Identification of putative expansin-like genes from the pine wood nematode, Bursaphelenchus xylophilus, and evolution of the expansin gene family within the Nematoda

AbstractWe report the cloning and characterisation of genes encoding expansin-like proteins from the pine wood nematodes, Bursaphelenchus xylophilus and B. mucronatus. A small family of genes is present in both species and the Bursaphelenchus genes are most similar to expansins and expansin-like proteins from the potato cyst nematode Globodera rostochiensis and root-knot nematodes. Molecular modelling suggests that the genes could encode a protein with a structure similar to that of functionally characterised expansins. Expression analysis showed that the Bursaphelenchus expansin-like genes are expressed solely in the pharyngeal gland cells, implying a role in the host-parasite interaction, most likely in assisting migration through the plant. Some G. rostochiensis and root-knot nematode expansins are composed of a carbohydrate-binding domain coupled to an expansin domain but no carbohydrate binding domain is present on any of the Bursaphelenchus sequences. We suggest a model for evolution of the expansin gene family within the plant-parasitic nematodes of the Tylenchida and Aphelenchida.

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Identification of a putative expansin gene expressed in the subventral glands of the cereal cyst nematode Heterodera avenae

Nematology ◽

10.1163/156854111x614674 ◽

2012 ◽

Vol 14 (5) ◽

pp. 571-577 ◽

Cited By ~ 6

Author(s):

Haibo Long ◽

Deliang Peng ◽

Wenkun Huang ◽

Yanke Liu ◽

Huan Peng

Keyword(s):

Cyst Nematode ◽

Developmental Expression ◽

Heterodera Avenae ◽

Parasitic Nematodes ◽

Secretory Proteins ◽

Expansin Gene ◽

Genes Encoding ◽

Juvenile Stages ◽

Stage Process ◽

Parasitism Genes

Parasitism genes encoding secretory proteins expressed in the pharyngeal glands of plant-parasitic nematodes play important roles in the parasitic process. A new expansin gene (Ha-expb1) expressed in the subventral glands of the sedentary cyst nematode, Heterodera avenae, was cloned. Southern blot analysis suggested that Ha-expb1 is a member of a multigene family. The deduced protein Ha-EXPB1 consists of a signal peptide, a CBM II and an expansin domain, and was significantly similar to expansins and expansin-like proteins from the potato cyst nematode, Globodera rostochiensis, and the pine wood nematodes, Bursaphelenchus spp. In situ hybridisation showed that Ha-expb1 transcript specifically accumulated in the two subventral gland cells of the second-stage juveniles. Developmental expression confirmed that its transcript abundances were high in the motile juvenile stages and low in the sedentary stage of the nematode, implying a role in the early parasitic-stage process, most likely in aiding migration within the plant.

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Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus

International Journal of Molecular Sciences ◽

10.3390/ijms20010215 ◽

2019 ◽

Vol 20 (1) ◽

pp. 215 ◽

Cited By ~ 6

Author(s):

Qi Xue ◽

Xiao-Qin Wu ◽

Wan-Jun Zhang ◽

Li-Na Deng ◽

Miao-Miao Wu

Keyword(s):

Developmental Stages ◽

Cysteine Proteinase ◽

Cathepsin L ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Parasitic Nematodes ◽

Pine Wood Nematode ◽

Pine Wood ◽

Real Time Quantitative Pcr ◽

Rapid Amplification

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

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Influence of cellulase gene knockdown by dsRNA interference on the development and reproduction of the pine wood nematode, Bursaphelenchus xylophilus

Nematology ◽

10.1163/138855409x12469541205044 ◽

2010 ◽

Vol 12 (2) ◽

pp. 225-233 ◽

Cited By ~ 28

Author(s):

Xin-Yue Cheng ◽

Su-Ming Dai ◽

Luo Xiao ◽

Bing-Yan Xie

Keyword(s):

Host Plants ◽

Plant Cell Wall ◽

Biological Effects ◽

Cellulase Activity ◽

Bursaphelenchus Xylophilus ◽

Parasitic Nematodes ◽

Transcriptional Gene Silencing ◽

Pine Wood Nematode ◽

Cellulase Gene ◽

Pine Wood

Abstract Cellulase genes are very important for plant-parasitic nematodes to move and feed within their host plants. The pine wood nematode, Bursaphelenchus xylophilus, causes destructive damage by killing pine trees. In this study, by employing dsRNA interference technology, knockdown of a cellulase gene (Bx-eng-1) of B. xylophilus was achieved and the biological effects of RNAi on the nematode were observed. The result showed that, after 24 h soaking, dsRNA of the Bx-eng-1 gene was effectively delivered into the nematode causing a post-transcriptional gene silencing and decrease in cellulase activity. Moreover, the number of F1 generation offspring was reduced significantly when the dsRNA-treated nematodes were cultured on fungal mats. We consider that cellulase is important to B. xylophilus because it not only hydrolyses cellulose of plant cell wall for its parasitism and penetration in host plants, but also influences its feeding, development and propagation on fungal mats.

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Construction of artificial microRNA expression vectors for inhibition of Minc16281 gene in root-knot nematode Meloidogyne incognita

The Journal of Agriculture and Development ◽

10.52997/jad.8.04.2019 ◽

2019 ◽

Vol 18 (4) ◽

pp. 62-69

Author(s):

Phong V. Nguyen

Keyword(s):

Meloidogyne Incognita ◽

Parasitic Nematode ◽

Expression Vectors ◽

Parasitic Nematodes ◽

Plant Parasitic Nematodes ◽

Root Knot Nematode ◽

Gene Encoding ◽

Soybean Field ◽

Genes Encoding ◽

Plant Parasitic

Effectors have been identified to play a very important role in the parasitism of plant-parasitic nematode. To cope with this type of pathogen, many approaches of silencing genes encoding for effectors have been studied and promise to be an effective tool to create plant varieties resistant to plant-parasitic nematodes. In this study, the Minc16281 gene encoding a pioneer effector with unknown function was determined and cloned from a Meloidogyne incognita population isolated from soybean field (ID: MH315945.1). The nucleotide sequence of this gene showed 97% identity to its homolog in GenBank (ID: JK287445.1) used as the control strain in our research. To generate host-induced gene silencing constructs which can potentially silence the expression of Minc16281 gene, two artificial microRNAs were synthesized based on the miR319a structure of Arabidopsis thaliana and inserted into an expression vector in soybean. These microRNAs can be introduced into soybean to investigate the function of Minc16281 on parasitism of root-knot nematode.

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Silencing of cyp-33C9 Gene Affects the Reproduction and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus

International Journal of Molecular Sciences ◽

10.3390/ijms20184520 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4520 ◽

Cited By ~ 1

Author(s):

Xiuwen Qiu ◽

Lili Yang ◽

Jianren Ye ◽

Wei Wang ◽

Tiantian Zhao ◽

...

Keyword(s):

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Economic Losses ◽

Parasitic Nematodes ◽

Pine Wood Nematode ◽

Egg Hatch ◽

Pine Wood ◽

Large Numbers ◽

Cytochrome P450 Genes ◽

Pine Trees

Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus. Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus. This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN.

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Molecular detection of nematicidal crystalliferousBacillus thuringiensisstrains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

Canadian Journal of Microbiology ◽

10.1139/w08-074 ◽

2008 ◽

Vol 54 (10) ◽

pp. 812-822 ◽

Cited By ~ 21

Author(s):

Gholamreza Salehi Jouzani ◽

Ali Seifinejad ◽

Abbas Saeedizadeh ◽

Amin Nazarian ◽

Majid Yousefloo ◽

...

Keyword(s):

Protein Band ◽

Pcr Analysis ◽

Parasitic Nematodes ◽

Cry Genes ◽

Root Knot Nematode ◽

Free Living ◽

Cry Gene ◽

Gene Profiles ◽

Pcr Product ◽

Genes Encoding

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode ( Meloidogyne incognita ) and two free-living nematodes ( Chiloplacus tenuis and Acrobeloides enoplus ). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 × 108 CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 × 108 CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.

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Application of RNA Interference to Root-Knot Nematode Genes Encoding Esophageal Gland Proteins

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0615 ◽

2005 ◽

Vol 18 (7) ◽

pp. 615-620 ◽

Cited By ~ 112

Author(s):

M.-N. Rosso ◽

M. P. Dubrana ◽

N. Cimbolini ◽

S. Jaubert ◽

P. Abad

Keyword(s):

Rna Interference ◽

Parasitic Nematodes ◽

Reverse Genetic ◽

Plant Parasitic Nematodes ◽

Root Knot Nematode ◽

Double Stranded Rna ◽

Obligate Parasites ◽

Esophageal Gland ◽

Genes Encoding ◽

Plant Parasitic

Plant parasitic nematodes have been, so far, refractory to transformation or mutagenesis. The functional analysis of nematode genes relies on the development of reverse genetic tools adapted to these obligate parasites. Here, we describe the application of RNA interference (RNAi) to the root-knot nematode Meloidogyne incognita for the knock-down of two genes expressed in the subventral esophageal glands of the nematode and potentially involved in parasitism, the calreticulin (Mi-crt) and the polygalacturonase (Mi-pg-1) genes. Incubation in 1% resorcinol for 4 h induced double-stranded RNA uptake through the alimentary track of the nematodes and led to up to 92% depletion of Mi-crt transcripts. Timecourse analysis of the silencing showed different temporal patterns for Mi-crt and Mi-pg-1. The silencing of Mi-crt was optimal 20 h after soaking, whereas the silencing of Mi-pg-1 was optimal 44 h after soaking. For the two genes, the silencing effect was highly time-limited, since no transcript depletion was detectable 68 h after soaking.

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Cloning and Characterization of Pectate Lyases Expressed in the Esophageal Gland of the Pine Wood Nematode Bursaphelenchus xylophilus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0280 ◽

2006 ◽

Vol 19 (3) ◽

pp. 280-287 ◽

Cited By ~ 75

Author(s):

Taisei Kikuchi ◽

Hajime Shibuya ◽

Takuya Aikawa ◽

John T. Jones

Keyword(s):

Pectate Lyase ◽

Bursaphelenchus Xylophilus ◽

Amino Acid Sequences ◽

Parasitic Nematodes ◽

Pine Wood Nematode ◽

Pine Wood ◽

Polysaccharide Lyase ◽

Pectate Lyases ◽

Esophageal Gland ◽

And Migration

Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xy-lophilus. The deduced amino acid sequences of these pectate lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55°C and pH 8 to 10 like other pectate lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.

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The influence of the actionmycete,Pasteuria penetrans, on the host–parasite relationship of the plant-parasitic nematode,Meloidogyne javanica

Parasitology ◽

10.1017/s0031182000081270 ◽

1986 ◽

Vol 93 (3) ◽

pp. 571-580 ◽

Cited By ~ 9

Author(s):

A. F. Bird

Keyword(s):

Giant Cells ◽

Parasitic Nematodes ◽

Pasteuria Penetrans ◽

Root Knot Nematode ◽

Plant Host ◽

Parasite Relationship ◽

Short Time ◽

Host Parasite ◽

Relationship Of ◽

Plant Parasitic

SUMMARYThe actinomycete,Pasteuria penetrans, is a specific endoparasite of various plant-parasitic nematodes. When parasitizing the root-knot nematode (Meloidogyne javanical) the nematode's capacity to reproduce is destroyed and feeding on and stimulation of the plant host are unaffected even though the bulbous body of the female nematode becomes filled with spores. Parasitism by the actinomycete does not alter the rate of growth in the early stages of nematode development although the non-parasitized female grows more rapidly than the parasitized one for a short time immediately after the final moult.Pasteuriadid not invade or inactivate the anterior oesophageal region of the femaleMeloidogyneor influence the morphology of the giant cells induced by these nematodes in their hosts' roots. The influence of this parasite on the developmental physiology of its nematode host is discussed.

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Nematicidal Activity of Cry1Ea11 from Bacillus thuringiensis BRC-XQ12 Against the Pine Wood Nematode (Bursaphelenchus xylophilus)

Phytopathology ◽

10.1094/phyto-05-17-0179-r ◽

2018 ◽

Vol 108 (1) ◽

pp. 44-51 ◽

Cited By ~ 6

Author(s):

Tianpei Huang ◽

Qunxin Lin ◽

Xiaoli Qian ◽

Ying Zheng ◽

Junmin Yao ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Bursaphelenchus Xylophilus ◽

Laser Scanning Microscopy ◽

Nematicidal Activity ◽

Confocal Laser ◽

Parasitic Nematodes ◽

Pine Wood Nematode ◽

Plant Parasitic Nematodes ◽

Pine Wood ◽

Plant Parasitic

The nematicidal activity of 92 Bacillus thuringiensis strains against the pine wood nematode Bursaphelenchus xylophilus, one of the world’s top 10 plant-parasitic nematodes, was determined. The insecticidal crystal proteins (ICPs) from Bacillus thuringiensis BRC-XQ12 were the most toxic to Bursaphelenchus xylophilus, with a lethal concentration 50 (LC50) of 32.13 μg/ml. Because the ICPs expressed by Bacillus thuringiensis BRC-XQ12 were closest to Cry1Ea6 and B. thuringiensis BRC-XQ12 contained four kinds of cry1 subgenes (cry1Aa, cry1Cb, cry1Ea, and cry1Ia), Cry1Ea was most likely to be the key active component against the nematode. The 3,516-bp cry1Ea11 gene from BRC-XQ12, as designated by the B. thuringiensis δ-endotoxin nomenclature committee, was expressed in Escherichia coli. Purified Cry1Ea11 showed an LC50 of 32.53 and 23.23 μg/ml at 24 and 48 h, with corresponding virulence equations of Y = 32.15X + 1.38 (R2 = 0.9951) and Y = 34.29X + 3.16 (R2 = 0.9792), respectively. In order to detect the pathway of B. thuringiensis Cry1Ea11 into Bursaphelenchus xylophilus, the nematode was fed with NHS-rhodamine-labeled GST-Cry1Ea11. The results of confocal laser-scanning microscopy showed that the 159-kDa GST-Cry1Ea11 could be detected in the stylet and the esophageal lumen of the pine wood nematode, indicating that GST-Cry1Ea11 could enter into the nematode through the stylet. As far as we know, no Cry1 proteins have been shown to have activity against plant-parasitic nematodes before. These results demonstrate that Cry1Ea11 is a promising nematicidal protein for controlling pine wilt disease rendered by B. xylophilus, further dramatically broadening the spectrum of Bacillus thuringiensis ICPs.

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