Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi.

RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted beta-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plant-parasitic nematode. Knockout of the beta-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.

Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.

We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.

Cloning of a Putative Pectate Lyase Gene Expressed in the Subventral Esophageal Glands of Heterodera glycines.

We report the cloning of a Heterodera glycines cDNA that has 72% identity at the amino acid level to a pectate lyase from Globodera rostochiensis. In situ hybridizations showed that the corresponding gene (Hg-pel-1) is expressed in the subventral esophageal gland cells of second-stage juveniles. The deduced amino acid sequence of the H. glycines cDNA shows homology to class III pectate lyases of bacterial and fungal origin.

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP.

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.

Functional screening yields a new beta-1,4-endoglucanase gene from Heterodera glycines that may be the product of recent gene duplication.

Clones with secreted cellulolytic activity were identified when a cDNA library constructed from poly A(+) RNA of preparasitic second-stage juveniles of Heterodera glycines, the soybean cyst nematode, was expressed in the Escherichia coli SOLR strain and overlaid with a carboxymethylcellulose (CMC) substrate. Twenty CMC-degrading clones were analyzed, and all were either identical or strongly similar to a beta-1,4-endoglucanase gene (HG-eng-2), previously isolated from H. glycines. A subgroup of identical "HG-eng-2-like" clones had considerable differences in the 5' untranslated region compared with HG-eng-2 and were designated HG-eng-3. One H. glycines genomic clone contained HG-eng-2 and HG-eng-3 full-length genes, separated by a distance of approximately 8 kb, and a second genomic clone contained two copies of HG-eng-2, separated by approximately 6.5 kb, suggesting the presence of endoglucanase gene clusters in H. glycines. The HG-eng-2 and HG-eng-3 genes were in opposite transcriptional orientation, with considerable nucleotide differences in their 5' flanking regions. The highly conserved nucleotide sequence in the introns and exons and their close proximity within the genome suggest that HG-eng-2 and HG-eng-3 are the products of recent gene duplication and inversion.

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

Isolation of Beta-1,4-Endoglucanase Genes from Globodera tabacum and their Expression During Parasitism.

Two beta-1,4-endoglucanase (EGase) cDNAs were isolated from Globodera tabacum, the tobacco cyst nematode, and have been designated as GT-eng-1 and GT-eng-2. GT-eng-1 and GT-eng-2 encode precursor proteins with a predicted secretion signal sequence, cellulolytic catalytic domain, and a linker domain. The protein product GT-ENG-1 contains an additional 95 amino acid carboxy terminal sequence with strong similarity to type II cellulose binding domains. Riboprobes and polyclonal antibodies raised to recombinant cyst nematode EGases were used to follow expression patterns of EGase transcripts and proteins throughout the nematode life cycle. EGase transcripts and proteins were specifically detected within the subventral esophageal gland cells of G. tabacum second-stage juveniles (J2) within eggs prior to hatching, in preparasitic J2, and in parasitic J2 that had invaded tobacco roots. EGase transcripts and proteins were not detected in G. tabacum after the molt to the sedentary J3, J4, and adult female life stages. Interestingly, EGase transcription and translation resumed in the subventral esophageal glands of late J4 males. It is hypothesized that secreted EGases play a major role to facilitate intracellular migration of G. tabacum within tobacco roots.

Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.

Developmental expression of secretory beta-1,4-endoglucanases in the subventral esophageal glands of Heterodera glycines.

Two beta-1,4-endoglucanases (EGases), Hg-eng-1 and Hg-eng-2, were recently cloned from the soybean cyst nematode, Heterodera glycines, and their expression was shown in the subventral esophageal glands of hatched second-stage juveniles (J2). We examined the expression of these EGases in the subventral glands of all post-embryonic life stages of H. glycines by in situ hybridization and immunolocalization. The first detectable accumulation of EGase mRNAs occurred in the subventral glands of unhatched J2. EGase transcripts remained detectable in J2 after hatching and during subsequent root invasion. However, in late parasitic J2 and third-stage juveniles (J3), the percentage of individuals that showed EGase transcripts decreased. In female fourth-stage juveniles and adult females, EGase transcripts were no longer detected in the subventral glands. EGase hybridization signal reappeared in unhatched males coiled within the J3 cuticle, and transcripts were also present in the subventral glands of migratory adult males. Immunofluorescence labeling showed that EGase translation products are most abundantly present in the subventral glands of preparasitic J2, migratory parasitic J2, and adult males. The presence of EGases predominantly in the migratory stages suggests that the enzymes are used by the nematodes to soften the walls of root cells during penetration and intracellular migration.

In planta localization of a beta-1,4-endoglucanase secreted by Heterodera glycines.

Polyclonal sera specific to beta-1,4-endoglucanases (cellulases) synthesized in the subventral esophageal gland cells of the soybean cyst nematode, Heterodera glycines, were used to provide the first identification of a nematode esophageal gland protein that is secreted into host plant tissue. Sera generated to proteins encoded by Hg-eng-1 and Hg-eng-2 (endoglucanases) did not cross-react with soybean root proteins on Western blots (immunoblots) or in immunofluorescence microscopy of noninoculated (control) soybean root sections. In cross sections of soybean roots at 24 h after inoculation of roots with second-stage juveniles of H. glycines, HG-ENG-1 was localized within the nematode's subventral gland cells and was not detected in root tissue. HG-ENG-2 was localized within the subventral gland cells and was secreted from the juvenile's stylet into root cortical tissue at 24 h after inoculation of roots with second-stage juveniles of H. glycines. HG-ENG-2 was localized along the juvenile's migratory path through the root cortex.

Cloning of a trans-spliced glyceraldehyde-3-phosphate-dehydrogenase gene from the potato cyst nematode Globodera rostochiensis and expression of its putative promoter region in Caenorhabditis elegans.

Reverse genetics to determine the relative importance of individual pathogenicity factors of the potato cyst nematode Globodera rostochiensis depends, apart from an efficient transformation protocol for this obligatory plant parasite, on the availability of an efficient promoter. PCR-based cloning was used to isolate a cDNA encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, a crucial enzyme in glycolysis and gluconeogenesis; this gene was designated gpd) and its 5'-flanking region. The cDNA includes 1047 nucleotides encoding an open reading frame that shows high homology with GAPDHs from Caenorhabditis elegans and other species. Analysis of the 745 bp 5'-flanking region of the gpd gene showed no homology with a similar region in C. elegans. In this region several eukaryotic promoter elements are present. 5' Rapid amplification of cDNA ends revealed this gene was trans-spliced with a SL1 spliced leader. The 5'-flanking region of the gpd gene was fused to green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. Green fluorescent protein expression, under the transcriptional control of the 5'-flanking region of gpd, was mainly observed in body wall muscles of transgenic animals. This putative promoter region of GAPDH could be a valuable tool to drive gene expression in transgenic G. rostochiensis and other related plant-parasitic nematode species.

Genomic organization of four beta-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications.

The genomic organization of genes encoding beta-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5'-GCellipsisAG-3' in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5'-GAUAAA-3'-both rare occurences in eukaryotic genes. The 5'- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.

Endogenous cellulases in animals: isolation of beta-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes.

beta-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3' end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.

In-situ Hybridization to Messenger RNA in Heterodera glycines.

A method is presented for in-situ hybridization to mRNA in second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA of a cellulase gene that was known to be expressed in the subventral esophageal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections with a vibrating razor blade to make the inside of the nematodes accessible for probing. These nematode fragments then were hybridized in suspension with riboprobe, and labeled with an alkaline phosphatase-conjugated antibody to digoxigenin. Staining with nitroblue tetrazolium and bromo-chloro-indolyl phosphate revealed a highly specific hybridization signal to mRNA within the cytoplasm of the subventral gland cells, using this specific antisense probe. This in-situ hybridization protocol will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications.

Potato root diffusate-induced secretion of soluble, basic proteins originating from the subventral esophageal glands of potato cyst nematodes.

ABSTRACT In preparasitic second-stage juveniles (J(2)) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses of 30, 31a/b, 32, 39, and 49 kDa were recognized on Western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J(2) and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J(2) showed that the svp's were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of svp's through the metacorporal pump chamber was confirmed by the presence of svp's in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.

Secretory granule proteins from the subventral esophageal glands of the potato cyst nematode identified by monoclonal antibodies to a protein fraction from second-stage juveniles.

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.