Isolation and localisation of an annexin gene (gp-nex) from the potato cyst nematode, Globodera pallida

AbstractA full length cDNA clone was isolated by screening a mixed stage Globodera pallida expression library using the monospecific polyclonal antibody IACR-PC320. Sequence comparison indicated that the predicted 332 amino acid protein encoded by the cDNA was a member of the annexin gene family named gp-nex and very similar to annexins found in other nematode species. Annexins are calcium-dependent phospholipid binding proteins characterised by four repeated domains approximately 70 amino acids in length. The predicted amino acid sequence of the protein did not contain an N-terminal secretion signal peptide; however, the protein was shown to be present in excretory/secretory products from G. pallida second stage juveniles treated with the neurotransmitter 5 methoxy-N, N dimethyl tryptamine. Gp-nex encodes a protein of 35 kDa and was immunolocalised in the amphids, genital primordium and in the constraining muscles above and below the metacorpus pump chamber of G. pallida second stage juveniles. It is also present in eggs and adult females of the two species of the potato cyst nematodes.

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Immunolocalization of a putative cuticular collagen protein in several developmental stages of Meloidogynearenaria, Globodera pallida and G. rostochiensis

Journal of Helminthology ◽

10.1079/joh200194 ◽

2002 ◽

Vol 76 (1) ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

I.M. de O. Abrantes ◽

R.H.C. Curtis

Keyword(s):

Developmental Stages ◽

Globodera Pallida ◽

Parasitic Nematodes ◽

Cdna Expression Library ◽

In Planta ◽

Cyst Nematodes ◽

Expression Library ◽

Second Stage ◽

Collagen Protein ◽

Cuticular Surface

AbstractThe monoclonal antibody IACR-CCNj.3d has previously been used to isolate a gene (gp-col-8) with strong similarity to cuticular collagen from a mixed stage Globodera pallida cDNA expression library. The antibody has also been shown to label specifically the amphidial canal of pre-parasitic second stage juveniles (J2) of several plant nematode species without any reactivity on the cuticular surface, indicating that this protein is either not present or is inaccessible on the cuticular surface. This paper investigates the cross-reactivity of Mab IACR-CCNj.3d with Meloidogyne arenaria and the localization of the putative collagen protein on the cuticular surface of parasitic stages in planta and on the cuticular surface of juveniles inside eggs. The antigen was shown to be present in all developmental stages of the two species of potato cyst nematodes and M. arenaria. The antibody bound strongly to the amphidial canal and hypodermis of pre-parasitic J2 and adult females. The antigen was present on the cuticular surface of the sausage-shaped J2 in planta and of first stage juveniles (J1) inside the eggs. The presence of collagen on the surface of the cuticle of moulting stages of plant parasitic nematodes has been observed for the first time. It is clear that this protein has a role in the construction of the cuticle of the first stage juveniles and parasitic second stage juveniles, during moulting inside the eggs and in the root tissue, respectively.

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Integrative Morphometric and Molecular Approach to Update the Impact and Distribution of Potato Cyst Nematodes Globodera rostochiensis and Globodera pallida (Tylenchida: Heteroderidae) in Algeria

Pathogens ◽

10.3390/pathogens10020216 ◽

2021 ◽

Vol 10 (2) ◽

pp. 216

Author(s):

Aouicha Djebroune ◽

Gahdab Chakali ◽

Eugénia de Andrade ◽

Maria João Camacho ◽

Leidy Rusinque ◽

...

Keyword(s):

Nematode Species ◽

Globodera Rostochiensis ◽

Globodera Pallida ◽

Morphological Identification ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Additional Information ◽

Second Stage ◽

Species Specific ◽

The Impact

Morphological and molecular studies were conducted to characterize the specific identity of 36 isolates of potato cyst nematodes (PCNs) recovered from soil samples collected in several potato producing areas of Algeria. Morphometric data revealed that 44% of isolates contained Globodera pallida alone, 28% contained Globodera rostochiensis alone and 28% mixtures of the two species. Morphometric values of cysts and second-stage juveniles were generally distributed with slight differences in the expected ranges for both Globodera species. Inter- and intraspecific morphometric variability in nematode isolates was noted. Molecular analysis using conventional multiplex PCR with species-specific primers and TaqMan real-time PCR confirmed the morphological identification. In addition, the distribution of both potato cyst nematode species throughout various parts of the country was investigated. In the central areas, the isolates of G. pallida alone dominate, whereas isolates of G. rostochiensis alone are more frequent in the southern areas. In the eastern regions, mixed isolates are more representative. Most isolates examined in the western areas are mixtures of the two species or G. rostochiensis alone. Comparatively, G. pallida remains the most widely distributed species in its geographic range. This study confirms the presence of two PCN species, G. pallida and G. rostochiensis, in Algeria and provides additional information on their biogeographic distribution.

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Cloning of fibA, Encoding an Immunogenic Subunit of the Fibril-Like Surface Structure ofPeptostreptococcus micros

Journal of Bacteriology ◽

10.1128/jb.181.8.2485-2491.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2485-2491 ◽

Cited By ~ 6

Author(s):

B. H. A. Kremer ◽

J. J. E. Bijlsma ◽

J. G. Kusters ◽

J. de Graaff ◽

T. J. M. van Steenbergen

Keyword(s):

Amino Acid ◽

Surface Structure ◽

Signal Sequence ◽

Open Reading Frames ◽

Periodontal Pathogen ◽

Immunogold Electron Microscopy ◽

Expression Library ◽

Gram Positive ◽

Secretion Signal ◽

Specific Serum

ABSTRACT Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogenPeptostreptococcus micros. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (RgSm) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the RgSm type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a λTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designatedfibA, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of fibA exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.

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Occurrence and molecular characterization of cyst nematode species (Globodera spp.) associated with potato crops in Colombia

PLoS ONE ◽

10.1371/journal.pone.0241256 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0241256

Author(s):

Daniela Vallejo ◽

Diego A. Rojas ◽

John A. Martinez ◽

Sergio Marchant ◽

Claudia M. Holguin ◽

...

Keyword(s):

Management Strategies ◽

Nematode Species ◽

Globodera Pallida ◽

Rrna Gene ◽

Potato Cyst Nematodes ◽

28S Rrna ◽

Cyst Nematodes ◽

Nucleotide Substitutions ◽

Internal Transcribed Spacer Region ◽

The Status

Potato cyst nematodes (PCN) from the genus Globodera spp. cause major losses in the potato (Solanum tuberosum) industry worldwide. Despite their importance, at present little is known about the status of this plant pathogen in cultivated potatoes in Colombia. In this study, a total of 589 samples collected from 75 geographic localities in nine potato producing regions of Colombia (Cundinamarca, Boyacá, Antioquia, Nariño, Santander, Norte de Santander, Tolima, Caldas and Cauca) were assayed for the presence of potato cyst nematodes. Fifty-seven percent of samples tested positive for PCN. Based on phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene, all populations but one were identified as Globodera pallida. Sequences of G. pallida from Colombia formed a monophyletic group closely related to Peruvian populations, with the lowest average number of nucleotide substitutions per site (Dxy = 0.002) and net nucleotide substitutions per site (Da = 0.001), when compared to G. pallida populations from Europe, South and North America. A single sample formed a well-supported subclade along with G. rostochiensis and G. tabacum from Japan, USA and Argentina. To our knowledge this is the first comprehensive survey of Globodera populations from Colombia that includes genetic data. Our findings on species diversity and phylogenetic relationships of Globodera populations from Colombia may help elucidate the status and distribution of Globodera species, and lead to the development of accurate management strategies for the potato cyst nematodes.

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A Synonymous Mutation in Yersinia enterocolitica yopE Affects the Function of the YopE Type III Secretion Signal

Journal of Bacteriology ◽

10.1128/jb.187.2.707-715.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 707-715 ◽

Cited By ~ 22

Author(s):

Kumaran S. Ramamurthi ◽

Olaf Schneewind

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Type Iii Secretion ◽

Predicted Amino Acid Sequence ◽

Nucleotide Position ◽

Reporter Protein ◽

Single Nucleotide ◽

Type Iii ◽

Secretion Signal ◽

Nucleotide Mutation

ABSTRACT Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE, when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE1-7-Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ, codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ, the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE1-7-Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.

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The effects of α-solanine and zinc sulphate on the behaviour of potato cyst nematodes Globodera rostochiensis and G. pallida

Nematology ◽

10.1163/15685411-00002927 ◽

2015 ◽

Vol 17 (9) ◽

pp. 1105-1111 ◽

Cited By ~ 1

Author(s):

Vincas Būda ◽

Rasa Čepulytė-Rakauskienė

Keyword(s):

Host Plants ◽

Zinc Sulphate ◽

Globodera Rostochiensis ◽

Globodera Pallida ◽

The Other ◽

Potato Cyst Nematodes ◽

Water Control ◽

Cyst Nematodes ◽

Second Stage ◽

Testing Period

Two behavioural assays were carried out: one on the attraction of potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, to solanaceae-specific secondary metabolite α-solanine, the other on the effect of ZnSO4 (a compound known to suppress chemoreceptors). The first assay demonstrated that α-solanine was attractive to second-stage juveniles (J2) of PCN; G. pallida was attracted to concentrations of 10−4 M and 10−5 M, whereas G. rostochiensis was attracted to 10−5 M. Globodera pallida reacted faster than G. rostochiensis to the same concentration of α-solanine. As α-solanine is produced by host plants of PCN, this compound is attributed to kairomones. The response to α-solanine of nematodes pre-exposed to a 3 mM ZnSO4 solution was significantly suppressed compared to that of water control. The effect was observed throughout the whole testing period (30 min). This is the first evidence that both α-solanine and ZnSO4 can affect the behaviour of hatched J2 of PCN.

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Comparative genomics among three cyst nematode species reveals distinct evolutionary histories among effector families and an irregular distribution of effector-associated promoter motifs

10.1101/2021.11.01.466756 ◽

2021 ◽

Author(s):

Joris J.M. van Steenbrugge ◽

Sven van den Elsen ◽

Martijn Holterman ◽

Jose L. Lozano-Torres ◽

Vera Putker ◽

...

Keyword(s):

Host Plant ◽

Reference Genome ◽

Nematode Species ◽

Cyst Nematode ◽

Globodera Pallida ◽

Heterodera Schachtii ◽

Cyst Nematodes ◽

Promoter Regions ◽

Long Read ◽

Additional Reference

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are an essential handle for PCN control. However, the poor delineation of G. pallida pathotypes hampers the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCN after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression (DOG boxes) were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Re-sequencing of PCN populations with deviant virulence characteristics will allow for the linking of these characteristics with the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

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Immunochemical studies on pathotypes of the potato cyst nematodes, Globodera rostochiensis and G. pallida

Parasitology ◽

10.1017/s0031182000055475 ◽

1985 ◽

Vol 90 (3) ◽

pp. 471-483 ◽

Cited By ~ 4

Author(s):

P. C. Fox ◽

H. J. Atkinson

Keyword(s):

Nematode Species ◽

Cyst Nematode ◽

Globodera Pallida ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Crossed Immunoelectrophoresis ◽

Specific Antigens ◽

And Cluster Analysis ◽

Species Specific ◽

Micro Organisms

The potential of antigenic differences for discriminating pathotypes of the potato cyst nematodes Globodera pallida and G. rostochiensis has been examined by the use of an antiserum raised to a homogenate of potato cyst nematode larvae. Species-specific antigens were detected among reference pathotypes but more variability was detected among field populations, and cluster analysis was used to interpret the precipitation are pattern produced by Laurell crossed-immunoelectrophoresis. A division into species was seen with this analysis but no definite pathotype groupings were detected. Cross-reaction with other cyst-nematode species was limited to general non-specific precipitation. The antigens were all proteinaceous, did not arise from micro-organisms within the cyst and were mainly hydrophilic with an acidic isoelectric point. Peptidase and acid phosphatase activity was detected in some precipitation arcs but this was not species specific.

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An evaluation of the implications of virulence in non-European populations of Globodera pallida and G. rostochiensis for potato cultivation in Europe

Nematology ◽

10.1163/138855411x587112 ◽

2012 ◽

Vol 14 (1) ◽

pp. 1-13 ◽

Cited By ~ 29

Author(s):

Sue Hockland ◽

Björn Niere ◽

Eric Grenier ◽

Vivian Blok ◽

Mark Phillips ◽

...

Keyword(s):

Genetic Variability ◽

South America ◽

Integrated Management ◽

Nematode Species ◽

Globodera Pallida ◽

Plant Health ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Resistant Cultivars ◽

Council Directive

The potato cyst nematodes Globodera pallida and G. rostochiensis are listed in the EU Plant Health Directive 2000/29/EC and are also subject to the new EU Council Directive 2007/33/EC on the control of potato cyst nematodes, requiring unilateral suppression of these pests in Europe. At the same time there is also pressure to increase world trade in potatoes. Such pressure has to be balanced by the risks involved in the associated spread of these pests and subsequent problems in management. Populations of the potato cyst nematodes from outside Europe, in particular South America, which is considered the origin of G. pallida and G. rostochiensis, pose a risk to those European countries where limited genetic variability of these nematode species has been recorded. The development and usage of resistant cultivars under such conditions has formed a pivotal role in integrated management programmes in Europe. Molecular studies have shown that populations of G. pallida and G. rostochiensis from South America have a different genetic composition from those in Europe. The introduction of such populations would pose a threat to the use of resistant cultivars as a major tool in reducing the potential spread and damage caused by these species. At present, an inability to link precisely genetic variability to the virulence characteristics of a specific nematode population, and quickly identify the virulence status of intercepted populations for inspection purposes, strengthens the case for using plant health legislation to prevent their introduction.

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Development and validation of real-time PCR assays based on novel molecular markers for the simultaneous detection and identification of Globodera pallida, G. rostochiensis and Heterodera schachtii

Nematology ◽

10.1163/15685411-00003086 ◽

2017 ◽

Vol 19 (7) ◽

pp. 789-804 ◽

Cited By ~ 5

Author(s):

Sylvie Gamel ◽

Aude Letort ◽

Didier Fouville ◽

Laurent Folcher ◽

Eric Grenier

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Simultaneous Detection ◽

Nematode Species ◽

Globodera Pallida ◽

Heterodera Schachtii ◽

Cyst Nematodes ◽

Detection And Identification ◽

Template Dna

Considering the growing trade of seed potato, reliable diagnostic protocols are required for the detection of regulated nematode species. In this study, a specific and sensitive multiplex Taqman-based real-time PCR method was developed in order to detect and identifyGlobodera pallida,G. rostochiensisandHeterodera schachtii. The newly designed primers and probes enabled the detection of all the target populations tested and with no cross-reaction for closely related non-target species (55 populations tested). The limit of detection (LOD) was one juvenile forG. rostochiensisandG. pallidaand five juveniles forH. schachtii. For monitoring potato cyst nematodes, this analytical tool would extend the number of cyst investigated as five juveniles can be detected among 50 cysts in a sample. Furthermore, this multiplex assay detects DNA of the three targeted species in template DNA obtained directly from float material after nematode extraction from soil.

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