematode, Aphelenchoides besseyi, is widely distributed in rice planting areas worldwide and causes serious economic losses. Cathepsin genes have been demonstrated to have importance in studying the reproduction, development, pathogenicity, and control methods of plant nematodes. In this paper, a novel cathepsin B gene, Ab-cb-1, was found and cloned. The Ab-cb-1 gene was 1347 bp in length and encodes 369 amino acids. The Ab-CB-1 protein contains characteristic occluding loops but no signal peptide. A homology analysis showed that Ab-CB-1 had the highest identity value (64%) to the known amino acid sequence of cathepsin B-like cysteine protease 6 from Toxocara canis. When Ab-cb-1 was expressed in a prokaryotic system, the protein massed approximately 45 kDa and could decompose carrot callus. Ab-cb-1 mRNA was localized in the nematode intestine. The relative expression level of Ab-cb-1 in the A. besseyi Ab-S24 population, which had high reproductivity, was approximately 6.9 times that in the Ab-N10 population, which had low reproductivity, and the difference was significant (p<0.05). The Ab-cb-1 expression level was highest in females; the expression levels in males, juveniles and eggs were 30%, 12.2% and 5% of that in females, respectively, and the differences were significant among all four stages (p<0.05). Nematodes of the Ab-S24 population were treated with Ab-cb-1 dsRNA for 12 h, 24 h, 36 h and 48 h, and their reproduction decreased with increasing time. These results demonstrated that Ab-CB-1 was a digestive enzyme with hydrolytic protease properties and that Ab-cb-1 played an important role in the reproduction of A. besseyi.

Identification and function of FAR protein family genes from a transcriptome analysis of Aphelenchoides besseyi

Motivation The rice white tip nematode (RWTN) Aphelenchoides besseyi is a migratory plant parasitic nematode that infects the aboveground parts of plants. Fatty acid- and retinoid-binding (FAR) proteins are nematode-specific proteins that are involved in many important biological processes. Genes encoding FAR proteins have been identified in many species of nematodes, which indicated that nematodes may produce more than one type of FAR protein. The main goal of this study is to find new molecular targets including new far genes that will help control RWTN, and reduce the economic damage caused by RWTN. Results Two RWTN populations with different levels of pathogenicity and reproduction were sequenced and analyzed with next-generation sequencing. 17 087 transcripts were annotated using six databases and 1696 differentially expressed genes (DEGs) were identified between the two RWTN populations. Seven new Ab-far genes were identified from the transcriptome data of the two RWTN populations which is the first to identify multiple far genes in plant parasitic nematodes. This study is the first to identify far genes in the nervous system of nematodes and the first to report a transcriptome sequencing analysis of different RWTN populations. The results help elucidate the genes related to parasitism and pathogenicity and also contribute to the identification of new target genes and development of new methods to control RWTN. Availability and implementation Our data are publicly available at Sequence Read Archive (SRA) database and GenBank database. Supplementary information Supplementary data are available at Bioinformatics online.

Nematicidal protease genes screened from a soil metagenomic library to control Radopholus similis mediated by Pseudomonas fluorescens pf36

Controlling Radopholus similis, an important phytopathogenic nematode, is a challenge worldwide. Herein, we constructed a metagenomic fosmid library from the rhizosphere soil of banana plants, and six clones with protease activity were obtained by functionally screening the library. Furthermore, subclones were constructed using the six clones, and three protease genes with nematicidal activity were identified: pase1, pase4, and pase6. The pase4 gene was successfully cloned and expressed, demonstrating that the protease PASE4 could effectively degrade R. similis tissues and result in nematode death. Additionally, we isolated a predominant R. similis-associated bacterium, Pseudomonas fluorescens (pf36), from 10 R. similis populations with different hosts. The pase4 gene was successfully introduced into the pf36 strain by vector transformation and conjugative transposition, and two genetically modified strains were obtained: p4MCS-pf36 and p4Tn5-pf36. p4MCS-pf36 had significantly higher protease expression and nematicidal activity (p < 0.05) than p4Tn5-pf36 in a microtiter plate assay, whereas p4Tn5-pf36 was superior to p4MCS-pf36 in terms of genetic stability and controlling R. similis in growth pot tests. This study confirmed that R. similis is inhibited by the associated bacterium pf36-mediated expression of nematicidal proteases. Herein, a novel approach is provided for the study and development of efficient, environmentally friendly, and sustainable biocontrol techniques against phytonematodes.