Journal of Heredity Advance Access originally published online on July 19, 2007
Journal of Heredity 2007 98(6):611-619; doi:10.1093/jhered/esm050
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Sequence Polymorphism of 2 Pioneer Genes Expressed in Phytoparasitic Nematodes Showing Different Host Ranges
From the National Institute of Agronomical Research, Agrocampus Rennes, UMR1099 BiO3P (Biology of Organisms and Populations applied to Plant Protection), F-35653 LE RHEU, France
Address correspondence to Eric Grenier at the address above, or e-mail: eric.grenier{at}rennes.inra.fr; or to Alexandra Blanchard at the address above, or e-mail: alexandra.blanchard{at}rennes.inra.fr.
In order to identify genes involved in pathogenicity, we compared the closely related species Globodera pallida (GP) and Globodera "mexicana" (GM) that have different host ranges and are able to produce viable and fertile hybrids. Three pioneer genes were previously identified as differentially expressed between GP and GM: GPLIA7 and GPLIB3 were found to be more highly expressed in GP, whereas GMLIVG9 was found more highly expressed in GM. In this study, we showed that Ia7 and IVg9 genes probably encode products secreted by the subventral oesophageal glands and the dorsal oesophageal gland, respectively. No Blast homolog was found in the databases, but a metridin-like ShK (Stichodactyla helianthus) toxin domain was identified in the Ia7 sequence. Analysis of the full-length sequences of these 2 genes between GP and GM revealed a high level of interspecies variability (8% for the Ia7 transcript and 17% for the IVg9 transcript) and a high proportion (90%) of nonsynonymous mutations among the substitutions observed. This suggested that these 2 pioneer genes are under strong diversifying selection pressures and therefore may be involved in pathogenicity. Further investigations of the sequence polymorphism of Ia7 and IVg9 genes were conducted in GP x GM hybrid lines that were selected in laboratory conditions for their different ability to develop on potato and black nightshade. As similar sequences were obtained for all the hybrid lines tested independently of their pathogenicity status, no correlation could be established between IA7 and IVG9 amino acid changes and the host range differences observed between GP and GM.
Plant parasitic nematodes such as cyst nematodes produce an assortment of parasitism proteins in order to infect plants (Davis et al. 2004; Vanholme et al. 2004). Both host range specificity and suppression of host plant resistance are thought to be controlled by some of these nematode parasitism genes. Progress have been made in recent years on the isolation of parasitism genes that may also constitute virulence genes (Semblat et al. 2001; Bekal et al. 2003; Neveu et al. 2003), but little is known about which nematode parasitism genes are responsible for the success of the interaction. In plant parasitic nematodes, no sequenced genome is yet available but several initiatives are underway. We can therefore expect that considerable progress in determining the genetic basis of the parasitism in nematodes will be made in the future as it has been achieved using a comparative genomic approach with other pathogens (Da silva et al. 2002; Paulsen et al. 2002). However, these studies have also shown that apart from some unique genes specific to particular species, point mutations may also contribute to the differences in the host specificity observed. The role of point mutations that change the amino acid sequence of a protein should therefore be considered; for example, a single amino acid change in Escherichia coli FimH adhesion leads to the loss of activity and an altered host specificity for this bacteria (Pouttu et al. 1999).
Cyst nematodes of agronomic interest in the genus Globodera infest only Solanaceous plants. Among them, Globodera pallida (GP) and Globodera "mexicana" (GM)—which was described in the unpublished Campos-Vela's thesis in 1967—are 2 closely related species (Bossis and Mugniéry 1993; Thiéry and Mugniéry 1996) that display distinct host ranges. GP is able to develop on potato (Solanum tuberosum) but not on black nightshade (Solanum nigrum), whereas G. mexicana is able to develop on black nightshade but not on potato. The tomato (Lycopersicum esculentum) is a common host for the 2 species. The incapacity of GM to develop on S. tuberosum resides in its inability to induce a feeding site, even after penetrating the roots (Thiéry et al. 1997). Interestingly, GP and GM are able to produce viable and fertile hybrids (Mugniéry et al. 1992). The progeny obtained in controlled conditions loose their ability to develop on potato, but this ability can be quickly restored after 1 or 2 backcrosses with the GP parent (Thiéry et al. 1997). Depending on the plant host used to rear these hybrids, we were able to select in the laboratory populations that are able to develop on both potato and black nightshade or only on potato.
Grenier et al. (2002) investigated the transcriptome differences between G. pallida and G. "mexicana" by suppressive and subtractive hybridization (SSH) and identified several unique sequences of potato cyst nematode. Three of them, GPLIA7, GPLIB3, and GMLIVG9, were pioneer sequences without any homologs in databases and were then classified as potato cyst nematode–specific genes. The GPLIA7 and GPLIB3 transcripts were detected as more highly expressed in GP, whereas GMLIVG9 transcript was overexpressed in GM. These results were confirmed in reverse Northern blots. Establishing the function of the proteins encoded by these pioneer genes is a difficult task. It has been shown that the genes expressed in nematode salivary glands have a role in parasitism as they correspond to secreted products that can interact with plant proteins (Williamson and Gleason 2003; Davis et al. 2004).
Thus, in this study, which is complementary to the quantitative SSH approach performed on the GP and GM transcriptomes, we carried out a qualitative investigation of the 3 pioneer genes, GPLIA7, GPLIB3, and GMLIVG9. In a first step, experiments including in situ hybridization and analysis of the full-length cDNA sequence were conducted to assign a function to these sequences. In a second step, the gene structures and sequence variability among GP, GM, and the hybrid lines selected in the laboratory were compared. Putative implications of the mutations observed in host range specificity are discussed.
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Biological Material
The G. pallida population Guiclan and the G. "mexicana" population Santa Ana used in this study were, respectively, reared on S. tuberosum and S. nigrum. Juveniles (J2s) were obtained by soaking cysts first in water for 4 days at 4 °C and then in potato root diffusate at room temperature. The J2s were stored in water at 4 °C for in situ hybridization or at –80 °C for nucleic acid extraction.
As sex determination for these nematodes is epigenetic, females and males were produced independently. Hybrid lines were obtained in controlled conditions after the selection scheme presented in Figure 1. A first cross was generated with GP female and GM male to produce hybrid lines of the first generation (G1). Then successive backcrosses, using GP females and several hybrid males, were performed to generate hybrid lines of generations 2–3 on tomato. For each cross, females were produced after in vitro inoculation of one juvenile in one root of tomato, and then one male of GM or G1 or G2 was added to each plate containing a single female. Finally, half of the progeny obtained in G3 was multiplied on potato, the other half on black nightshade. Starting from 33 individual G3 cysts, we were able to obtain 9 hybrid lines on potato but none on black nightshade. Each potato line generated was tested for its ability to develop on S. nigrum.
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mRNA In Situ Hybridization
The clones GPLIA7, GPLIB3, and GMLIVG9 were used to design specific primers (Table 1) to generate in situ hybridization probes. The first step was to amplify the insert of each clone before dig labeling each probe (sense and antisense) by asymmetric PCR procedure. Insert amplification was performed in 50 µl with 30 ng of insert, 0.4 µM of each primer, 1x buffer, 0.2 mM of each dNTP, 2 U of Taq DNA polymerase (Promega, Charbonniéres, France) and 1.5 mM MgCl2, for 4 min at 94 °C, 40 cycles at 94 °C for 30 s, Tm for 30 s, 72 °C for 1 min, and a final elongation step of 4 min at 72 °C. Asymmetric PCR was performed in 40 µl with 2 µl of the previous purified PCR product, 2.5 µM of a primer (Fwd or Rev), 1x buffer, 1.5 mM MgCl2, 0.75x Dig-dUTP/dNTP mix (Roche Applied Science, Mannheim, Germany) and 2 U of Taq DNA polymerase (Promega) for 4 min at 94 °C, 34 cycles at 94 °C for 30 s, Tm for 1 min, 72 °C for 90 s, and a final elongation step of 4 min at 72 °C. In situ hybridization was performed as described by De Boer et al. (1998) with slight differences. Freshly hatched J2s were fixed in fixation buffer (including paraformaldehyde) for 18 h at 4 °C and 4 h at room temperature. The nematodes were cut with a razor blade and the sections were permeabilized with proteinase K (0.5 mg/ml) for 30 min at 22 °C before freezing at –80 °C. Hybridizations were performed at 45 °C overnight with purified sense or antisense single-strand cDNA probes. The signal was detected using alkaline phosphatase immunostaining (NBT-BCIP, Boehringer, Ingelheim, Germany; Sigma-Aldrich, Lyon, France).
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Isolation of the Full Length of the Ia7 and IVg9 Transcripts
RNA Isolation
Total RNA was extracted from 50 to 100 mg of J2s with the RNeasy Mini kit (Sigma) according to the manufacturer's instructions. The RNAs were treated with DNase I (10 U, Ambion, Courtaboeuf, France) for 15 min at 37 °C and then purified by phenol/chloroform/isoamyl alcohol (25/24/1) extraction. The RNAs were precipitated with 3 M sodium acetate and absolute ethanol, and stored at –80 °C.
mRNA Reverse Transcription
Reverse transcription was performed in a mix containing 2 µg of total RNA, 0.25 µM of d(T)25 primer and 8.5 µl of RNase-free water. The samples were heated for 10 min at 70 °C and set on ice. 1x superscript buffer, 2.5 mM MgCl2, 0.25 µM of each dNTP and 0.01 µM of dithiothreitol were then added. The sample was heated for 1 min at 42 °C. Superscript III (Invitrogen, Cergy, France 200 U/µl) was added and the mix was incubated at 42 °C for 50 min, and at 70 °C for 15 min. One microliter of RNase mix (Boehringer) was added and the mixture was incubated for 30 min at 37 °C.
5' RACE
Transcripts 5' ends were isolated using the 5' RACE System for Rapid Amplification of cDNA Ends (Invitrogen) according to the manufacturer's instructions. The first strand cDNA was obtained from total RNAs using 0.4 µM of specific primer GPLIA7Rev2 or IVG9Fwd (Table 1). Nested PCR was performed in 50 µl with 1x buffer, 1.5 mM of MgCl2, 0.2 mM of each dNTP, 2.5 U of Taq DNA polymerase (Promega), 5 µl of cDNA, 0.4 µM of AAP (5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG -3', Invitrogen), and 0.4 µM of the internal specific primer (GPLIA7rev2 or IVG9Fwd; Table 1). PCR conditions were 96 °C for 1 min, 35 cycles at 96 °C for 20 s, 53 °C for 30 s and 72 °C for 30 s, with a final elongation step at 72 °C for 5 min.
The PCR products were purified (GeneElute PCR Clean-Up kit, Sigma) and cloned in pGEM-T Easy Vector System I (Promega) according to the manufacturer's instructions.
Characterization of the Ia7 and IVg9 Genomic Sequences
DNA Isolation
Genomic DNA was extracted from the eggs (separated from the cysts) of 500 rehydrated cysts. The frozen eggs were crushed in eppendorf tubes with a piston. The DNA was extracted with lysis buffer (0.1 M Tris, pH 8, 10 mM ethylenediaminetetraacetic acid, 2% Sodium dodecyl sulfate) and proteinase K, and then incubated at 65 °C for 1 h. The DNA was centrifuged and purified with 140 µl of 5 M NaCl and 64 µl of 10% hexadecyltrimethylammonium bromide, incubated for 10 min at 65 °C and then with a phenol/chloroform (1:1) mix. The DNA was precipitated with ammonium acetate 5 M overnight at 4 °C. A mix containing 30% of PEG 6000 and isopropanol (99.8%) was added to achieve DNA precipitation. The sample was treated with 1 µl of RNase A (500 µg/ml) for 2 h at 37 °C.
Ia7 and IVg9 Gene Amplifications
The 5'IA7/3'IA7 and 5'IVG9.2/3'IVG9.2 (Table 1) primers designed from the Ia7 and IVg9 cDNA sequences were used to, respectively, amplify the Ia7 and IVg9 genes in G. pallida, G. "mexicana" and in the hybrid populations.
The Ia7 gene was amplified by PCR using 50 ng of DNA, 1x buffer, 1.5 mM of MgCl2, 0.2 mM of each dNTP, 0.4 µM of each primer, and 1.25 U of GoTaq flexi (Promega). PCR was performed after the program: 96 °C for 1 min, 35 cycles at 94 °C for 20 s, 59 °C for 5 s, 57 °C for 5 s, 55 °C for 10 s, and 68 °C for 3 min, with a final elongation step at 68 °C for 10 min. The IVg9 gene was amplified in the same conditions except for the MgCl2 concentration that was 3 mM.
All the PCR products were purified using Sephadex G50 and then directly sequenced one time by Macrogen (Korea, http://www.macrogen.com) using sense and antisense primers.
Sequence Analysis
WU-Blast2 Parasite Genomes Database Query on European Molecular Biology Laboratory-European Bioinformatics Institute (www.ebi.ac.uk/blast2/parasites.html), and Blast on National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/) or nemaBLAST (http://www.nematode.net/) were used to search for sequence homologies. The EXPASY translate tool (http://us.expasy.org/tools/dna.html) was used to predict amino acid sequences. Sequence alignments were performed with Multalin algorithm (Corpet 1998; http://prodes.toulouse.inra.fr/multalin/multalin.html). The sequence signatures were detected using SIGNALP (Bendtsen et al. 2004; www.cbs.dtu.dk/services/SignalP/), and INTERPROSCAN (Quevillon et al. 2005; http://www.ebi.ac.uk/InterProScan/) and the properties of the proteins were identified using PROTPARAM (Gasteiger et al. 2005; http://us.expasy.org/tools/protparam.html).
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Spatial Expression of the Transcripts GPLIA7, GPLIB3, and GMLIVG9
Grenier et al. (2002) identified GPLIA7, GPLIB3, and GMLIVG9 as differentially expressed by SSH (Table 1). The spatial expression patterns of these 3 transcripts were studied using in situ hybridization. Approximately 70–80% of the nematodes were labeled in each experiment. One of them, GPLIB3, was located in the intestine (Figure 2A) of the GP nematodes using the IB3Fwd digoxigenin-labeled probe (see primers in Table 1).
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The GPLIA7 transcript was located in esophageal glands of the GP nematodes (Figure 2B) using the IA7Rev digoxigenin-labeled probe. After 3 days of incubation, the labeling extended to the median bulb of the J2s (Figure 2C) where the valve of the ventral glands is connected to the lumen of the esophagus, clearly indicating a localization in the ventral esophageal glands.
Using the IVG9Fwd digoxigenin-labeled probe, an accumulation of GMLIVG9 transcripts was clearly observed in the dorsal gland of the GM nematodes (Figure 2D).
The 3 complementary probes generated using IB3Rev, IA7Fwd2, and IVG9Rev primers were used as negative controls (data not shown) and produced no signal. After these observations, we decided to focus further investigations on only the GPLIA7 and GMLIVG9 transcripts that showed a localization in the nematode salivary glands and therefore correspond to secreted products that are probably implicated in the parasitism process.
Isolation of the Full-Length Ia7 and IVg9 cDNA
As a polyA tail was observed in both GPLIA7 and GMLIVG9 inserts, specific primers (Table 1) were designed in order to obtain the full-length cDNAs using 5' RACE PCR technique. The GPLIA7Rev2 and IVG9Fwd primers (Table 1) were used to obtain the 5' end of the Ia7 and IVg9 transcripts in GP and GM, respectively. The PCR products were cloned and sequenced one time using sense and antisense primers to build the full-length transcripts in silico using the SSH clone insert sequences.
The full-length Ia7 cDNA was then estimated to be 389 bp in length for G. pallida with an identified open reading frame (ORF) of 222 bp. Two primers (5'IA7 and 3'IA7, Table 1) flanking the ORF were designed to check the in silico construction. A fragment of approximately 250 bp was amplified confirming the construction. The Ia7 cDNA encoded a deduced protein of 73 aa (Figure 3A) with a calculated molecular weight of 8 kDa. A peptide signal of 25 aa was predicted by SignalP and a metridin-like ShK (Stichodactyla helianthus) toxin domain was identified by the INTERPROSCAN program from the amino acids 39–73 (Figure 3A).
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The full-length IVg9 cDNA built in silico represented in GM a product of 471 bp with an ORF of 285 bp encoding a putative 95 aa protein with a molecular weight of 11 kDa and a peptide signal of 24 aa (Figure 3B). In order to check the in silico construction, the 5'IVG9.2 and the 3'IVG9.2 primers were designed (Table 1). A PCR product of more than 300 bp was obtained in agreement with predicted sequence. No specific domains were identified using INTERPROSCAN program.
BLAST searches revealed no significant homology (E value threshold 10–4) in databases for both full-length transcripts.
Sequence Variability of Ia7 and IVg9 Genes in GP and GM
Amplifications of the Ia7 and IVg9 genes were obtained from GP and GM genomic DNAs. For Ia7, a band of about 250 bp was observed on an agarose gel from both GP and GM suggesting that this gene was intronless. The high quality of the sequences enabled analysis without a cloning step. Sequences obtained for GP and GM were of the same size. Out of the 222 bp of the ORF, 18 substitutions (8% of sites) were observed between GP and GM (Figure 4A) resulting in 15 variable aa in the predicted protein sequence between the 2 species (89% of nonsynonymous substitutions) (Figure 3A). The mutations are equally distributed along the amino acid sequence with only one accumulation of 3 nonsynonymous substitutions from the amino acids 29–31.
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The IVg9 gene was found to be 830 bp long for GP and 712 bp long for GM. The ORFs were 285 bp for GP and 300 bp for GM, encoding predicted proteins of 95 and 100 amino acids, respectively (Figure 3B). Comparing GP and GM sequences, 50 variable sites were identified (17.5% of sites) resulting in 26 altered amino acid positions in the predicted protein sequences. Similar to Ia7, 90% of the substitutions observed are nonsynonymous substitutions. Three insertion deletions were observed between the GM and GP IVg9 sequences (Figure 4B): 2 insertions of 15 and 3 nucleotides were observed in the GM IVg9 sequence and 1 deletion of 3 nucleotides was observed in the C-terminal part of the GM gene. Compared with the transcript sequence, this gene appeared to contain 2 introns. The introns had the same distribution along the IVg9 gene sequence in both GP and GM (Figure 4B) indicating that these are orthologs. The first intron was 102 bp in length for both species and displayed 4% variability. The second was 428 bp in length for GP and 295 bp in length for GM and showed 8.5% variability and 6 indels (4 indels of only one nucleotide).
Pathogenicity of the Hybrid Lines
We obtained 9 G3 hybrid lines (HL) all derived from the same individual cross between a GM male and a GP female. All these HL were obtained from nematodes that were reared and multiplied on potato. Attempts to rear HL on black nightshade were all unsuccessful. As few material of each hybrid line was available, the host range of the 9 potato hybrid lines was investigated through an in vitro inoculation test on S. nigrum. Considering the level of development of GM on S. nigrum during this test (30%), and the fact that the hybrid lines only contained 20% of GM AFLP markers (Grenier E, personal communication), the hybrid lines development was expected to reach at best 6% on black nightshade. Potato HL displayed various host ranges: 3 were unable to develop on black nightshade and were called simple pathogens (HLs), the others were able to develop on S. nigrum but only 2 of them (HLd) with a level similar to that expected (Table 2).
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Characterization of the Ia7 and IVg9 Genes in Hybrid Lines
As the sequence variations observed between GP and GM for both Ia7 and IVg9 genes strongly affected the amino acid sequences and therefore putatively the function of these proteins in GM and GP, we decided to investigate the sequence variability of these 2 genes in the hybrid lines derived from these species. The Ia7 and IVg9 genes were amplified using the same primers as for GP and GM (see amplification in Figure 5B and 5D) in 3 different hybrids lines: an HL showing no development on S. nigrum (HL 86) and 2 HLs representing the inferior and superior level of development observed on S. nigrum (HL 80 and HL 96). For the Ia7 gene, a PCR product of about 250 bp was amplified (Figure 5A). Sequences obtained for the 3 HL (one sequencing on both strands for each HL) were also of high quality enabling analysis without a cloning step. These sequences were identical to the GP sequence except for one nucleotide difference at position 192 (Figure 4A). For the IVg9 gene, a PCR product of about 850 bp was amplified from the 3 hybrid lines (Figure 5C). In the coding region of this gene, no variation was found among the hybrid lines. The IVg9 sequences of the HL correspond to the GP sequence except for 2 nucleotides at the position 267 and 831 (Figure 4B) that appeared to be synonymous substitutions in the deduced proteins.
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Consequently, we can consider that for both genes the hybrid sequences were inherited from the GP parent and appeared independent of the ability of the hybrids to develop on potato or on potato and black nightshade.
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The study of the genetic differences between 2 closely related species of Globodera that have different host ranges is an opportunity to reveal potential pathogenicity factors that act specifically against a host during the plant invasion by the nematode. Two genes are described in this study as potential pathogenicity factors of Globodera nematodes both of which encode putative-secreted proteins. However, no significant matches in databases to homologs of these genes were found. So their function in host specificity remained unknown.
The in situ hybridization experiments localized Ia7 and IVg9 to esophageal glands and Ib3 to the intestine. This suggests that the IA7 and IVG9 proteins are secreted during the plant invasion and thus play a role in the plant–nematode interaction, whereas the role of the IB3 protein in parasitism remains less clear. The accumulation of Ia7 transcripts was clearly observed in the ventral glands and not in the dorsal gland. With longer exposure times, the transcripts were also localized in the valve of the glands that is connected to esophagus in the median bulb of the J2s. In contrast, the IVg9 transcripts were localized in the dorsal gland of the nematodes. The accumulation of those transcripts in the 2 different types of salivary gland could be an indication that these 2 genes act at different stages of parasitism. Von Mende (1997) reported that ventral glands are metabolically active earlier than the dorsal one. The sequences of the Ia7 and IVg9 transcripts indicated the presence of a secretion signal in the corresponding predicted proteins as a supplementary indication that these 2 proteins are secreted. Moreover, the small sizes (molecular weight: 8 kDa for IA7 and 11 kDa for IVG9) of the predicted proteins are compatible with the exclusion size of the lumen of the nematode stylet and feeding tubes (Böckenhoff and Gründler 1994).
Database searches with the full-length sequences of Ia7 and IVg9 did not reveal significant homologies with any sequences. The function of these genes remained unknown, although the bioinformatics analyses did identify specific motifs in the Ia7 sequence where a metridin-like ShK toxin (Stichodactyla helianthus K channel toxin) domain was identified. Such a domain was found in some toxins, but also in several Caenorhabditis elegans proteins without any known function (Castadena et al. 1995; Tudor et al. 1998).
The orthology of the GP and GM IVg9 sequences was confirmed by finding the same intron/exon boundaries and a low level of intron variability between GP and GM sequences (4–8%). Indeed, numerous studies sustained the high conservation of the intron/exon structure among clades (Rokas et al. 1999; Wada et al. 2002; Rogozin et al. 2003) even if some studies contradicted this rule (Goetze 2006). Consideration of the orthology of the GP and GM Ia7 sequences is more difficult as the IA7 gene is intron less. However, as the variability of the Ia7 coding region (8%) is less than half of that observed for IVg9 (17%), we assume that we are also comparing orthologs in the case of Ia7.
The variability of the Ia7 and IVg9 gene sequences between GP and GM was approximately 8% for Ia7 and 18% for IVg9. This inter species variability appeared very high compared with other parasitism genes such as cellulase or RanBPM genes that showed 1–2% of variability between the same species (Grenier et al. 2002; Blanchard et al. 2005). As a high proportion of the mutations observed correspond to nonsynonymous substitutions (nearly 90%), we suggest that the Ia7 and IVg9 genes have both evolved under strong selective pressure. Some recent studies have reported that genes encoded some surface proteins of parasitic bacteria that were directly in contact with the host were under positive selection (Tsai et al. 2006; Sawires and Songer 2006). Moreover, it has also been shown that the hypervariability of some hrp genes (hypersensitive response and pathogenicity genes) involved in pathogenicity can be found at the intra specific level for the plant parasitic bacteria of various Xanthomonas campestris pathovars (Weber and Koebnik 2005) suggesting a potential role in host specificity. Genes under positive selection are the key to the adaptation to a habitat or niche (Chen et al. 2006). We can therefore hypothesize that this could also occur for hosts and that nematode parasitism genes encoding proteins implicated in plant–nematode interactions will be under positive selection to facilitate the adaptation of the species on different host plants.
In this study, several results indicated that the pioneer genes identified could be pathogenicity genes: differential expression between GP and GM, their putative secretion, the absence of homologs in the extensive C. elegans databases, the high sequence variability, and the high rate of nonsynonymous substitutions. In an attempt to further characterize the function of these genes, studying, for example, the expression of the genes and the impact of the mutations observed, we used GP x GM hybrid lines with various pathogenicities and postulated that the variability of the Ia7 and IVg9 genes in these hybrid lines might be correlated to their host specificity. The Ia7 and IVg9 sequences obtained in all the hybrid lines tested were the same as that of GP that was the recurrent parent during the crossing experiment. Some PCR performed in other hybrid lines obtained in G2 instead G3 (data not shown) displayed the same results. Thus, as no sequence difference was observed among hybrid lines tested, we were unable to show any association of the mutations observed in these 2 genes in host range specificity. However, as the hybrid lines were able to develop on potato we cannot exclude the possibility that the mutations observed in Ia7 or IVg9 GM genes preclude the development of GM on potato or that the mutations observed in Ia7 and IVg9 GP genes allow the nematodes to develop on potato. Clearly, a new crossing experiment should now be designed to obtain new hybrid lines representing all the expected various pathogenicities on black nightshade in order to answer these questions.
We described an original method to study candidate parasitism genes through the evaluation of their polymorphism in populations and lines that differentially develop on various plants. We strongly support this kind of investigation using other putative parasitism genes to test the hypothesis of a host specificity implication to complement functional analyses of corresponding proteins.
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We thank the "Santé des Plantes et Environnement" department of INRA for funding.
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Corresponding Editor: Ron C. Woodruff
Received November 10, 2006
Accepted May 17, 2007
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Bekal S, Niblack TL, Lambert KN. A chorismate mutase from the soybean cyst nematode Heterodera glycines shows polymorphisms that correlate with virulence. Mol Plant Microbe Interact (2003) 16:439–446.[CrossRef][Web of Science][Medline]
Bendtsen JD, Nielsen H, Von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol (2004) 340:783–795.[CrossRef][Web of Science][Medline]
Blanchard A, Esquibet M, Fouville D, Grenier E. Ranbpm homologue genes characterized in the cyst nematodes Globodera pallida and Globodera ‘mexicana’. Physiol Mol Plant Pathol (2005) 67:15–22.[CrossRef]
Böckenhoff A, Gründler FMW. Studies on the nutrient uptake of the beet cyst nematode. H. schachtii by in situ microinjection of fluorescent probes into the feeding structures in Arabidopsis thaliana. Parasitology (1994) 109:249–254.
Bossis M, Mugniéry D. Specific status of six Globodera parasites of Solanaceous plants studied by means of two-dimensional gel electrophoresis with a comparison of gel patterns by a computed system. Fundam Appl Nematol (1993) 16:47–56.
Campos-Vela A. Taxonomy, life cycle and host range of Heterodera mexicana. Sp. (Nematoda: Heteroderidae) [thesis]. (1967) Madison (WI): University of Wisconsin. 70.
Castadena O, Sotolongo V, Amor AM, Stöcklin R, Anderson AJ, Harvey AL, Engström A, Wernstedt C, Karlson E. Characterization of a potassium channel toxin from the caribbean sea anemaone Stichodactyla helianthus. Toxicon (1995) 33:603–613.[Medline]
Chen SL, Hung CS, Xu J, Reigstad CS, Magrini V, Sabo A, Blasiar D, Bieri T, Meyer RR, Ozersky P, et al. Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach. Proc Natl Acad Sci USA (2006) 103(15):5977–5982.
Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res (1998) 16:10881–10890.[CrossRef]
Da Silva ACR, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB, Monteiro-Vitorello CB, Sluys MAV, Almeida NF, Alves LMC, et al. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature (2002) 417:459–463.[CrossRef][Medline]
Davis EL, Hussey RS, Baum TJ. Getting to the roots of parasitism by nematodes. Trends Parasitol (2004) 20:134–141.[CrossRef][Web of Science][Medline]
De Boer JM, Yan YT, Smant G, Davis EL, Baum TJ. In-situ hybridization to messenger RNA in Heterodera glycines. J Nematol (1998) 30:309–312.[Medline]
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy Server. In: The proteomics protocols handbook—Walker JM, ed. (2005) Towaota (NJ): Humana Press. 571–607.
Goetze E. Elongation factor 1-alpha in marine copepods (Calanoida: Eucalanidae): phylogenetic utility and unique intron structure. Mol Phylogenet Evol (2006) 40:880–886.[CrossRef][Web of Science][Medline]
Grenier E, Blok VC, Jones JT, Fouville D, Mugniéry D. Identification of gene expression differences between Globodera pallida and G. mexicana by suppression subtractive hybridization. Mol Plant Pathol (2002) 3:217–226.[CrossRef]
Mugniéry D, Bossis M, Pierre JS. Hybridations entre Globodera rostochiensis (Wollenweber), G. pallida (Stone), G. virginiae (Miller & Gray), G. solanacearum (Miller & Gray) et G. mexicana (Campos-Vela). Description et devenir des hybrids. Fundam Appl Nematol (1992) 15:375–382.
Neveu C, Abad P, Castagnone-Sereno P. Molecular cloning and characterization of an intestinal cathepsin L protease from the plant-parasitic nematode Meloidogyne incognita. Physiol Mol Plant Pathol (2003) 63:159–165.[CrossRef]
Paulsen IT, Seshadri R, Nelson KE, Eisen JA, Heidelberg JF, Read TD, Dodson RJ, Umayam L, Brinkac LM, Beanan MJ, et al. The Brucellasuis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc Natl Acad Sci USA (2002) 99:13148–13153.
Pouttu R, Puustinen T, Virkola R, Hacker J, Klemm P, Korhonen TK. Amino acid residue Ala-62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis-associated Escherichia coli to collagens. Mol Microbiol (1999) 31:1747–1757.[CrossRef][Web of Science][Medline]
Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R. InterProScan: protein domains identifier. Nucleic Acids Res (2005) 33:116–120.
Rogozin IB, Wolf YI, Sorokin AV, Mirkin BG, Koonin EV. Remarkable interkingdom conservation of intron positions and massive, lineage-specific intron loss and gain in eukaryotic evolution. Curr Biol (2003) 13:1512–1517.[CrossRef][Web of Science][Medline]
Rokas A, Kathirithamby J, Holland PW. Intron insertion as a phylogenetic character: the engrailed homeobox of Strepsiptera does not indicate affinity with Diptera. Insect Mol Biol (1999) 8:527–530.[CrossRef][Web of Science][Medline]
Sawires YS, Songer JG. Clostridium perfringens: insight into virulence evolution and population structure. Anaerobe (2006) 12:23–43.[CrossRef][Web of Science][Medline]
Semblat JP, Rosso MN, Hussey RS, Abad P, Castagnone-Sereno P. Molecular cloning of a cDNA encoding an amphid-secreted putative avirulence protein from the root-knot nematode Meloidogyne incognita. Mol Plant Microbe Interact (2001) 14:72–79.[CrossRef][Web of Science][Medline]
Thiéry M, Mugniéry D. Interspecific rDNA restriction fragment length polymorphism in Globodera species, parasites of Solanaceaous plants. Fundam Appl Nematol (1996) 19:471–479.
Thiéry M, Mugniéry D, Bossis M, Sosa-Moss C. Résultats de croisements entre Globodera pallida Stone et. G. mexicana Campos-Vela: héritabilité du développement sur pomme de terre et notion d'espèce. Fundam Appl Nematol (1997) 20:551–556.
Tsai YH, Orsi RH, Nightingale KK, Wiedmann M. Listeria monocytogenes internalins are highly diverse and evolved by recombination and positive selection. Infect Genet Evol (2006) 6:378–389.[CrossRef][Web of Science][Medline]
Tudor JE, Pennington MW, Norton R. Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin. Eur J Biochemy (1998) 251:133–141.[CrossRef]
Vanholme B, De Meutter J, Tytgat T, Van Montagu M, Coomans A, Gheysen G. Secretions of plant-parasitic nematodes: a molecular update. Gene (2004) 332:13–27.[CrossRef][Web of Science][Medline]
Von Mende N. Invasion and migration behaviour of sedentary nematodes. In: Cellular and molecular aspects of plant-nematodes interactions—Fenoll C, Grundler FMW, Ohl SA, eds. (1997) Dordrecht (The Netherlands): Kluwer Academics Publishers. 51–64.
Wada H, Kobayashi M, Sato R, Satoh N, Miyasaka H, Shirayama Y. Dynamic insertion-deletion of introns in deuterostome EF-1 alpha genes. J Mol Evol (2002) 54:118–128.[CrossRef][Web of Science][Medline]
Weber E, Koebnik R. Positive selection of the Hrp pilin HrpE of the plant pathogen Xanthomonas. J Bacteriol (2006) 188:1405–1410.
Williamson VM, Gleason CA. Plant-nematode interactions. Curr Opin Plant Biol (2003) 6:327–333.[CrossRef][Web of Science][Medline]
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