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SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ACKNOWLEDGEMENTS
  5. REFERENCES

A solid-phase subtractive strategy was used to clone parasitism gene candidates (PGCs) expressed in the oesophageal gland cells of Meloidogyne incognita. Nematode intestinal first-strand cDNA was synthesized directly on magnetic beads and used to enrich for gland-specific sequences by high stringency hybridization to gland-cell mRNA. A gland-specific cDNA library was created from the nonhybridizing gland-cell mRNA by long-distance reverse transcription polymerase chain reaction. Subtraction of the gland cDNA library (1000 clones) with previously cloned M. incognita parasitism genes removed 89 cDNA clones and promoted efficient identification of new PGCs. Sequencing of 711 cDNA clones from the subtracted library revealed that deduced protein sequences of 67 cDNAs were preceded by a signal peptide for secretion, a key criterion for parasitism genes. In situ hybridization with probes from the cDNA clones encoding signal peptides showed that seven cDNA clones were specifically expressed in the subventral gland cells and four in the dorsal gland cell of M. incognita. BLASTP analyses revealed the predicted proteins of five cDNAs to be novel sequences. The six PGCs with similarities to known proteins included a pectate lyase, three beta-1,4-endoglucanases and two chorismate mutases. This subtractive protocol provides an efficient and reliable approach for identifying PGCs encoding oesophageal gland cell secretory proteins that may have a role in M. incognita parasitism of plants.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ACKNOWLEDGEMENTS
  5. REFERENCES

Root-knot nematodes, Meloidogyne species, are among nature's most successful parasites. They parasitize more than 2000 plant species from diverse plant families and represent a tremendous threat to crop production world-wide (Sasser, 1980). These biotrophic pathogens have evolved highly specialized and complex feeding relationships with their hosts. A successful nematode–host interaction requires molecular signals from the parasite to modify, directly or indirectly, plant root cells into elaborate feeding cells, called giant-cells, which are the sole source of nutrients needed for nematode development and reproduction.

Plant-parasitic nematodes release proteinaceous secretions through a hollow, protrusible stylet into plant cells when feeding. These secretions are encoded by parasitism genes expressed in large and transcriptionally active oesophageal gland cells (Hussey, 1989). The profound cellular modifications induced by Meloidogyne species to form the giant-cells are the result of an alteration in root cell gene expression and phenotype that is driven by the molecular signals secreted through the nematode's stylet (Davis et al., 2000; Gheysen and Fenoll, 2002). An understanding of the nature of the parasitism genes and the function of their products in parasitism is slowly beginning to emerge, aided by the availability of new molecular tools. (Davis et al., 2000, 2004; Jasmer et al., 2003). Most interesting, only a few of the identified parasitism gene candidates (PGCs) of phytoparasitic nematodes have homologues in Caenorhabditis elegans and the majority are novel genes that encode proteins with unknown functions (Davis et al., 2004). Comparative and subtractive gene expression profiles have provided a sampling of parasitism genes in plant-parasitic nematodes, but these data suggest that a more comprehensive approach is necessary to obtain a complete profile of the parasitism genes (Ding et al., 1998; Lambert et al., 1999; Qin et al., 2000).

The approach of directly microaspirating the contents of oesophageal gland cells of parasitic nematode stages to generate cDNA libraries of gland cell-expressed genes has recently provided an array of PGCs that profile the entire nematode parasitic cycle (De Boer et al., 2002; Gao et al., 2001a, 2003; Huang et al., 2003; Wang et al., 2001). Expressed sequence tag (EST) analyses of a gland cell cDNA library generated from Heterodera glycines was combined with high-throughput in situ expression localization of clones encoding secretory proteins to obtain a profile of 51 new PGCs (Gao et al., 2003), which brings the total PGCs cloned from this cyst nematode to over 60. A similar study of parasitism genes in Meloidogyne incognita identified 37 PGCs expressed in the oesophageal gland cells throughout the parasitic cycle (Huang et al., 2003). However, parasitism genes encoding cellulases and chorismate mutase previously cloned from root-knot nematodes (Lambert et al., 1999; Rosso et al., 1999) were not identified in the first M. incognita gland cell cDNA library (Huang et al., 2003). To obtain a comprehensive profile of parasitism genes expressed in the oesophageal glands of M. incognita, we used a more efficient solid-phase subtractive strategy to construct a new gland cell-specific cDNA library for identifying additional oesophageal gland-expressed PGCs in M. incognita.

The subtractive approach used here was modified from Aasheim et al. (1997). Poly(A)+ RNA from the cytoplasm of gland cells aspirated from 53 viable nematodes covering the full range of parasitic stages or the contents from the intestinal cells of 30 parasitic nematodes was bound to 125 µg of Dynabeads oligo(dT)25 magnetic beads (Dynal, Lake Success, NY) and eluted with 15 µL of diethyl pyrocarbonate (DEPC)-treated d2H2O at 70 °C for 2 min as previously described (Gao et al., 2003; Huang et al., 2003). In the case of intestinal region, the final step of eluting poly(A)+ RNA was omitted, and instead, the intestinal mRNA/oligo(dT)25 Dynabeads complex was washed twice in 1× first-strand buffer (50 mm Tris-HCl pH 8.3, 75 mm KCl, 6 mm MgCl2), and resuspended in 100 µL of cDNA synthesis buffer (1× first-strand buffer, 2 mm DTT, 1 mm dNTPs, 1 µL of RNase inhibitors [40 units/µL; Promega], 2 µL of Superscript II reverse transcriptase [200 units/µL; Gibco-BRL]). First-strand cDNA was synthesized directly on the magnetic beads using Dynabeads oligo(dT)25 as primers by reverse-transcriptase polymerase chain reaction (RT-PCR) in a 0.5-mL microcentrifuge tube. The microcentrifuge tube was placed horizontally in an air incubator with a rotating wheel (40 r.p.m.) at 42 °C to keep beads in suspension, and incubated for 2 h to ensure efficient enzymatic reactions. After synthesis of the first-strand cDNA, the magnetic beads were washed twice with 2 mm EDTA at 95 °C for 3 min in order to denature the poly(A)+ RNA, which was removed from the first-strand cDNA coupled to Dynabeads by magnetic separation.

The intestinal first-strand cDNA coupled to Dynabeads was hybridized to gland cell mRNA to remove any common housekeeping and structural genes expressed in the gland cells (Fig. 1). The intestinal first-strand cDNA coupled to the beads was collected with a magnet and resuspended in 90 µL of hybridization buffer (120 mm NaH2PO4 pH 6.8, 820 mm NaCl, 1 mm EDTA, 0.1% SDS and 1 µL of RNase inhibitors [40 units/µL]). Fifteen microlitres of poly(A)+ RNA isolated from the cytoplasm of gland cells was added, and the mixture was layered with 100 µL of sterilized mineral oil. The cap of the microcentrifuge tube was sealed in Parafilm™ to prevent evaporation. The mass ratio of the intestinal first-strand cDNA to the gland cell mRNA in this subtraction step was estimated to be approximately 10 : 1, based on the amount of contents microaspirated from the intestinal region and gland cells of parasitic stages of M. incognita. An initial denaturation step at 95 °C for 3 min was followed by hybridization at 65 °C for 24 h in a rotary hybridization oven (Stovall Life Science, Greensboro, NC) in order to keep the beads suspended (60 r.p.m.) during the hybridization reaction. The hybridized mRNA/cDNA–Dynabead complex was captured by magnetic separation, and the mineral oil was removed. The supernatant containing unhybridized mRNA was collected and subjected to a second hybridization reaction with the recycled cDNA–Dynabeads. The magnetic beads were recycled by eluting the hybridized mRNA twice in 1 mL of H2O at 95 °C for 3 min and washing the magnetic beads twice in 1× washing buffer (150 mm LiCl, 10 mm Tris-HCl pH 8.0, 1 mm EDTA). After three cycles of hybridization, unhybridized gland-cell-specific mRNA in the final supernatant was captured with fresh oligo(dT)25-Dynabeads (50 µg) and eluted with 5 µL of DEPC-treated d2H2O at 70 °C for 2 min and used as template to create a gland-specific cDNA library by long-distance (LD) RT-PCR (SMART cDNA Synthesis System, Clontech Laboratories, Palo Alto, CA). Gland cell first-strand cDNA synthesis was conducted in 0.5-mL reaction tubes in a 10-µL volume of the following mixture: 4 µL of subtracted gland-cell mRNA sample, 0.5 µL of 10 µm cDNA Synthesis primer (5′-AAGCAGTGGTAACAACGCAGAGTACT(30)N−1N-3′) (N−1 = A, G or C) (N-3 = A, C, G or T) (Clontech), 0.5 µL of 10 µm SMART II oligonucleotide (5′-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3′), 2 µL of 5× first-strand buffer, 1.0 µL of 20 mm DTT, 1.0 µL of 10 mm 50× dNTP, 1.0 µL of Superscript II reverse transcriptase (200 units/µL). The tubes were incubated at 42 °C for 1 h, and 90 µL of TE buffer [10 mm Tris-HCl pH 7.6, 1 mm EDTA] was added. Ten microlitres of diluted first-strand reaction solution, 2 µL of 10 mm dNTP mix, 1 µL of TaqPlusLong 10× low-salt buffer, and 1 µL of TaqPlusLong DNA polymerase (Stratagene, La Jolla, CA), 2 µL of PCR primer (5′-AAGCAGTGGTAACAACGCAGAGT-3′) (Clontech) were used in a 100-µL volume LD PCR reaction. LD PCR was performed with hot start followed by 24 cycles at 94 °C (20 s), 65 °C (30 s) and 72 °C (4 min). Negative controls of DEPC-treated water were performed at each reaction step above. To monitor the efficiency of the subtraction, one aliquot of gland-cell LD PCR cDNA was used to amplify the corresponding fragment (410 bp) of the M. incognita house-keeping histone H1 gene (GenBank accession no. BQ548185) using the specific primers MH1F (5′-AGGAAAAGAAAGGGGCTAGCC-3′) and MH1R (5′-GGCTTGACTGTCTTCTTAGCTG-3′). We were not able to amplify the histone H1 gene in the cDNA pool while positive controls were easily detectable, suggesting that most house-keeping genes in the gland cells were subtracted. Prior to creating the gland-cell LD PCR cDNA library, an aliquot of the gland-cell LD PCR cDNA products was used as template to amplify the corresponding predicted products with the primers derived from the 37 previously cloned M. incognita PGCs (Huang et al., 2003). We were able to identify in the cDNA pool 32 of the 37 previously identified PGCs, indicating that the gland cell cDNA pool was of high quality.

image

Figure 1. Schematic presentation of solid-phase subtractive strategy.

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Another aliquot of the gland-cell LD PCR products was purified with QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and ligated into the pGEM-T Easy vector (Promega, Madison, WI) at a mass ratio of 3 : 1 (plasmid : cDNA) at 4 °C overnight. Ligation products were precipitated with 10 mm glycogen and 100% ethanol, followed by a wash with 70% ethanol. The purified ligation products were heat-shock transformed into Escherichia coli XL10-GLOD ultracompetent cells (Stratagene). EcoRI digestions were used to check the inserts in the pGEM-T Easy vector. The efficiency of the heat-shock transformation was 5.0 × 109 clones per microgram of vector. One thousand white colonies (on blue-white selection) of the gland cell subtracted cDNA library were hand-picked and transferred to 96-well MICROTEST III Tissue Culture plates (Becton Dickinson, Franklin Lakes, NJ) containing 200 µL of 10% glycerol Luria-Bertani with ampicillin, and incubated overnight at 37 °C prior to colony dot-blotting on to sterile Hybond-XL nylon membranes (70 × 105 mm; Amersham Pharmacia Biotech, Piscataway, NJ). Gel analysis of 20 clones selected randomly from the gland-cell LD PCR cDNA library created from the LD PCR cDNA pool showed that insert sizes ranged from 0.3 to 3.0 kb.

Hybridization probes were made by amplifying cDNA fragments from the 37 previously cloned parasitism genes by PCR (Huang et al., 2003). PCR products were cut from gel, purified with the QIAquick gel extraction kit (Qiagen), and the RTS RadPrime DNA labelling system (Gibco_BRL, Grand Island, NY) was used to random prime label the mixture of gel-purified PCR products with 32P-dCTP. Colony dot-blotting and high-stringency hybridization (65 °C overnight) were performed according to standard procedures (Sambrook et al., 1989), and membranes were washed twice with 0.1× SSC (1× SSC is 150 mm NaCl plus 15 mm sodium citrate), and 0.1% sodium dodecyl sulphate at 65 °C. Hyperfilm (Amersham Pharmacia Biotech) was developed after overnight exposure at −80 °C. Hybridizing with the 37 previously cloned M. incognita PGCs (Huang et al., 2003) identified only 89 positive cDNA clones and thus promoted efficient identification of new PGCs from the non-hybridizing gland-specific cDNA clones.

Gland cell cDNA clones that did not hybridize to the 37 previously cloned M. incognita PGC probes were randomly selected for 5′-end single pass cDNA sequencing using SMART-1 primer (5′-GGTAACAACGCAGAGTACGCG-3′). Sequencing reactions were prepared using an Applied Biosystems cycle sequencing kit. Sequences were collected on an ABI 3700 autosequencer (Applied Biosystems, Foster City, CA) and processed as previously described (Huang et al., 2003). High-quality sequences were obtained for 711 cDNA clone sequences. BLASTP (or BLASTX) analyses (Altschul et al., 1997) were conducted on the predicted protein sequences encoded by all cDNA with novel sequences designated as ‘pioneers’. The deduced protein sequences of 67 cDNAs from 711 cDNA sequences were predicted by SignalP to contain N-terminal signal peptides to direct the proteins into the secretory pathway (Nielsen et al., 1997). The presence of the predicted signal peptides identified these proteins as candidates for being secreted by M. incognita and potentially having a biological function in the nematode–host interaction.

The 67 cDNA clones with predicted signal peptides were screened by high-throughput in situ mRNA hybridization to confirm gland expression in M. incognita specimens as previously described (Huang et al., 2003). Specific forward and reverse primers for each gland-cell cDNA clone predicted to encode a signal peptide were used to synthesize digoxigenin (DIG)-labelled antisense cDNA probe (200–300 nt) by asymmetric PCR. PCR reactions were performed in a 20-µL reaction mixture with PCR DIG labelling mix (Boehringer Mannheim, Mannheim, Germany) in the asymmetric PCR instead of dNTP in the normal PCR. Specimens were observed with a compound light microscope to detect cDNA probes that hybridized within the nematode. The in situ analyses identified 11 distinct cDNA clones that specifically hybridized to transcripts accumulating within the subventral (seven clones) or dorsal (four clones) oesophageal gland cells of M. incognita (Table 1). These clones can be regarded as PGCs because they are expressed specifically in the oesophageal gland cells and encode proteins with signal peptides. PSORT II analyses predicted ten of the deduced proteins of these 11 PGCs to be extracellular, and one (clone 4F05B) to be localized in the endoplasmic reticulum (Nakai et al., 1999). Two of the predicted products of the PGCs (1C05B and 4F05B) contain transmembrane domains based on TMHMM computer analysis (http://www.cbs.dtu.dk/services/TMHMM/) and may not actually be secreted.

Table 1.  Summary of 11 parasitism gene candidates encoding proteins preceded by a signal peptide for secretion and expressed exclusively with the oesophageal gland cells of Meloidogyne incognita.
CloneAccession no.FL/ORF (bp)Protein homologyBLASTP score/E valueGland expression*
Pre-J2Par-J2J3-A
  • *

    In situ hybridization of cDNA probes to mRNA specifically within the single dorsal oesophageal gland cell (DG) or the two subventral oesophageal gland cells (SvG) in preparasitic second-stage juveniles (Pre-J2), parasitic J2 (Par-J2) or later stages (J3-A) of Meloidogyne incognita.

  • New sequences submitted to GenBank with the exception of AF100549, which was already in the databases.

  • Size of the full-length clone with predicted open reading frame (ORF) size.

  • §

    No homology (E value < 0.005) to any protein in the public databases.

  • Not detected.

1C05BAY422829617/375Pioneer§DGDG
1C11BAF1005491690/1518Mi-eng-1—M. incognita877/0SvGSvG
1D08BAY422830547/513Pioneer DGDG
2B02BAY327873963/840 Pectate lyase—Globodera rostochiensis149/4e−35SvGSvG
2G06BAY422834720/571Chorismate mutase—M. javanita331/4e−90SvGSvG
4F05BAY422831763/471Pioneer DGDG
5A12BAY4228361706/1518Mi-eng-1a—M. incognita863/0SvGSvG
5C03BAY422832589/273Pioneer DGDG
6D09BAY422835660/573Chorismate mutase—M. javanita320/9e−87SvGSvG
8E08BAY4228371242/1080Cellulase—M. incognita587/e−167SvGSvG
8E10BAY4228331217/945Putative oesophageal gland cell secretory protein 10—M. incognita154/2e−36SvGSvGSvG

Full-length cDNA sequences with predicted open reading frames for the PGCs ranged in size from 547 to 1706 bp (Table 1). The predicted open reading frames were determined by the presence of (i) translation initiation and termination signals, (ii) a polyadenylation signal sequence and (iii) a putative signal peptide at the amino-terminal end of the predicted protein. 3′-RACE was used to obtain full-length cDNAs for three PGCs (4F05B, 8E08B and 8E10B) where only gene fragments were initially cloned. To obtain these full-length cDNAs, mixed parasitic stages (150 µL) of M. incognita were frozen in 1.5-mL microcentrifuge tubes with liquid nitrogen and ground with a smooth-end metal bar. mRNA was purified with oligo (dT)25 magnetic beads and eluted with 20 µL DEPC-treated water. Reverse transcription and 3′-RACE were carried out with SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. Universal Primer Mix (5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGT-3′ and 5′-CTAATACGACTCACTATAGGGC-3′) and 5′ end gene-specific primer 4F05BF (5′-TCACAATTATGGGCAGTTGGG-3′), 8E08BF (5′-GGGGAAGGTTTCTATAACAG-3′), or 8E10BF (5′-AAGGATGACGAAGAAGAGAATGG-3′) were used for PCR amplification. The amplified PCR products were cloned into pGEM-T Easy vector for sequencing.

PSI-BLASTP analyses revealed six PGC products as homologues with proteins with known functions in other organisms. The deduced protein of subventral gland-specific clone 8E10B had high similarity to a putative oesophageal gland cell secretory protein (GenBank Accession Number AF531170) previously cloned from M. incognita (Huang et al., 2003). Domain analysis using the SMART database (http://www.smart.embl-heidelberg.de) revealed the predicted protein of the clone 8E10B (like its homologue) contained coiled-coil domains, but its function remains unknown. Clone 1C11B encodes a β-1,4-endoglucanase containing a catalytic domain and a cellulose-binding domain (CBD) separated by a linker and was previously cloned (MI-ENG-1) from M. incognita (Rosso et al., 1999). Two other cellulase genes (5A12B and 8E08B) were novel and bring the total of subventral gland cell-synthesized β-1,4-endoglucanases in M. incognita to three. The predicted β-1,4-endoglucanase of clone 5A12B had 98% identity to the catalytic domain of MI-ENG-1 and 97% identity to the CBD of MI-ENG-1 (Rosso et al., 1999). However, clone 8E08B encoded a distinctly different β-1,4-endoglucanase, which lacks CBD and has only 89% amino acid identity to the catalytic domain of MI-ENG-1. The β-1,4-endoglucanases are used by M. incognita to hydrolyse the β-1,4 glycosidic bonds of cellulose in the cell walls during penetration and their intercellular migration within plant roots (Bera-Maillet et al., 2000; Rosso et al., 1999; Wang et al., 1999). A different group of cell wall-digesting enzymes is represented by clone 2B02B. Clone 2B02B encodes a pectate lyase, which is the second pectinase gene cloned from M. incognita. The predicted pectate lyase of 2B02B had only 30% identity to the MI-PEL-1, which was previously cloned from M. incognita (Huang et al., 2003). The pectate lyases are also expressed in the subventral oesophageal gland cells of plant-parasitic nematodes with a presumed function in nematode migration in plant roots (Davis et al., 2000; Doyle and Lambert, 2002; Popeijus et al., 2000).

Two other PGCs (2G06B and 6D09B) that are expressed in the subventral oesophageal gland cells of preparasitic and parasitic M. incognita second-stage juveniles encode chorismate mutase. Chorismate mutase was previously cloned from the root-knot nematode M. javanica (Lambert et al., 1999). This protein is a key branch-point enzyme in the shikimate pathway leading to the synthesis of phenylalanine and tyrosine in plants. Transgenic expression of the nematode chorismate mutase gene (Mj-cm-1) in soybean hairy roots results in a phenotype of reduced and aborted lateral roots (Doyle and Lambert, 2002). Homologues of chorismate mutases have also been identified in the cyst nematodes, H. glycines and Globodera pallia (Bekal et al., 2003; Gao et al., 2003; Jones et al., 2003). Interestingly, chorismate mutase is expressed in the dorsal gland cell of H. glycines, but it is expressed in the subventral gland cells of Meloidogyne species and Globodera pallida (Gao et al., 2003; Jones et al., 2003). Recently, chorismate mutase genes of H. glycines were shown to exhibit polymorphisms that correlate with virulence on resistance soybean cultivars (Bekal et al., 2003).

Only two of the PGCs (clones 2G06B and 6D09B encoding chorismate mutases) were trans-spliced with a leader sequence. The leader sequences were spliced 35 bp upstream from the initiating methionine codon and were identical to the SL1M splice leader previously described from M. incognita (Ray et al., 1994). The transcript of a chorismate mutase gene (Mj-cm-1) cloned from M. javanica, which shares 94–97% sequence identity at the amino acid level with the two M. incognita chorismate mutase genes identified here, also contains the SL1M (Lambert et al., 1999). The transcripts of two previously cloned pectate lyase genes from Meloidogyne spp. also contained the SL1M, which was spliced 9 bp from the initiating methionine codon (Doyle and Lambert, 2002; Huang et al., 2003). However, the transcript of 2B02B encoding a pectate lyase contained no SL1M, or derivatives thereof, beyond 34 bp upstream of the putative translation initiation codon.

Five PGCs had no similarity (E value < 0.005) to functionally annotated genes in the databases, as did 70% of the PGCs previously cloned from M. incognita (Huang et al., 2003). These ‘pioneer’ sequences will provide a significant challenge to determine their roles in Meloidogyne spp. parasitism of plants. Nevertheless, domain analyses of these pioneer sequences may provide clues for functional analysis. For example, the coiled-coil domain in the predicted protein of 8E10B suggests a possible function in a structural protein or in binding to host molecules, because coiled-coil domains in proteins fulfil a variety of functions (Lupas, 1996). Currently, expression of parasitism genes in host cells and RNA-mediated interference (RNAi) are emerging to be feasible techniques, which will allow us to study the function of these PGCs in the M. incognita–plant interaction (Mazarei et al., 1998; Urwin et al., 2002).

The immobilized cDNA subtraction technique used here provided an efficient method to remove common mRNAs in the aspirated M. incognita gland cell cytoplasm for the isolation of genes specifically expressed in the gland cells. The solid-phase subtractive strategy used here for constructing the gland cell cDNA library has several advantages over the protocols previously used to construct gland cell cDNA libraries (Gao et al., 2001; Huang et al., 2003), including (i) subtractive hybridization prior to PCR amplification to remove common genes in the gland cells for enrichment of gland-specific sequences; (ii) reduced size of the gland cell cDNA library for easier screening; (iii) the ability to re-use the driver cDNA coupled to magnetic beads several times for high stringency hybridizations; and (iv) the ability to generate labelled probes from the cDNA–magnetic beads for hybridization approaches (Aasheim et al., 1997). Identifying the complete profile of parasitism genes expressed in the oesophageal gland cells during the parasitic cycle of a nematode is of critical significance for understanding the molecular basis of nematode parasitism of plants and defining what makes a nematode a plant parasite (Hussey et al., 2002). The procedure outlined herein provides a strategy to clone rapidly parasitism genes encoding secretory proteins from plant-parasitic nematodes.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ACKNOWLEDGEMENTS
  5. REFERENCES

Support for this research was provided by the National Research Initiative Competitive Grants Program of the Cooperative State Research, Education, and Extension Service of the United States Department of Agriculture under Agreement no. 99-35302-8080, the Iowa Soybean Promotion Board, the Iowa Agriculture and Home Economics Experiment station (Project no. 3381), by Hatch ACT and State of Iowa, and by state and Hatch Funds allocated to the Georgia Agricultural Experiment Stations.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. ACKNOWLEDGEMENTS
  5. REFERENCES
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