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Characterisation of guanylyl cyclase genes in the soybean cyst nematode, Heterodera glycines
International Journal for Parasitology 32 (2002) 65–72 www.parasitology-online.com
Characterisation of guanylyl cyclase genes in the soybean cyst nematode, Heterodera glycines q Yitang Yan, Eric L. Davis* Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA Received 25 June 2001; received in revised form 25 September 2001; accepted 25 September 2001
Abstract Parasitism by the soybean cyst nematode, Heterodera glycines, has become one of the major limiting factors in soybean production worldwide. A partial HG-gcy-1 cDNA clone was obtained by screening a H. glycines cDNA library with a probe derived from the HG-gcy1 genomic sequence, and HG-gcy-1 full-length cDNA was obtained by nested PCR and 5 0 rapid amplification of cDNA ends (5 0 RACE). Two additional, full-length guanylyl cyclase cDNA clones from H. glycines, named HG-gcy-2 and HG-gcy-3, were recovered directly by screening the H. glycines cDNA library with a probe derived from sequence of the HG-gcy-1 catalytic domain. The encoded proteins of all three HG-gcy genes had an extracellular ligand-binding domain, a single membrane-spanning domain, an intracellular protein kinase-like domain, and a guanylyl cyclase catalytic domain. The three HG-GCY proteins had conserved cysteine residues to form disulfide bridges within the extracellular domain similar to the predicted ligand-binding domains of other known membrane-bound guanylyl cyclases. mRNA in situ hybridisation detected the expression of HG-gcy-1 and HG-gcy-2 transcripts in specific and different sensory neurons within H. glycines specimens. HG-gcy-3 transcripts were not localised in H. glycines specimens by in situ hybridisation. The discovery of the three guanylyl cyclase genes in H. glycines is the first of its kind in a plant-parasitic nematode and may be representative of a conserved gene family used for chemosensory recognition in parasitic nematodes. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Caenorhabditis elegans; cDNA; Chemosensory receptor; In situ hybridisation; Heterodera glycines; Parasitic nematode; RNA splicing; Sensory neurons
1. Introduction Plant-parasitic nematodes cause greater than $77 billion in world-wide crop losses each year (Sasser and Freckman, 1987). The cyst nematodes (Heterodera and Globodera species) and root-knot nematodes (Meloidogyne species) are sedentary endoparasites of plant roots that account for a majority of nematode-associated crop losses (Sasser and Freckman, 1987). Motile, preparasitic second-stage juveniles (J2) of cyst and root-knot nematodes hatch from eggs in soil and follow gradients of chemical and environmental cues emanating from the host rhizosphere to locate host plant roots (Dusenbery, 1987). The J2 penetrate plant roots completely, establish a permanent feeding site within the root vascular tissue, feed, grow, and molt three more q Nucleotide sequence data reported in this paper are available in GenBank accessions: AF331456 (HG-gcy-1 genomic DNA), AF095746 (HG-gcy-1 cDNA), AF283665 (HG-gcy-2 cDNA), AF283664 (HG-gcy-3 cDNA). * Corresponding author. Tel.: 11-919-515-6692; fax: 11-919-515-7716. E-mail address: [email protected] (E.L. Davis).
times to a swollen and sedentary adult life stage (Davis et al., 2000). Several generations that produce thousands of offspring can occur during one crop growing season, creating an enormous parasitic load on host plants. The soybean cyst nematode, Heterodera glycines, has become one of the major limiting factors in soybean production in the United States and other major soybean production areas (Riggs and Wrather, 1992). Rotations to non-host crops, modifications of cultural practices, the use of nematode-resistant cultivars, and the application of nematicides all provide some reduction in H. glycines damage to soybean, but more efficient nematode management tactics are in great demand. It has been suggested that disorientation of plant-parasitic nematodes by affecting their ability to detect and discriminate (host) chemical signals in their environment might present a powerful means to develop novel nematode management tactics (Perry, 1996). Nematodes can direct their movement toward chemical signals (chemotaxis) emanating from their food source (Bargmann and Mori, 1997). A series of chemosensory-related genes have been isolated in odorant (odr) mutants of the model nematode,
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00315-0
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Caenorhabditis elegans, that have altered response to specific chemical stimuli (Bargmann and Mori, 1997). The odr10 gene of C. elegans encodes a seven-transmembrane (chemoreceptor) protein (Sengupta et al., 1996), the largest gene family identified in C. elegans (Bargmann, 1998). The odr-1 gene of C. elegans encodes a guanylyl cyclase that functions in signalling pathways that affect olfaction and odour discrimination (L’Etoile and Bargmann, 2000). Guanylyl cyclases [GTP pyrophosphate-lyase, EC 4.6.1.2] are a family of enzymes that catalyse the conversion of GTP to cGMP (Lucas et al., 2000). The enzyme family is comprised of both membrane-bound and soluble isoforms. Guanylyl cyclase and cGMP-mediated signalling cascades play an essential role in the regulation of diverse physiological processes including olfaction, muscle motility, electrolyte homeostasis, and retinal phototransduction (Lucas et al., 2000; Meyer et al., 2000). Membrane-bound guanylyl cyclases exhibit highly-conserved domain structure and characteristics of some types of chemoreceptors, including (1) an N-terminal extracellular binding domain that (in some cases) binds to defined ligands, (2) a single transmembrane domain, (3) a cytoplasmic juxtamembrane domain, (4) a protein kinase-like domain, (5) a hinge region, and (6) a C-terminal catalytic domain (Lucas et al., 2000). A guanylyl cyclase (gcy) gene family containing at least 29 members has been identified in C. elegans (Yu et al., 1997). A subset of this gene family contained membranebound guanylyl cyclases that were demonstrated to be expressed in specific sensory neurons, suggesting that guanylyl cyclases may compliment the seven-transmembrane proteins as chemoreceptors in nematodes (Yu et al., 1997). Partial genomic sequence of a guanylyl cyclase gene was recently obtained while sequencing the 5 0 -flanking region of the HG-eng-1 (endoglucanase) gene of H. glycines (Yan et al., 1998). Here we report the first full-length cloning and localisation of expression of three guanylyl cyclase genes in a parasitic nematode.
2. Materials and methods 2.1. Nematodes Soybean cyst nematode, H. glycines, inbred line ‘OP50’ (Dong and Opperman, 1997) was cultured on roots of soybean (Glycine max cv. Lee 68) in a greenhouse. Heterodera glycines cysts were recovered from soil and roots of greenhouse cultures by flotation and sieving, the cysts were gently crushed to release and recover eggs, and the eggs were hatched at 288C to collect daily cohorts of preparasitic J2 (Goellner et al., 2000). Mild blending was used to release parasitic juvenile and adult stages of H. glycines from within soybean roots and parasitic nematodes were subsequently separated from debris with sucrose centrifugation (Goellner et al., 2000). Some fresh preparasitic J2 and parasitic nematode specimens were immediately frozen at 2808C to use
for DNA and RNA extractions. For mRNA in situ hybridisation experiments, some preparasitic J2 and mixed parasitic stages of H. glycines were fixed in 2% paraformaldehyde in M9 buffer (42 mM Na2HPO4, 22 mM KH2PO4, 85 mM NaCl, 1 mM MgSO4, pH 7.0) overnight at 48C (Goellner et al., 2000). Fixed nematodes were collected, rinsed and stored in M9 buffer at 2808C before preparation for mRNA in situ hybridisation experiments. 2.2. Characterisation of the H. glycines guanylyl cyclase-1 genomic clone As indicated above, a 14.6 kb H. glycines genomic clone containing both a guanylyl cyclase gene and a HG-eng-1 gene (Fig. 1A) was initially identified after sequencing a clone that was obtained by screening a H. glycines lambda Fix II (Stratagene) genomic library with a HG-eng-1 probe (Yan et al., 1998). BamHI and NotI digestion divided the 14.6 kb genomic clone into 4.8 kb, 3.8 kb, and 6.0 kb fragments that were subcloned into the pCR2.1 TA vector (Invitrogen). A series of nested primers were used to sequence the H. glycines genomic DNA in the 4.8 kb and 3.8 kb subclones to obtain complete genomic sequence of H. glycines guanylyl cyclase gene-1 (HG-gcy-1). 2.3. cDNA clones of H. glycines guanylyl cyclases Probe designed from the 3 0 end of the HG-gcy-1 gene was used to screen a H. glycines cDNA library (7.8 £ 10 6 pfu/ml) in Lambda Uni-Zap vector (Stratagene) that was previously constructed from mRNA of preparasitic J2 (Smant et al., 1998). Primers designed from the coding sequence of the 3 0 end of the HG-gcy-1 genomic clone, Cycle3UP (5 0 - GGAGG AACGG ACCAA AGAGT -3 0 ) and Cycle4LOW (5 0 GCTGG CCGTG TTCAC TGAGT -3 0 ), were used to synthesise a labelled cDNA probe (Fig. 1B). The cDNA probe was synthesised with the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals) as described by the manufacturer using H. glycines J2 cDNA library as template. XL-Blue MRF’ E. coli cells were infected with H. glycines J2 cDNA phage library stock and plated on growth media (Sambrook et al., 1989), and about 10,000 independent plaques developed overnight at 378C per 150 mm-diameter plate. Plaque lifts on Hybonde-N nylon membrane (Amersham Pharmacia Biotech) were prehybridised at 658C for 2 h, hybridised with the DIG-labelled HG-gcy-1 cDNA probe at 658C overnight as described by the manufacturer (Roche Molecular Biochemicals), and washed 2 £ for 15 min with 0.5 £ SSC, 0.1% SDS at 658C under constant agitation. Plaques that hybridised with the HG-gcy-1 probe were visualised using alkaline phosphatase-conjugated anti-DIG antibody and NBT/X-phosphate substrate solution as described by the manufacturer (Roche Molecular Biochemicals). Forty individual positive plaques were recovered from the plated H. glycines cDNA library for sequence analysis. Since only partial cDNA clones of HG-gcy-1 were recovered from plaque screening, full-length HG-gcy-1 cDNA
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Fig. 1. (A) The 14.6 kb fragment of Heterodera glycines genomic DNA that contained full-length guanylyl cyclase 1 (HG-gcy-1) and endoglucanase 1 (HGeng-1) genes in opposite transcriptional orientations (lower bold arrows). The 14.6 kb fragment had been cloned in the T3/T7 sites of the lambda Fix II vector (Stratagene), and Bam H1 and Not 1 restriction digests of the insert produced three fragments (dotted arrows) that were subcloned for DNA sequencing. (B) The steps to compile the full-length cDNA clone (3762 bp) of Heterodera glycines guanylyl cyclase 1 (HG-gcy-1) included a partial (1327 bp) HG-gcy-1 cDNA clone that was isolated from a H. glycines cDNA library with a labelled probe produced by PCR using primers (Cycle 4LOW and Cycle3UP) that were complimentary to the coding regions of the 3 0 end of HG-gcy-1 genomic DNA. Primer pairs based upon upstream HG-gcy-1 genomic sequence were used with first-strand cDNA template from H. glycines to obtain 1941 and 819 bp cDNA fragments to reconstruct the HG-gcy-1 cDNA, and primer Cycle K was used in 5 0 rapid amplification of cDNA ends (5 0 RACE) to complete (383 bp) the cDNA sequence.
was obtained by PCR using the primer sets outlined in Fig. 1B and template first-strand cDNA that was synthesised from total RNA of H. glycines preparasitic J2. Total RNA was prepared from 1 ml of packed H. glycines preparasitic J2 using a cesium trifluoracetate RNA extraction kit (Amersham Pharmacia Biotech). First-strand cDNA was synthesised at 428C for 50 min using random hexamers and SuperScript II reverse transcriptase according to manufacturer’s instructions (GIBCO-BRL). Using first-strand H. glycines cDNA as template, the following primer sets (Fig. 1B) were used to obtain the full coding region of HG-gcy-1 cDNA using PCR: CycleA (5 0 - ATTTG TGGGG TGCTA CTGTGG -3 0 ) and CycleB (5 0 - GGTGC GTCTG AGCCG ATAAAG -3 0 ), CycleE (5 0 - TCGGC TTCCT CTTTG CTA -3 0 ) and CycleF (5 0 - TAGCA CCCCA CAAAT ACAG -3 0 ). To obtain complete 5 0 end sequence of HG-gcy cDNA, the 5 0 RACE (Rapid Amplification of cDNA Ends) System (GIBCO-BRL) was used with H. glycines total RNA and the following gene-specific primers; CycleK (5 0 - CCGTT
GGGTG CCGTT AGC -3 0 ) for HG-gcy-1, Cycle2E (5 0 CGGGC AAAAG ACAAA TCGG AC -3 0 ) for HG-gcy-2, and Cycle3E (5 0 - TTCGT CTGCC CACAA CTGCT CC 3 0 ) for HG-gcy-3. 2.4. mRNA in situ hybridisation of H. glycines guanylyl cyclases Digoxygenin (DIG)-labelled cDNA probes used for mRNA in situ hybridisations in H. glycines specimens were synthesised by asymmetric PCR according to manufacturer’s instructions (Roche Molecular Biochemicals). Primer sets to produce gene-specific probes using linear template of each isolated H. glycines guanylyl cyclase cDNA included: HG-gcy-1 primers Cycle3UP and Cycle4LOW (see above and Fig. 1) to generate a 516 bp probe; HG-gcy-2, primers (5 0 -AGGAACGGACCAAAGAGT-3 0 ) and (5 0 -CAAGTGGGGAATGGTGAA-3 0 ) to generate a 397 bp probe; HG-gcy-3, primers (5 0 -ATGTGTGGCGGGAGTTGT-3 0 ) and (5 0 - TTTCTCCGAT TTGTCACT-3 0 )
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to generate a 321 bp probe. Each DIG-labelled product was purified using the QIAquicke PCR Purification Kit (Qiagen) and confirmed for size and quantity by agarose gel electrophoresis. Complimentary antisense and (control) sense DIG-labelled cDNA probes for each H. glycines guanylyl cyclase gene were synthesised and tested on nematode specimens. Specimens of H. glycines were fixed and processed for in situ hybridisation as described previously (De Boer et al., 1998). Processed nematode sections were prehybridised at 458C for 1 h and incubated overnight at 458C under constant agitation in denatured DIG-labelled gcy-specific probe that was added at a dilution of 1:10. Hybridisation of DIGlabelled gcy cDNA probes to transcripts with H. glycines specimens was visualised by incubation in alkaline-phosphatase conjugated anti-DIG antibody (1:1000 dilution) and NBT/BCIP substrate colour reaction. Treated H. glycines specimens were mounted on glass slides with a cover slip and in situ hybridisations were observed with differential interference contrast optics on a Zeiss Axiophot microscope. 3. Results 3.1. Genomic organisation of H. glycines guanylyl cyclase gene-1 The full-length genomic DNA sequence of HG-gcy-1 (GenBank accession AF331456) was contained on the same 14.6 kb lambda clone as the full-length HG-eng-1 (Yan et al., 1998) gene (Fig. 1A). The genes were in opposite transcriptional orientation and the transcriptional start points for both genes were separated by about 900 kb. No standard TATA box was found upstream of HG-gcy-1 coding sequence. The full-length HG-gcy-1 genomic DNA sequence was 7127 nucleotides and contained 24 introns. Within HG-gcy-1, intron size ranged from 47 bp to 205 bp and exon size ranged from 72 to 526 bp. All 24 intron border sequences conformed to the GT-AG consensus splice signal (Blumenthal and Steward, 1997). Introns 1–10 were located within the predicted extracellular domain. Introns 12–18 and introns 19–24 were located within the predicted kinase-like and catalytic domains, respectively. Intron splice sites did not correlate with the borders of any predicted protein domain. 3.2. cDNA clones of H. glycines guanylyl cyclases The full-length cDNA of HG-gcy-1 was derived from the partial cDNA clone obtained by H. glycines J2 cDNA library screening, and PCR using the primer sets described in Fig. 1B with template first-strand cDNA generated from H. glycines RNA. Interestingly, sequence analysis (Genetics Computer Group, Inc) suggested some discontinuity in the predicted open reading frames (ORF) of some of the HGgcy-1 cDNA fragments obtained. Frame-shift analysis and comparison with the intron splice consensus sequence
within HG-gcy-1 genomic DNA determined that some introns were present in the HG-gcy-1 cDNA fragments. HG-gcy-1 introns 1 and 2 occurred in the 819 bp cDNA fragment (Fig. 1B), intron 11 was found in the 1941 bp cDNA fragment, and intron 18 was present in the 1327 bp cDNA fragment. The three HG-gcy-1 cDNA fragments that contained introns may have represented intermediate mRNA processing products (Blumenthal and Steward, 1997). A continuous ORF was assembled by removing the four intron sequences from the HG-gcy-1 cDNA fragments. The full-length HG-gcy-1 cDNA (GenBank accession AF095746) was 3762 bp and encoded a predicted protein of 1511 amino acids. BLASTx analysis indicated that the most similar match to HG-GCY-1 was the guanylyl cyclase gene in C. elegans cosmid clone U41109 (Fig. 2). HG-GCY1 contained predicted extracellular, single transmembrane, kinase-like, and catalytic domains that were typical of membrane-bound guanylyl cyclases (Lucas et al., 2000). Two additional, unique H. glycines guanylyl cyclase cDNA clones were identified from sequencing of the 40 clones that hybridised the HG-gcy-1 probe in H. glycines J2 cDNA library screening. One of the 40 cDNA clones contained the full-length cDNA (3499 bp) of HG-gcy-2 (GenBank accession AF283665), and another cDNA clone contained the full-length cDNA (3824 bp) of HG-gcy-3 (GenBank accession AF283664). HG-gcy-2 and HG-gcy-3 encoded proteins of 1112 amino acids and 1174 amino acids, respectively. BLASTx determined that full-length HG-gcy-2 and HG-gcy-3 had their highest similarities with HG-gcy-1. All three H. glycines gunaylyl cyclases contained a secretion signal peptide as predicted by SignalP (Henrik et al., 1997). Comparisons of the peptide domains of HG-GCY-1 to HG-GCY-2, HG-GCY-3, and homologous C. elegans and human guanylyl cyclases (Fig. 2) were conducted using BESTFIT (Genetics Computer Group, Inc.). Amino acid similarity to HG-GCY-1 in the guanylyl cyclase catalytic domain ranged from 64.4% (human) to 80.9% (C. elegans U41109), from 44.9% (human) to 54.7% (HG-gcy-2) in the kinase-like domain, and from no significant similarity (human) to 42.2% (HG-gcy-3) in the extracellular domain. The extracellular domain of the three H. glycines guanylyl cycalses each contained six conserved cysteine residues (Fig. 3) in positions consistent with the extracellular domains of membrane-bound guanylyl cyclase genes from C. elegans and humans (Yu et al., 1997). Full-length cDNA clones were obtained for HG-gcy-1, HG-gcy-2, and HG-gcy-3 using 5 0 RACE. The 5 0 untranslated (5 0 UTR) regions of HG-gcy-1, HG-gcy-2, and HGgcy-3 differed significantly in sequence and were 77 bp, 11 bp, and 33 bp in length, respectively. All three H. glycines guanylyl cyclase cDNA clones had variants of the consensus polyadenylation and cleavage signal sequence (AAUAAA) commonly found in C. elegans (Blumenthal and Steward, 1997). HG-gcy-1 and HG-gcy-2 had AAUGAA, and HG-gcy-3 had AUUAAA, polydenylation and cleavage signals, respectively. Interestingly, a
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Fig. 2. Percent amino acid similarity of the five predicted protein coding domains (A–E) of the three Heterodera glycines guanylyl cyclases (HG-gcy-1, gcy-2, and gcy-3) and homologous guanylyl cyclases from Caenorhabditis elegans and humans. Percent similarity was calculated using the BESTFIT program in the GCG package and all values presented are compared to HG-gcy-1. GenBank accessions for H. glycines gcy cDNA are listed and predicted cysteine-cysteine disulfide bonds at conserved amino acid residues in the extracellular domains are represented at the top of the figure.
number of independent HG-gcy-1 clones identified from the 40 positive cDNA library clones had unique polyadenylation sites. Each of the independent HG-gcy-1 cDNA clones matched exactly to the coding sequence of HG-gcy-1 genomic DNA, but the position of the poly A(1) tail on some HG-gcy-1 cDNA clones ranged from 13–138 bp downstream of the predicted polyadenylation and cleavage signal sequence. 3.3. mRNA in situ hybridisation of H. glycines guanylyl cyclases The antisense cDNA probe of HG-gcy-1 hybridised specifically to transcripts within neurons that comprise the nerve ring of preparasitic J2 of H. glycines (Fig. 4). The HG-gcy-1 transcript could only be detected within the preparasitic J2 life stage. HG-gcy-2 transcripts were detected with the antisense probe specifically within the sensory neurons of the amphids and caudal region of H. glycines preparasitic J2. HG-gcy-2 antisense probe hybridised to transcripts within the developing ovaries of H. glycines females but was not detected in any other structure within parasitic stages of H. glycines. No HG-gcy-3 transcripts were able to be localised within any developmental stage of H. glycines. No hybridisation signal was detected in H. glycines using the (control) sense probes of each H. glycines guanyl cylase gene. 4. Discussion To our knowledge, the isolation of guanylyl cyclase genes
in H. glycines is the first of its kind in a parasitic nematode. The involvement of guanylyl cyclases in a number of cell signalling processes (Lucas et al., 2000), most notably chemosensation in C. elegans (Bargmann and Mori, 1997; Yu et al., 1997), make them an attractive target of investigation in parasites. It is interesting that the initial discovery of the HG-gcy-1 gene was due to its relatively close (within 900 bp) proximity to the HG-eng-1 (endoglucanase) gene of H. glycines (Yan et al., 1998). It has been hypothesised that the endoglucanase genes of cyst nematodes may have been acquired via ancient horizontal gene transfer from prokaryotes (Davis et al., 2000; Smant et al., 1998; Yan et al., 1998), yet the gcy genes of H. glycines appear to have strong similarity and genomic features of nematode (C. elegans) genes. Not only is the nucleotide and peptide sequence similarity of the H. glycines gcy genes most similar to gcy genes of C. elegans, but the consensus intron splice signal sequences are highly conserved between gcy genes of H. glycines and genes of C. elegans (Blumenthal and Steward, 1997). It may be considered that the HG-gcy-1/HG-eng-1 border represents one potential point of insertion of horizontally-transferred genes in H. glycines. The HG-gcy-1 gene (7127 bp) and cDNA (3762 bp) are relatively large, which is consistent with known membrane-bound forms of guanylyl cyclase (Lucas et al., 2000). The strong similarity in coding sequence between H. glycines and C. elegans gcy genes suggests that guanylyl cyclases may be an evolutionarily conserved family of proteins among nematodes. The sizes of some of the HGgcy-1 introns are large (.150 bp) compared with the average intron size (44–52 bp) in C. elegans (Blumenthal and Stew-
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Fig. 3. Alignment of the amino acid sequence of the predicted extracellular domains of three Heterodera glycines guanylyl cyclases (HG-gcy-1, gcy-2, and gcy3) and two homologous Caenorhabditis elegans guanylyl cyclases (T21319 and U41109) showing the conserved positions of cysteine residues (in boxes) that may form disulfide bonds to provide the three-dimensional ligand-binding site conserved in receptor guanylyl cyclases. Sequence alignments were constructed using the PILEUP program of the GCG package and the predicted secretion signal peptides (in bold) of HG-gcy-1 and HG-gcy-2 were identified according to Henrik et al. (1997).
ard, 1997), although the significance of this observation is not understood. The absence of a TATA box in the 5 0 -flanking region of the HG-gcy-1 coding sequence is unusual since the presence of multiple clones of HG-gcy-1 in the H. glycines cDNA library suggests that this gene is highly expressed in preparasitic J2. Three H. glycines guanylyl cyclases were isolated by screening a cDNA library with a single probe derived from the HG-gcy-1 catalytic domain. It is uncertain if a relatively large family of gcy genes exists in H. glycines as it does in C. elegans (Yu et al., 1997), or if H. glycines contains relatively few gcy genes as have been identified in mammals (Lucas et al., 2000). It seems likely that more exhaustive screening of H. glycines cDNA libraries with different gcy probes would identify additional HG-gcy family members. When making these interpretations, it should be considered that potential intermediates in RNA processing (i.e. the intron sequences found in some HG-gcy1 cDNA clones) and relatively extreme variants in polyadenylation site may exist among HG-gcy transcripts. The significance of these observations is unclear since these phenomena are rarely reported for nematode genes (Blumenthal and Steward, 1997).
The strong similarity of some protein domains of the H. glycines guanylyl cyclases to homologous domains in C. elegans and even humans suggest that guanylyl cyclases represent an evolutionarily-conserved family of cell signalling molecules (Lucas et al., 2000). The strongest similarities were in the catalytic and kinase-like domains, and the least similarity was observed among the extracellular domains. All reported membrane-bound guanylyl cyclases contain a series of conserved cysteine residues in the extracellular domain (Lucas et al., 2000), including the H. glycines guanylyl cyclases described here. It has been suggested that disulfide bonds formed between these conserved cysteine residues create a conformation required for ligand-binding within the extracellular domain of membrane-bound guanylyl cyclases (Yu et al., 1997). Amino acid sequence divergence within other regions of the extracellulalr domains of guanylyl cyclases may represent adaptations for ligand-binding (chemosensory recognition) specificity. It has not been resolved if membrane-bound guanylyl cyclases act as direct receptors (and transducers) of external chemical stimuli in nematodes, as secondary signalling molecules in the olfactory pathways of nematodes, or
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both. Genetic dissection and identification of the odr-1 gene of C. elegans indicates that the odr-1 guanylyl cyclase functions in olfactory discrimination and signalling downstream of odorant receptors (L’Etoile and Bargmann, 2000). The HG-gcy-1 gene is expressed specifically within neurons of the nerve ring of preparasitic J2 of H. glycines, suggesting that HG-GCY-1 may also function in downstream signalling events (olfactory or other). In contrast, the localisation of HG-gcy-2 transcripts to sensory neurons within the amphids and tail region of preparasitic H. glycines J2 was similar to
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the localisation of expression of some guanylyl cyclases within sensory neurons in C. elegans, including sensory neurons (i.e. AWC) reported to be directly involved in chemoreception (Yu et al., 1997). The loss of detectable HG-gcy expression in sensory neurons, and upregulation of HG-gcy-2 in ovarian cells of parasitic stages of H. glycines, suggests both developmental regulation and multiple functions of guanylyl cyclases within the nematode. In Drosophila, specific guanylyl cyclase transcripts are present in unfertilised eggs and embryos in early developmental stages (McNeil et al., 1995), and this may relate to a potential function of HG-GCY-2 in developing H. glycines oocytes, eggs, and embryos. The lack of mRNA in situ detection of HG-gcy-3 in H. glycines specimens was likely due to technical difficulties since the presence of HG-gcy-3 in the J2 cDNA library confirms its expression within the nematode. Overexpressed HG-gcy proteins may be used to generate antisera for more detailed localisation of guanylyl cyclases within the nematode and for potential ligand-binding assays. Exposure of nematodes to HG-GCY antisera and RNA interference (Fire et al., 1998) of HG-gcy genes may also be employed for potential functional assays of their effects on chemoreception by H. glycines. The conservation of membrane-bound guanylyl cyclase sequence and structure within nematodes and among widely-diverged organisms is encouraging with respect to identifying these type of cell signalling molecules in other parasites. Whether guanylyl cyclases play direct roles in perception of host stimuli or downstream roles in signal processing, it is clear that at least subsets of guanylyl cyclases are not only important for olfaction, but for discrimination in odorant recognition (L’Etoile and Bargmann, 2000; Lucas et al., 2000; Meyer et al., 2000). How guanylyl cyclases compliment and interact with other well-characterised chemoreceptors (i.e. seven-transmembrane domain receptor proteins) is unknown, but understanding these processes may lead to novel strategies to interfere in host recognition by parasites.
Acknowledgements This research was supported by the North Carolina Agricultural Research Service and the North Carolina Biotechnology Center under grant # 9905-ARG-0033.
References Fig. 4. In situ hybridisation of digoxygenin-labelled Heterodera glycines guanylyl cyclase (gcy) cDNA probes (dark staining) to transcripts expressed within H. glycines specimens. (A) Hybridisation of HG-gcy-1 within neurons of the nerve ring of a preparasitic H. glycines second-stage juvenile (J2); (B) Hybridisation of HG-gcy-2 within sensory neurons of the amphids of a preparasitic H. glycines J2; (C) Hybridisation of HG-gcy-2 within sensory neurons in the tail of a preparasitic H. glycines J2; (D) Hybridisation of HG-gcy-2 within cells of the ovaries of a young female of H. glycines.
Bargmann, C.I., Mori, I., 1997. Chemotaxis and thermotaxis. In: Riddle, D.L., Blumenthal, T., Meyer, B.J., Priess, J.R. (Eds.). C. elegans II, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 717–37. Bargmann, C.I., 1998. Neurobiology of the Caenorhabditis elegans genome. Science 282, 2028–33. Blumenthal, T., Steward, K., 1997. RNA processing and gene structure. In: Riddle, D.L., Blumenthal, T., Meyer, B.J., Priess, J.R. (Eds.). C. elegans
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II, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 117–46. Davis, E.L., Hussey, R.S., Baum, T.J., Bakker, J., Schots, A., Rosso, M.N., Abad, P., 2000. Nematode parasitism genes. Annu. Rev. Phytopathol. 38, 341–72. De Boer, J.M., Yan, Y., Smant, G., Davis, E.L., Baum, T.J., 1998. In situ hybridization to messenger RNA in Heterodera glycines. J. Nematol. 30, 309–12. Dong, K., Opperman, C.H., 1997. Genetic analysis of parasitism in the soybean cyst nematode Heterodera glycines. Genetics 46 (4), 1311–8. Dusenbery, D.B., 1987. Prospects for exploring sensory stimuli in nematode control. In: Veech, J.A., Dickson, D.W. (Eds.). Vistas on Nematology, Society of Nematologist, Hyattsville, MD, pp. 131–5. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–11. Goellner, M., Smant, G., de Boer, J.M., Baum, T.J., Davis, E.L., 2000. Isolation of beta-1,4-endoglucanase genes of Globodera tabacum and their expression during parasitism. J. Nematol. 32, 154–65. Henrik, N., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engin. 10, 1–6. L’Etoile, N.D., Bargmann, C.I., 2000. Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1. Neuron 25, 575–86. Lucas, K.A., Pitari, G.M., Kazerounian, S., Ruiz-Stewart, I., Park, J., Schulz, S., Chepenik, K.P., Waldman, S.A., 2000. Guanylyl cyclases and signalling by cyclic GMP. Pharmacol. Rev. 52, 375–413. McNeil, L., Chinkers, M., Forte, M., 1995. Identification, characterization, and developmental regulation of a receptor guanylyl cyclase expressed
during early stages of Drosophila development. J. Biol. Chem. 270, 7189–96. Meyer, M.R., Angele, A., Kremmer, E., Kaupp, U.B., Mu¨ ller, 2000. A cGMP-signalling pathway in a subset of olfactory sensory neurons. Proc. Natl. Acad. Sci. USA 97, 10595–600. Perry, R.N., 1996. Chemoreception in plant parasitic nematodes. Annu. Rev. Phytopathol. 34, 181–99. Riggs, R.D., Wrather, J.A. (Eds.), 1992. Biology and management of the soybean cyst nematode APS Press, St. Paul, MN. Sambrook, J.E., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY. Sasser, J.N., Freckman, D.W., 1987. A world perspective on nematology: the role of the society. In: Veech, J.A., Dickson, D.W. (Eds.). Vistas on Nematology, Society of Nematologist, Hyattsville, MD, pp. 7–14. Sengupta, P., Chou, J.H., Bargmann, C.I., 1996. odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell 84, 899–909. Smant, G., Stokkermans, J., Yan, Y., de Boer, J.M., Baum, T., Wang, X., Hussey, R.S., Davis, E.L., Gommers, F.J., Henrissat, B., Helder, J., Schots, A., Bakker, J., 1998. Endogenous cellulases in animals: cloning of expressed beta-1,4-endoglucanase genes from two species of plantparasitic cyst nematodes. Proc. Natl. Acad. Sci. USA 95, 4906–11. Yan, Y., Smant, G., Stokkermans, J., Qin, L., Helder, J., Baum, T., Schots, A., Davis, E., 1998. Genomic organization of four beta-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications. Gene 220, 61–70. Yu, S., Avery, L., Baude, E., Garbers, D.L., 1997. Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors. Proc. Natl. Acad. Sci. USA 94, 3384–7.
Characterisation of guanylyl cyclase genes in the soybean cyst nematode, Heterodera glycines q Yitang Yan, Eric L. Davis* Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA Received 25 June 2001; received in revised form 25 September 2001; accepted 25 September 2001
Abstract Parasitism by the soybean cyst nematode, Heterodera glycines, has become one of the major limiting factors in soybean production worldwide. A partial HG-gcy-1 cDNA clone was obtained by screening a H. glycines cDNA library with a probe derived from the HG-gcy1 genomic sequence, and HG-gcy-1 full-length cDNA was obtained by nested PCR and 5 0 rapid amplification of cDNA ends (5 0 RACE). Two additional, full-length guanylyl cyclase cDNA clones from H. glycines, named HG-gcy-2 and HG-gcy-3, were recovered directly by screening the H. glycines cDNA library with a probe derived from sequence of the HG-gcy-1 catalytic domain. The encoded proteins of all three HG-gcy genes had an extracellular ligand-binding domain, a single membrane-spanning domain, an intracellular protein kinase-like domain, and a guanylyl cyclase catalytic domain. The three HG-GCY proteins had conserved cysteine residues to form disulfide bridges within the extracellular domain similar to the predicted ligand-binding domains of other known membrane-bound guanylyl cyclases. mRNA in situ hybridisation detected the expression of HG-gcy-1 and HG-gcy-2 transcripts in specific and different sensory neurons within H. glycines specimens. HG-gcy-3 transcripts were not localised in H. glycines specimens by in situ hybridisation. The discovery of the three guanylyl cyclase genes in H. glycines is the first of its kind in a plant-parasitic nematode and may be representative of a conserved gene family used for chemosensory recognition in parasitic nematodes. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Caenorhabditis elegans; cDNA; Chemosensory receptor; In situ hybridisation; Heterodera glycines; Parasitic nematode; RNA splicing; Sensory neurons
1. Introduction Plant-parasitic nematodes cause greater than $77 billion in world-wide crop losses each year (Sasser and Freckman, 1987). The cyst nematodes (Heterodera and Globodera species) and root-knot nematodes (Meloidogyne species) are sedentary endoparasites of plant roots that account for a majority of nematode-associated crop losses (Sasser and Freckman, 1987). Motile, preparasitic second-stage juveniles (J2) of cyst and root-knot nematodes hatch from eggs in soil and follow gradients of chemical and environmental cues emanating from the host rhizosphere to locate host plant roots (Dusenbery, 1987). The J2 penetrate plant roots completely, establish a permanent feeding site within the root vascular tissue, feed, grow, and molt three more q Nucleotide sequence data reported in this paper are available in GenBank accessions: AF331456 (HG-gcy-1 genomic DNA), AF095746 (HG-gcy-1 cDNA), AF283665 (HG-gcy-2 cDNA), AF283664 (HG-gcy-3 cDNA). * Corresponding author. Tel.: 11-919-515-6692; fax: 11-919-515-7716. E-mail address: [email protected] (E.L. Davis).
times to a swollen and sedentary adult life stage (Davis et al., 2000). Several generations that produce thousands of offspring can occur during one crop growing season, creating an enormous parasitic load on host plants. The soybean cyst nematode, Heterodera glycines, has become one of the major limiting factors in soybean production in the United States and other major soybean production areas (Riggs and Wrather, 1992). Rotations to non-host crops, modifications of cultural practices, the use of nematode-resistant cultivars, and the application of nematicides all provide some reduction in H. glycines damage to soybean, but more efficient nematode management tactics are in great demand. It has been suggested that disorientation of plant-parasitic nematodes by affecting their ability to detect and discriminate (host) chemical signals in their environment might present a powerful means to develop novel nematode management tactics (Perry, 1996). Nematodes can direct their movement toward chemical signals (chemotaxis) emanating from their food source (Bargmann and Mori, 1997). A series of chemosensory-related genes have been isolated in odorant (odr) mutants of the model nematode,
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00315-0
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Caenorhabditis elegans, that have altered response to specific chemical stimuli (Bargmann and Mori, 1997). The odr10 gene of C. elegans encodes a seven-transmembrane (chemoreceptor) protein (Sengupta et al., 1996), the largest gene family identified in C. elegans (Bargmann, 1998). The odr-1 gene of C. elegans encodes a guanylyl cyclase that functions in signalling pathways that affect olfaction and odour discrimination (L’Etoile and Bargmann, 2000). Guanylyl cyclases [GTP pyrophosphate-lyase, EC 4.6.1.2] are a family of enzymes that catalyse the conversion of GTP to cGMP (Lucas et al., 2000). The enzyme family is comprised of both membrane-bound and soluble isoforms. Guanylyl cyclase and cGMP-mediated signalling cascades play an essential role in the regulation of diverse physiological processes including olfaction, muscle motility, electrolyte homeostasis, and retinal phototransduction (Lucas et al., 2000; Meyer et al., 2000). Membrane-bound guanylyl cyclases exhibit highly-conserved domain structure and characteristics of some types of chemoreceptors, including (1) an N-terminal extracellular binding domain that (in some cases) binds to defined ligands, (2) a single transmembrane domain, (3) a cytoplasmic juxtamembrane domain, (4) a protein kinase-like domain, (5) a hinge region, and (6) a C-terminal catalytic domain (Lucas et al., 2000). A guanylyl cyclase (gcy) gene family containing at least 29 members has been identified in C. elegans (Yu et al., 1997). A subset of this gene family contained membranebound guanylyl cyclases that were demonstrated to be expressed in specific sensory neurons, suggesting that guanylyl cyclases may compliment the seven-transmembrane proteins as chemoreceptors in nematodes (Yu et al., 1997). Partial genomic sequence of a guanylyl cyclase gene was recently obtained while sequencing the 5 0 -flanking region of the HG-eng-1 (endoglucanase) gene of H. glycines (Yan et al., 1998). Here we report the first full-length cloning and localisation of expression of three guanylyl cyclase genes in a parasitic nematode.
2. Materials and methods 2.1. Nematodes Soybean cyst nematode, H. glycines, inbred line ‘OP50’ (Dong and Opperman, 1997) was cultured on roots of soybean (Glycine max cv. Lee 68) in a greenhouse. Heterodera glycines cysts were recovered from soil and roots of greenhouse cultures by flotation and sieving, the cysts were gently crushed to release and recover eggs, and the eggs were hatched at 288C to collect daily cohorts of preparasitic J2 (Goellner et al., 2000). Mild blending was used to release parasitic juvenile and adult stages of H. glycines from within soybean roots and parasitic nematodes were subsequently separated from debris with sucrose centrifugation (Goellner et al., 2000). Some fresh preparasitic J2 and parasitic nematode specimens were immediately frozen at 2808C to use
for DNA and RNA extractions. For mRNA in situ hybridisation experiments, some preparasitic J2 and mixed parasitic stages of H. glycines were fixed in 2% paraformaldehyde in M9 buffer (42 mM Na2HPO4, 22 mM KH2PO4, 85 mM NaCl, 1 mM MgSO4, pH 7.0) overnight at 48C (Goellner et al., 2000). Fixed nematodes were collected, rinsed and stored in M9 buffer at 2808C before preparation for mRNA in situ hybridisation experiments. 2.2. Characterisation of the H. glycines guanylyl cyclase-1 genomic clone As indicated above, a 14.6 kb H. glycines genomic clone containing both a guanylyl cyclase gene and a HG-eng-1 gene (Fig. 1A) was initially identified after sequencing a clone that was obtained by screening a H. glycines lambda Fix II (Stratagene) genomic library with a HG-eng-1 probe (Yan et al., 1998). BamHI and NotI digestion divided the 14.6 kb genomic clone into 4.8 kb, 3.8 kb, and 6.0 kb fragments that were subcloned into the pCR2.1 TA vector (Invitrogen). A series of nested primers were used to sequence the H. glycines genomic DNA in the 4.8 kb and 3.8 kb subclones to obtain complete genomic sequence of H. glycines guanylyl cyclase gene-1 (HG-gcy-1). 2.3. cDNA clones of H. glycines guanylyl cyclases Probe designed from the 3 0 end of the HG-gcy-1 gene was used to screen a H. glycines cDNA library (7.8 £ 10 6 pfu/ml) in Lambda Uni-Zap vector (Stratagene) that was previously constructed from mRNA of preparasitic J2 (Smant et al., 1998). Primers designed from the coding sequence of the 3 0 end of the HG-gcy-1 genomic clone, Cycle3UP (5 0 - GGAGG AACGG ACCAA AGAGT -3 0 ) and Cycle4LOW (5 0 GCTGG CCGTG TTCAC TGAGT -3 0 ), were used to synthesise a labelled cDNA probe (Fig. 1B). The cDNA probe was synthesised with the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals) as described by the manufacturer using H. glycines J2 cDNA library as template. XL-Blue MRF’ E. coli cells were infected with H. glycines J2 cDNA phage library stock and plated on growth media (Sambrook et al., 1989), and about 10,000 independent plaques developed overnight at 378C per 150 mm-diameter plate. Plaque lifts on Hybonde-N nylon membrane (Amersham Pharmacia Biotech) were prehybridised at 658C for 2 h, hybridised with the DIG-labelled HG-gcy-1 cDNA probe at 658C overnight as described by the manufacturer (Roche Molecular Biochemicals), and washed 2 £ for 15 min with 0.5 £ SSC, 0.1% SDS at 658C under constant agitation. Plaques that hybridised with the HG-gcy-1 probe were visualised using alkaline phosphatase-conjugated anti-DIG antibody and NBT/X-phosphate substrate solution as described by the manufacturer (Roche Molecular Biochemicals). Forty individual positive plaques were recovered from the plated H. glycines cDNA library for sequence analysis. Since only partial cDNA clones of HG-gcy-1 were recovered from plaque screening, full-length HG-gcy-1 cDNA
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Fig. 1. (A) The 14.6 kb fragment of Heterodera glycines genomic DNA that contained full-length guanylyl cyclase 1 (HG-gcy-1) and endoglucanase 1 (HGeng-1) genes in opposite transcriptional orientations (lower bold arrows). The 14.6 kb fragment had been cloned in the T3/T7 sites of the lambda Fix II vector (Stratagene), and Bam H1 and Not 1 restriction digests of the insert produced three fragments (dotted arrows) that were subcloned for DNA sequencing. (B) The steps to compile the full-length cDNA clone (3762 bp) of Heterodera glycines guanylyl cyclase 1 (HG-gcy-1) included a partial (1327 bp) HG-gcy-1 cDNA clone that was isolated from a H. glycines cDNA library with a labelled probe produced by PCR using primers (Cycle 4LOW and Cycle3UP) that were complimentary to the coding regions of the 3 0 end of HG-gcy-1 genomic DNA. Primer pairs based upon upstream HG-gcy-1 genomic sequence were used with first-strand cDNA template from H. glycines to obtain 1941 and 819 bp cDNA fragments to reconstruct the HG-gcy-1 cDNA, and primer Cycle K was used in 5 0 rapid amplification of cDNA ends (5 0 RACE) to complete (383 bp) the cDNA sequence.
was obtained by PCR using the primer sets outlined in Fig. 1B and template first-strand cDNA that was synthesised from total RNA of H. glycines preparasitic J2. Total RNA was prepared from 1 ml of packed H. glycines preparasitic J2 using a cesium trifluoracetate RNA extraction kit (Amersham Pharmacia Biotech). First-strand cDNA was synthesised at 428C for 50 min using random hexamers and SuperScript II reverse transcriptase according to manufacturer’s instructions (GIBCO-BRL). Using first-strand H. glycines cDNA as template, the following primer sets (Fig. 1B) were used to obtain the full coding region of HG-gcy-1 cDNA using PCR: CycleA (5 0 - ATTTG TGGGG TGCTA CTGTGG -3 0 ) and CycleB (5 0 - GGTGC GTCTG AGCCG ATAAAG -3 0 ), CycleE (5 0 - TCGGC TTCCT CTTTG CTA -3 0 ) and CycleF (5 0 - TAGCA CCCCA CAAAT ACAG -3 0 ). To obtain complete 5 0 end sequence of HG-gcy cDNA, the 5 0 RACE (Rapid Amplification of cDNA Ends) System (GIBCO-BRL) was used with H. glycines total RNA and the following gene-specific primers; CycleK (5 0 - CCGTT
GGGTG CCGTT AGC -3 0 ) for HG-gcy-1, Cycle2E (5 0 CGGGC AAAAG ACAAA TCGG AC -3 0 ) for HG-gcy-2, and Cycle3E (5 0 - TTCGT CTGCC CACAA CTGCT CC 3 0 ) for HG-gcy-3. 2.4. mRNA in situ hybridisation of H. glycines guanylyl cyclases Digoxygenin (DIG)-labelled cDNA probes used for mRNA in situ hybridisations in H. glycines specimens were synthesised by asymmetric PCR according to manufacturer’s instructions (Roche Molecular Biochemicals). Primer sets to produce gene-specific probes using linear template of each isolated H. glycines guanylyl cyclase cDNA included: HG-gcy-1 primers Cycle3UP and Cycle4LOW (see above and Fig. 1) to generate a 516 bp probe; HG-gcy-2, primers (5 0 -AGGAACGGACCAAAGAGT-3 0 ) and (5 0 -CAAGTGGGGAATGGTGAA-3 0 ) to generate a 397 bp probe; HG-gcy-3, primers (5 0 -ATGTGTGGCGGGAGTTGT-3 0 ) and (5 0 - TTTCTCCGAT TTGTCACT-3 0 )
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to generate a 321 bp probe. Each DIG-labelled product was purified using the QIAquicke PCR Purification Kit (Qiagen) and confirmed for size and quantity by agarose gel electrophoresis. Complimentary antisense and (control) sense DIG-labelled cDNA probes for each H. glycines guanylyl cyclase gene were synthesised and tested on nematode specimens. Specimens of H. glycines were fixed and processed for in situ hybridisation as described previously (De Boer et al., 1998). Processed nematode sections were prehybridised at 458C for 1 h and incubated overnight at 458C under constant agitation in denatured DIG-labelled gcy-specific probe that was added at a dilution of 1:10. Hybridisation of DIGlabelled gcy cDNA probes to transcripts with H. glycines specimens was visualised by incubation in alkaline-phosphatase conjugated anti-DIG antibody (1:1000 dilution) and NBT/BCIP substrate colour reaction. Treated H. glycines specimens were mounted on glass slides with a cover slip and in situ hybridisations were observed with differential interference contrast optics on a Zeiss Axiophot microscope. 3. Results 3.1. Genomic organisation of H. glycines guanylyl cyclase gene-1 The full-length genomic DNA sequence of HG-gcy-1 (GenBank accession AF331456) was contained on the same 14.6 kb lambda clone as the full-length HG-eng-1 (Yan et al., 1998) gene (Fig. 1A). The genes were in opposite transcriptional orientation and the transcriptional start points for both genes were separated by about 900 kb. No standard TATA box was found upstream of HG-gcy-1 coding sequence. The full-length HG-gcy-1 genomic DNA sequence was 7127 nucleotides and contained 24 introns. Within HG-gcy-1, intron size ranged from 47 bp to 205 bp and exon size ranged from 72 to 526 bp. All 24 intron border sequences conformed to the GT-AG consensus splice signal (Blumenthal and Steward, 1997). Introns 1–10 were located within the predicted extracellular domain. Introns 12–18 and introns 19–24 were located within the predicted kinase-like and catalytic domains, respectively. Intron splice sites did not correlate with the borders of any predicted protein domain. 3.2. cDNA clones of H. glycines guanylyl cyclases The full-length cDNA of HG-gcy-1 was derived from the partial cDNA clone obtained by H. glycines J2 cDNA library screening, and PCR using the primer sets described in Fig. 1B with template first-strand cDNA generated from H. glycines RNA. Interestingly, sequence analysis (Genetics Computer Group, Inc) suggested some discontinuity in the predicted open reading frames (ORF) of some of the HGgcy-1 cDNA fragments obtained. Frame-shift analysis and comparison with the intron splice consensus sequence
within HG-gcy-1 genomic DNA determined that some introns were present in the HG-gcy-1 cDNA fragments. HG-gcy-1 introns 1 and 2 occurred in the 819 bp cDNA fragment (Fig. 1B), intron 11 was found in the 1941 bp cDNA fragment, and intron 18 was present in the 1327 bp cDNA fragment. The three HG-gcy-1 cDNA fragments that contained introns may have represented intermediate mRNA processing products (Blumenthal and Steward, 1997). A continuous ORF was assembled by removing the four intron sequences from the HG-gcy-1 cDNA fragments. The full-length HG-gcy-1 cDNA (GenBank accession AF095746) was 3762 bp and encoded a predicted protein of 1511 amino acids. BLASTx analysis indicated that the most similar match to HG-GCY-1 was the guanylyl cyclase gene in C. elegans cosmid clone U41109 (Fig. 2). HG-GCY1 contained predicted extracellular, single transmembrane, kinase-like, and catalytic domains that were typical of membrane-bound guanylyl cyclases (Lucas et al., 2000). Two additional, unique H. glycines guanylyl cyclase cDNA clones were identified from sequencing of the 40 clones that hybridised the HG-gcy-1 probe in H. glycines J2 cDNA library screening. One of the 40 cDNA clones contained the full-length cDNA (3499 bp) of HG-gcy-2 (GenBank accession AF283665), and another cDNA clone contained the full-length cDNA (3824 bp) of HG-gcy-3 (GenBank accession AF283664). HG-gcy-2 and HG-gcy-3 encoded proteins of 1112 amino acids and 1174 amino acids, respectively. BLASTx determined that full-length HG-gcy-2 and HG-gcy-3 had their highest similarities with HG-gcy-1. All three H. glycines gunaylyl cyclases contained a secretion signal peptide as predicted by SignalP (Henrik et al., 1997). Comparisons of the peptide domains of HG-GCY-1 to HG-GCY-2, HG-GCY-3, and homologous C. elegans and human guanylyl cyclases (Fig. 2) were conducted using BESTFIT (Genetics Computer Group, Inc.). Amino acid similarity to HG-GCY-1 in the guanylyl cyclase catalytic domain ranged from 64.4% (human) to 80.9% (C. elegans U41109), from 44.9% (human) to 54.7% (HG-gcy-2) in the kinase-like domain, and from no significant similarity (human) to 42.2% (HG-gcy-3) in the extracellular domain. The extracellular domain of the three H. glycines guanylyl cycalses each contained six conserved cysteine residues (Fig. 3) in positions consistent with the extracellular domains of membrane-bound guanylyl cyclase genes from C. elegans and humans (Yu et al., 1997). Full-length cDNA clones were obtained for HG-gcy-1, HG-gcy-2, and HG-gcy-3 using 5 0 RACE. The 5 0 untranslated (5 0 UTR) regions of HG-gcy-1, HG-gcy-2, and HGgcy-3 differed significantly in sequence and were 77 bp, 11 bp, and 33 bp in length, respectively. All three H. glycines guanylyl cyclase cDNA clones had variants of the consensus polyadenylation and cleavage signal sequence (AAUAAA) commonly found in C. elegans (Blumenthal and Steward, 1997). HG-gcy-1 and HG-gcy-2 had AAUGAA, and HG-gcy-3 had AUUAAA, polydenylation and cleavage signals, respectively. Interestingly, a
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Fig. 2. Percent amino acid similarity of the five predicted protein coding domains (A–E) of the three Heterodera glycines guanylyl cyclases (HG-gcy-1, gcy-2, and gcy-3) and homologous guanylyl cyclases from Caenorhabditis elegans and humans. Percent similarity was calculated using the BESTFIT program in the GCG package and all values presented are compared to HG-gcy-1. GenBank accessions for H. glycines gcy cDNA are listed and predicted cysteine-cysteine disulfide bonds at conserved amino acid residues in the extracellular domains are represented at the top of the figure.
number of independent HG-gcy-1 clones identified from the 40 positive cDNA library clones had unique polyadenylation sites. Each of the independent HG-gcy-1 cDNA clones matched exactly to the coding sequence of HG-gcy-1 genomic DNA, but the position of the poly A(1) tail on some HG-gcy-1 cDNA clones ranged from 13–138 bp downstream of the predicted polyadenylation and cleavage signal sequence. 3.3. mRNA in situ hybridisation of H. glycines guanylyl cyclases The antisense cDNA probe of HG-gcy-1 hybridised specifically to transcripts within neurons that comprise the nerve ring of preparasitic J2 of H. glycines (Fig. 4). The HG-gcy-1 transcript could only be detected within the preparasitic J2 life stage. HG-gcy-2 transcripts were detected with the antisense probe specifically within the sensory neurons of the amphids and caudal region of H. glycines preparasitic J2. HG-gcy-2 antisense probe hybridised to transcripts within the developing ovaries of H. glycines females but was not detected in any other structure within parasitic stages of H. glycines. No HG-gcy-3 transcripts were able to be localised within any developmental stage of H. glycines. No hybridisation signal was detected in H. glycines using the (control) sense probes of each H. glycines guanyl cylase gene. 4. Discussion To our knowledge, the isolation of guanylyl cyclase genes
in H. glycines is the first of its kind in a parasitic nematode. The involvement of guanylyl cyclases in a number of cell signalling processes (Lucas et al., 2000), most notably chemosensation in C. elegans (Bargmann and Mori, 1997; Yu et al., 1997), make them an attractive target of investigation in parasites. It is interesting that the initial discovery of the HG-gcy-1 gene was due to its relatively close (within 900 bp) proximity to the HG-eng-1 (endoglucanase) gene of H. glycines (Yan et al., 1998). It has been hypothesised that the endoglucanase genes of cyst nematodes may have been acquired via ancient horizontal gene transfer from prokaryotes (Davis et al., 2000; Smant et al., 1998; Yan et al., 1998), yet the gcy genes of H. glycines appear to have strong similarity and genomic features of nematode (C. elegans) genes. Not only is the nucleotide and peptide sequence similarity of the H. glycines gcy genes most similar to gcy genes of C. elegans, but the consensus intron splice signal sequences are highly conserved between gcy genes of H. glycines and genes of C. elegans (Blumenthal and Steward, 1997). It may be considered that the HG-gcy-1/HG-eng-1 border represents one potential point of insertion of horizontally-transferred genes in H. glycines. The HG-gcy-1 gene (7127 bp) and cDNA (3762 bp) are relatively large, which is consistent with known membrane-bound forms of guanylyl cyclase (Lucas et al., 2000). The strong similarity in coding sequence between H. glycines and C. elegans gcy genes suggests that guanylyl cyclases may be an evolutionarily conserved family of proteins among nematodes. The sizes of some of the HGgcy-1 introns are large (.150 bp) compared with the average intron size (44–52 bp) in C. elegans (Blumenthal and Stew-
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Fig. 3. Alignment of the amino acid sequence of the predicted extracellular domains of three Heterodera glycines guanylyl cyclases (HG-gcy-1, gcy-2, and gcy3) and two homologous Caenorhabditis elegans guanylyl cyclases (T21319 and U41109) showing the conserved positions of cysteine residues (in boxes) that may form disulfide bonds to provide the three-dimensional ligand-binding site conserved in receptor guanylyl cyclases. Sequence alignments were constructed using the PILEUP program of the GCG package and the predicted secretion signal peptides (in bold) of HG-gcy-1 and HG-gcy-2 were identified according to Henrik et al. (1997).
ard, 1997), although the significance of this observation is not understood. The absence of a TATA box in the 5 0 -flanking region of the HG-gcy-1 coding sequence is unusual since the presence of multiple clones of HG-gcy-1 in the H. glycines cDNA library suggests that this gene is highly expressed in preparasitic J2. Three H. glycines guanylyl cyclases were isolated by screening a cDNA library with a single probe derived from the HG-gcy-1 catalytic domain. It is uncertain if a relatively large family of gcy genes exists in H. glycines as it does in C. elegans (Yu et al., 1997), or if H. glycines contains relatively few gcy genes as have been identified in mammals (Lucas et al., 2000). It seems likely that more exhaustive screening of H. glycines cDNA libraries with different gcy probes would identify additional HG-gcy family members. When making these interpretations, it should be considered that potential intermediates in RNA processing (i.e. the intron sequences found in some HG-gcy1 cDNA clones) and relatively extreme variants in polyadenylation site may exist among HG-gcy transcripts. The significance of these observations is unclear since these phenomena are rarely reported for nematode genes (Blumenthal and Steward, 1997).
The strong similarity of some protein domains of the H. glycines guanylyl cyclases to homologous domains in C. elegans and even humans suggest that guanylyl cyclases represent an evolutionarily-conserved family of cell signalling molecules (Lucas et al., 2000). The strongest similarities were in the catalytic and kinase-like domains, and the least similarity was observed among the extracellular domains. All reported membrane-bound guanylyl cyclases contain a series of conserved cysteine residues in the extracellular domain (Lucas et al., 2000), including the H. glycines guanylyl cyclases described here. It has been suggested that disulfide bonds formed between these conserved cysteine residues create a conformation required for ligand-binding within the extracellular domain of membrane-bound guanylyl cyclases (Yu et al., 1997). Amino acid sequence divergence within other regions of the extracellulalr domains of guanylyl cyclases may represent adaptations for ligand-binding (chemosensory recognition) specificity. It has not been resolved if membrane-bound guanylyl cyclases act as direct receptors (and transducers) of external chemical stimuli in nematodes, as secondary signalling molecules in the olfactory pathways of nematodes, or
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both. Genetic dissection and identification of the odr-1 gene of C. elegans indicates that the odr-1 guanylyl cyclase functions in olfactory discrimination and signalling downstream of odorant receptors (L’Etoile and Bargmann, 2000). The HG-gcy-1 gene is expressed specifically within neurons of the nerve ring of preparasitic J2 of H. glycines, suggesting that HG-GCY-1 may also function in downstream signalling events (olfactory or other). In contrast, the localisation of HG-gcy-2 transcripts to sensory neurons within the amphids and tail region of preparasitic H. glycines J2 was similar to
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the localisation of expression of some guanylyl cyclases within sensory neurons in C. elegans, including sensory neurons (i.e. AWC) reported to be directly involved in chemoreception (Yu et al., 1997). The loss of detectable HG-gcy expression in sensory neurons, and upregulation of HG-gcy-2 in ovarian cells of parasitic stages of H. glycines, suggests both developmental regulation and multiple functions of guanylyl cyclases within the nematode. In Drosophila, specific guanylyl cyclase transcripts are present in unfertilised eggs and embryos in early developmental stages (McNeil et al., 1995), and this may relate to a potential function of HG-GCY-2 in developing H. glycines oocytes, eggs, and embryos. The lack of mRNA in situ detection of HG-gcy-3 in H. glycines specimens was likely due to technical difficulties since the presence of HG-gcy-3 in the J2 cDNA library confirms its expression within the nematode. Overexpressed HG-gcy proteins may be used to generate antisera for more detailed localisation of guanylyl cyclases within the nematode and for potential ligand-binding assays. Exposure of nematodes to HG-GCY antisera and RNA interference (Fire et al., 1998) of HG-gcy genes may also be employed for potential functional assays of their effects on chemoreception by H. glycines. The conservation of membrane-bound guanylyl cyclase sequence and structure within nematodes and among widely-diverged organisms is encouraging with respect to identifying these type of cell signalling molecules in other parasites. Whether guanylyl cyclases play direct roles in perception of host stimuli or downstream roles in signal processing, it is clear that at least subsets of guanylyl cyclases are not only important for olfaction, but for discrimination in odorant recognition (L’Etoile and Bargmann, 2000; Lucas et al., 2000; Meyer et al., 2000). How guanylyl cyclases compliment and interact with other well-characterised chemoreceptors (i.e. seven-transmembrane domain receptor proteins) is unknown, but understanding these processes may lead to novel strategies to interfere in host recognition by parasites.
Acknowledgements This research was supported by the North Carolina Agricultural Research Service and the North Carolina Biotechnology Center under grant # 9905-ARG-0033.
References Fig. 4. In situ hybridisation of digoxygenin-labelled Heterodera glycines guanylyl cyclase (gcy) cDNA probes (dark staining) to transcripts expressed within H. glycines specimens. (A) Hybridisation of HG-gcy-1 within neurons of the nerve ring of a preparasitic H. glycines second-stage juvenile (J2); (B) Hybridisation of HG-gcy-2 within sensory neurons of the amphids of a preparasitic H. glycines J2; (C) Hybridisation of HG-gcy-2 within sensory neurons in the tail of a preparasitic H. glycines J2; (D) Hybridisation of HG-gcy-2 within cells of the ovaries of a young female of H. glycines.
Bargmann, C.I., Mori, I., 1997. Chemotaxis and thermotaxis. In: Riddle, D.L., Blumenthal, T., Meyer, B.J., Priess, J.R. (Eds.). C. elegans II, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 717–37. Bargmann, C.I., 1998. Neurobiology of the Caenorhabditis elegans genome. Science 282, 2028–33. Blumenthal, T., Steward, K., 1997. RNA processing and gene structure. In: Riddle, D.L., Blumenthal, T., Meyer, B.J., Priess, J.R. (Eds.). C. elegans
72
Y. Yan, E.L. Davis / International Journal for Parasitology 32 (2002) 65–72
II, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 117–46. Davis, E.L., Hussey, R.S., Baum, T.J., Bakker, J., Schots, A., Rosso, M.N., Abad, P., 2000. Nematode parasitism genes. Annu. Rev. Phytopathol. 38, 341–72. De Boer, J.M., Yan, Y., Smant, G., Davis, E.L., Baum, T.J., 1998. In situ hybridization to messenger RNA in Heterodera glycines. J. Nematol. 30, 309–12. Dong, K., Opperman, C.H., 1997. Genetic analysis of parasitism in the soybean cyst nematode Heterodera glycines. Genetics 46 (4), 1311–8. Dusenbery, D.B., 1987. Prospects for exploring sensory stimuli in nematode control. In: Veech, J.A., Dickson, D.W. (Eds.). Vistas on Nematology, Society of Nematologist, Hyattsville, MD, pp. 131–5. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–11. Goellner, M., Smant, G., de Boer, J.M., Baum, T.J., Davis, E.L., 2000. Isolation of beta-1,4-endoglucanase genes of Globodera tabacum and their expression during parasitism. J. Nematol. 32, 154–65. Henrik, N., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engin. 10, 1–6. L’Etoile, N.D., Bargmann, C.I., 2000. Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1. Neuron 25, 575–86. Lucas, K.A., Pitari, G.M., Kazerounian, S., Ruiz-Stewart, I., Park, J., Schulz, S., Chepenik, K.P., Waldman, S.A., 2000. Guanylyl cyclases and signalling by cyclic GMP. Pharmacol. Rev. 52, 375–413. McNeil, L., Chinkers, M., Forte, M., 1995. Identification, characterization, and developmental regulation of a receptor guanylyl cyclase expressed
during early stages of Drosophila development. J. Biol. Chem. 270, 7189–96. Meyer, M.R., Angele, A., Kremmer, E., Kaupp, U.B., Mu¨ ller, 2000. A cGMP-signalling pathway in a subset of olfactory sensory neurons. Proc. Natl. Acad. Sci. USA 97, 10595–600. Perry, R.N., 1996. Chemoreception in plant parasitic nematodes. Annu. Rev. Phytopathol. 34, 181–99. Riggs, R.D., Wrather, J.A. (Eds.), 1992. Biology and management of the soybean cyst nematode APS Press, St. Paul, MN. Sambrook, J.E., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY. Sasser, J.N., Freckman, D.W., 1987. A world perspective on nematology: the role of the society. In: Veech, J.A., Dickson, D.W. (Eds.). Vistas on Nematology, Society of Nematologist, Hyattsville, MD, pp. 7–14. Sengupta, P., Chou, J.H., Bargmann, C.I., 1996. odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell 84, 899–909. Smant, G., Stokkermans, J., Yan, Y., de Boer, J.M., Baum, T., Wang, X., Hussey, R.S., Davis, E.L., Gommers, F.J., Henrissat, B., Helder, J., Schots, A., Bakker, J., 1998. Endogenous cellulases in animals: cloning of expressed beta-1,4-endoglucanase genes from two species of plantparasitic cyst nematodes. Proc. Natl. Acad. Sci. USA 95, 4906–11. Yan, Y., Smant, G., Stokkermans, J., Qin, L., Helder, J., Baum, T., Schots, A., Davis, E., 1998. Genomic organization of four beta-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications. Gene 220, 61–70. Yu, S., Avery, L., Baude, E., Garbers, D.L., 1997. Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors. Proc. Natl. Acad. Sci. USA 94, 3384–7.