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Published in Crop Sci. 44:265-273 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Genomic Polymorphism Identifies a Subtilisin-Like Protease near the Rhg4 Locus in Soybean

Naoma S. Nelsena, Zhigang Lib, April L. Warnera, Benjamin F. Matthewsc and Halina T. Knap*,a

a Dep. of Crop and Soil Environmental Science, 276 Poole Agricultural Center, Clemson Univ., Clemson, SC 29634-0359
b Agronomy College, Shanxi Agricultural Univ., Taigu, Shanxi Province 030801, P.R. China
c USDA-ARS-PSI, Soybean Genomics & Improvement Lab., Bldg. 006, Room 118, BARC-West, 10300 Baltimore Blvd., Beltsville, MD 20705-2350

* Corresponding author (hskrpsk{at}clemson.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Soybean [Glycine max (L.) Merr.] germplasm PI 437654 exhibits broad resistance to soybean cyst nematode (Heterodera glycines Ichinohe, SCN). Probes derived from PI 437654 bacterial artificial chromosome (BAC) clone 15G19 at the Rhg4 locus detected a restriction fragment length polymorphism (RFLP) between resistant and susceptible germplasms. Detailed RFLP analysis using restriction fragments from BAC clone 15G19 associated the polymorphism with an 8-kb BamHI fragment containing the promoter region and partial coding sequence of a novel soybean subtilisin-like protease, GmSUB1. Complete sequence of GmSUB1 was determined (GenBank AY277949). Regulatory elements for root gene expression, pathogen response, coordinated multiple-gene expression, and a novel 90-bp direct repeat were identified. GmSUB1 shows 74% similarity to Arabidopsis thaliana AIR3. Hybridization analyses indicate that PI 437654 contains only full-length copies of GmSUB1, whereas susceptible germplasm ‘Williams 82’ contains both full-length and truncated copies of the gene. A 4-fold increase in GmSUB1 copy number, and a corresponding 2- to 3-fold increase in steady state GmSUB1 mRNA levels, was observed in PI 437654 compared with Williams 82. Localization and polymorphism of GmSUB1 within the Rhg4 resistance region, and increases in GmSUB1 gene copy number and expression in PI 437654 compared with Williams 82 infers a functional role in the pathogen response. GmSUB1 is believed to be secreted into the extracellular matrix, and may function in reorganization of cell wall components during plant development and in the defense response.

Abbreviations: BAC, bacterial artificial chromosome • cDNA, complementary DNA • mRNA, messenger RNA • PCR, polymerase chain reaction • RFLP, restriction fragment length polymorphism • SCN, soybean cyst nematode


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PLANT RESISTANCE to pathogen attack requires an integrated series of events resulting in pathogen recognition, signal transduction, and cellular response. Interactions between plant resistance (R) proteins and pathogen avirulence (Avr) proteins provide the basis for pathogen recognition and initiation of the defense response in many defense pathways (Flor, 1971). However, recent studies indicate that a number of other proteins are also involved in regulating the activity of R proteins and mediating interactions between R and Avr (Bogdanove, 2002; Bonas and Lahaye, 2002; Axtell and Staskawicz, 2003). These proteins act together within the complex network of proteins involved in signaling and cellular response to provide resistance to the specific pathogen. The complexity of this system is represented by the large number of genes whose activity can affect the plant defense response.

In plants, subtilisin-like proteases, or subtilases, have been associated with a number of developmental processes, as well as cellular defense and stress responses. These enzymes are involved in epidermal surface formation (ALE1, Tanaka et al., 2001), stomatal development (SDD1, Berger and Altmann, 2000), microsporogenesis (LIM9, Taylor et al., 1997), processing of storage proteins during germination (C1, Boyd et al. 2002), lateral root development (AIR3, Neuteboom et al., 1999b), root nodule development (Ag12, Ribeiro et al., 1995; Cg12, Laplaze et al., 2000), and cellular differentiation during seed coat development (SCS1, Batchelor et al., 2000). There is also evidence that plant subtilases may be involved in signal transduction in response to environmental stresses. A Kex2p-like protease from tomato has been implicated in the processing of systemin, a peptide hormone responsible for mediating signal transduction in response to wounding (Schaller and Ryan, 1994).

In tomato, the P69 family of pathogenesis-related proteins is comprised of six closely related subtilisin-like proteases (Tornero et al., 1996a, 1997a; Jorda et al., 1999, 2000). Two genes in this family, P69B and P69C, are induced in response to pathogen attack or treatment with salicylic acid (Jorda et al., 1999). These proteases may play an active defensive role against the pathogen, or may function in reorganization of the extracellular matrix in response to pathogen attack. One of the proposed substrates of the P69 proteases is a leucine-rich repeat protein, LRP (Tornero et al., 1996b). This protein is associated with the extracellular matrix, and processing of LRP occurs in response to pathogen attack. The specific function of LRP is unknown, although it may be involved in molecular recognition within the extracellular matrix initiating signal transduction processes during pathogenesis.

In soybean, resistance to SCN is a multigenic and quantitative trait with at least two loci, rhg1 on linkage group (LG) G and Rhg4 on LG A2, providing a major portion of this resistance (Concibido et al., 1994; Mahalingam and Skorupska, 1995; Webb et al., 1995; Meksem et al., 2001). Receptor-like kinases identified at these loci are believed to play a role in SCN resistance (GenBank accession numbers AF506516 and AF506517, Meksem and Lightfoot, Isolated polynucleotides and polypeptides relating to loci underlying resistance to soybean cyst nematode and soybean sudden death syndrome and methods employing same, Patent pending). The soybean plant introduction PI 437654 is a primitive germplasm that exhibits resistance to most SCN populations and has evolved cellular mechanisms that restrict establishment of the nematode feeding site early after infection (Mahalingam and Skorupska, 1996). We constructed a BAC library from PI 437654 to facilitate cloning of genes associated with SCN resistance (Tomkins et al., 1999). Molecular markers linked to the Rhg4 resistance locus were then used to identify BAC clones from the PI 437654 library corresponding to this genomic region (Lewers et al., 2001). Here we describe the identification of a specific genomic polymorphism between resistant and susceptible soybean germplasms using these BAC clones. Characterization of the polymorphism shows that it involves a subtilisin-like protease, GmSUB1, that is differentially expressed between resistant and susceptible germplasms.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
Soybean genotypes ‘Essex’, ‘Faribault’, ‘Williams 82’, PI 437654, and PI 209332 were grown under greenhouse conditions in the Biosystems Research Complex at Clemson University, Clemson, SC. Young, trifoliate leaves were harvested for DNA and RNA isolation and stored at –80°C. For analysis of gene expression in roots, Williams 82 and PI 437654 seeds were sterilized with bleach (20% v/v for 15 min), rinsed in sterile water, and placed on germination paper in the dark at 28°C. After 5 d, seedlings were transferred to fresh germination paper and the roots were inoculated with approximately 1000 soybean cyst nematode eggs as described by Mahalingam et al. (1998). Inoculations were performed with an SCN population similar to HG-type 7 (Niblack et al., 2002). Inoculated seedlings were incubated in the dark at 28°C for 24 h. The roots were then rinsed with water to remove remaining nematodes and seedlings were placed on fresh germination paper and incubated for an additional 10 h at 28°C. SCN infection of the roots was confirmed by staining (Mahalingam et al., 1998). Whole roots were harvested and stored at –80°C.

DNA Isolation and Hybridization Analysis
Genomic DNA was isolated from soybean leaves according to the protocol of Delaporta et al. (1983) or by the Nucleon Phytopure Plant DNA extraction kit (Amersham Biosciences, Piscataway, NJ). Purified DNA was digested with the indicated restriction enzymes, and fragments were separated by electrophoresis on 0.8% (w/v) agarose gels. DNA was transferred to Magnacharge nylon membranes (Osmonics, Westborough, MA) by capillary blotting as described by Sambrook et al. (1989). BAC DNA was isolated with the QIAGEN Plasmid Mini kit (Qiagen, Valencia, CA), and plasmid DNA was isolated with the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA).

For RFLP analysis, probes were synthesized from BAC DNA by random prime labeling (Feinberg and Vogelstein, 1984). Hybridizations and washes were performed in siliconized glass bottles at 65°C in a hybridization incubator (Robbins Scientific Model 310, Robbins Scientific Corp., Sunnyvale, CA). The hybridization solution consisted of 6x SSPE [1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA (pH 7.7)], 10x Denhardt's solution (1x Denhardt's solution is 0.02%, w/v, Ficol; 0.02%, w/v, polyvinylpyrrolidone; 0.02%, w/v, bovine serum albumin), 1% (w/v) SDS, and 300 µg/mL of denatured salmon sperm DNA. Filters were washed in 2, 1, and 0.5x SSC (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate) with 1% (w/v) SDS at 65°C and were used to expose Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) using an intensifying screen. The genomic polymorphism was confirmed with a PCR-generated probe specific to the isolated 8.0-kb fragment using SUB Pair 5 F and SUB Pair 5 R primers (Table 1). PCR conditions were denaturation at 95°C for 10 min; 35 cycles of 95°C for 30 s, 58°C for 40 s, and 72°C for 45 s; and extension at 72°C for 7 min. For copy number analysis, PCR-generated probes for GmSUB1 and 26S ribosomal RNA were synthesized with SUB 1/SUB 2 primers and T7/SP6 primers (Table 1), respectively, under the above conditions.


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  		Table 1. Primers used for sequencing, probe synthesis, and gene expression analysis of subtilisin-like protease GmSUB1.

 
DNA Cloning and Sequence Analysis
PI 437654 BAC clone 15G19 was digested with BamHI, and fragments were cloned into pBlueScript II KS+ (Stratagene, San Diego, CA). The fragments were end-sequenced and used in Southern hybridization analyses with genomic DNA. Subclone p67-3B produced the specific polymorphism, and the 8-kb insert was further subcloned as HindIII, PstI, and DraI fragments into pBlueScript II KS+ for sequence analysis. Subclones p67-4 and p67-5 (containing inserts of 4.5 and 2 kb, respectively) contained sequences adjacent to p67-3B and were used for sequence extension by primer walking. DNA sequencing reactions were performed with the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) with T7 and T3 sequencing primers (Table 1, Operon, Alameda, CA), or gene-specific primers designed from sequence information (Table 1, Integrated DNA Technologies, Inc., Coralville, IA). Sequencing reactions were analyzed on an ABI 377 automated DNA sequencer (Applied Biosystems, Foster City, CA). DNA homology searches and sequence analysis were performed by means of the NCBI Blast Server (Altschul et al., 1990, http://www.ncbi.nlm.nih.gov/blast; verified 1 August 2003), ClustalW (Thomson et al., 1994, http://www.ebi.ac.uk/clustalw/; verified 4 September 2003), GENSCAN (Burge and Karlin, 1997, http://genes.mit.edu/GENSCAN.html; verified 1 August 2003), PLACE (Higo et al., 1999, http://www.dna.affrc.go.jp/htdocs/PLACE/; verified 1 August 2003), SignalP v2.0 (Nielsen and Krogh, 1998, http://www.cbs.dtu.dk/services/SignalP-2.0; verified 1 August 2003), and Translate Tool web sites (Appel et al., 1994, http://us.expasy.org/tools/dna.html; verified 1 August 2003).

RNA Expression Analysis
Total RNA was isolated from leaves and whole roots (uninfected and 10 h post-infection) of soybean germplasms PI 437654 and Williams 82 with the Qiagen RNeasy Plant Mini kit (Valencia, CA). RNA samples were treated with RQ1 DNase (Promega, Madison, WI) to remove contaminating genomic DNA. Reactions containing 0.5 U DNase per microgram RNA in 1x buffer were incubated at 37°C for 20 min and then cleaned by phenol extraction and precipitation. RNA integrity was confirmed by gel electrophoresis.

cDNA was generated from RNA samples with the SuperScriptII First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) following the manufacturer's instructions and using 3 µg RNA and 50-ng random hexamers per reaction. Control reactions without reverse transcriptase were run for all samples to confirm removal of contaminating genomic DNA. Following cDNA synthesis, samples were diluted 2-fold, and a test amplification with QuantumRNA Universal 18S Internal Standard primers (2:8 primer:competimer ratio, Ambion, Austin, TX) was performed to confirm uniform efficiency of cDNA synthesis in all samples.

Real-time PCR analysis was performed with the iCycler iQ Real Time PCR Detection System (Bio-Rad, Richmond, CA) and the SYBR Green PCR Core Reagents kit (Applied Biosystems, Foster City, CA). Reactions were performed in a volume of 50 µL containing 1x SYBR Green PCR Buffer, 2.5 mM MgCl2, 0.2 mM each dATP, dCTP, dGTP, 0.4 mM dUTP, 1 U AmpliTaq Gold DNA Polymerase, 20 nM fluorescein calibration dye (Bio-Rad, Hercules, CA), 1 µL template and primers. Each cDNA sample was tested in triplicate using GmSUB1-specific primers (500 mM each SUB Pair 6 F and SUB Pair 6 R, Table 1) and QuantumRNA Universal 18S Internal Standard primers (200 nM of a 2:8 primer:competimer ratio, Ambion, Austin, TX). PCR conditions were denaturation at 95°C for 3 min; 50 cycles of 95°C for 30 s, 58°C for 40 s, and 72°C for 45 s; and melt curve analysis from 55 to 95°C at 0.4°C increments. For each experiment, a gene-specific standard curve was prepared by plotting threshold cycle (CT) versus log starting quantity (SQ) of a 10-fold serial dilution with amplification products as templates. CT values of the cDNA samples were compared with the standard curve to determine mean starting quantity. Relative GmSUB1 mRNA levels were then determined by the formula GmSUB1 SQ/18S SQ for each sample. The results were then scaled by setting the PI 437654 uninfected root cDNA sample equal to 1. Data from a total of three independent sets of RNA were combined to produce the final results.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Specific Polymorphism within the Rhg4 Resistance Region
Within BAC clones spanning the Rhg4 resistance region (Lewers et al., 2001), two overlapping clones, 15G19 and 11K09, spanning approximately 90 kb produced an RFLP when hybridized to soybean genomic DNA (Fig. 1). In germplasms Essex, Faribault, and Williams 82, a unique fragment with a molecular weight of approximately 7.5 kb was observed. This fragment was not present in germplasms with SCN resistance, PI 437654 and PI 209332.



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  		Fig. 1. Southern hybridization analysis with PI 437654 BAC clone 15G19. Genomic DNA from susceptible germplasms Essex (1), Faribault (2), and Williams 82 (3), and resistant germplasms PI 209332 (4) and PI 437654 (5) digested with BamHI was hybridized with a random-prime generated probe from a PI 437654 BAC clone associated with the Rhg4 resistance locus. The 7.5-kb polymorphic fragment is indicated by the arrow.

 
A strategy was designed to identify the DNA sequences responsible for this genomic polymorphism. The BAC clone 15G19 insert was subjected to restriction enzyme digestion, and the generated fragments were used as probes in Southern hybridization analyses. An 8-kb BamHI fragment from BAC clone 15G19 produced an RFLP that corresponded to the polymorphism obtained with the entire BAC sequence (Fig. 2). This probe hybridized to two or three bands in each digest, suggesting that this sequence is present in low copy number in the genome (Fig. 2). In PI 437654 and PI 209332, the probe hybridized to the 8-kb BamHI fragment and to a higher molecular weight fragment. In Essex, Faribault, and Williams 82, the probe hybridized to both of these fragments, as well as the 7.5-kb polymorphic band (Fig. 2). Detailed restriction enzyme mapping and Southern hybridizations with PI 437654 BAC 15G19 or Williams 82 BAC 5G2 (previously assigned to the Rhg4 resistance region, Lewers et al., 2001) show that the 8-kb fragment is present in both of these BAC clones, and the 7.5-kb fragment originates from Williams 82 BAC 5G2 only. Additionally, mapping studies using a small population of F2 individuals (82 plants) from a cross of PI 437654 and Williams 82 showed less than 1% recombination between the genomic polymorphism and the BARC Sat_162 marker, which has been shown to be linked to the Rhg4 locus (data not shown, Cregan et al., 1999). The presence of only the 8.0-kb fragment in PI 437654 and both the 8.0 and 7.5-kb fragments in Williams 82 suggests that the resistant germplasm has only the full-length sequence at the Rhg4 locus, whereas the susceptible germplasm has both a full-length copy of this sequence at the locus, as well as a truncated copy of the homologous sequence.



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  		Fig. 2. Southern hybridization analysis confirming isolation of the DNA fragment producing the specific genomic polymorphism. Genomic DNA from soybean germplasms Essex (1), Faribault (2), Williams 82 (3), PI 209332 (4), and PI 437654 (5) digested with BamHI was hybridized with a PCR generated probe from an 8-kb BamHI fragment of a PI 437654 BAC clone associated with the Rhg4 resistance locus.

 
Sequence Analysis of the 8-kb Fragment Producing the Polymorphism
The 8-kb BamHI fragment from PI 437654 BAC 15G19 was subcloned as a series of HindIII, PstI, or DraI fragments to create overlapping clones that could be used for sequence analysis. These subclones were sequenced using T7 and T3 primers, and the sequences were aligned to create a contiguous sequence. Internal primers were then designed based on the sequence information (Table 1), and were used to confirm junctions between the subclones and to fill in gaps within the sequence. Sequence analysis indicated the 8-kb fragment contained the promoter region and partial coding sequence of a subtilisin-like protease, GmSUB1. The complete sequence of this gene was obtained by primer walking using two BamHI fragments (2 and 4.5 kb) subcloned from BAC 15G19 as sequencing templates. In total, 13 225 bp of sequence was obtained. This includes the complete sequence of GmSUB1, as well as partial sequence of another gene, which is located approximately 1.5 kb downstream of GmSUB1and lies in the opposite orientation. The sequence of GmSUB1 has been submitted to GenBank (accession number AY277949).

GmSUB1 contains 11 exons and encodes a predicted protein of 773 amino acid residues (Fig. 3). The coding region of GmSUB1 shows a high degree of sequence similarity with the subtilisin-like protease AIR3 of Arabidopsis thaliana at both the nucleotide (66% identity between mature transcripts) and protein levels (61% identity, 74% similarity; Fig. 3B). However, there is no significant sequence similarity with AIR3 outside of the coding region and no similarity between promoter regions of the two genes could be detected. Comparison of GmSUB1 with other soybean subtilisin-like proteases shows lower levels of sequence similarity [SCS1, 36% identity, 51% similarity (GenBank accession number AJ276710); SLP-2, 38% identity, 55% similarity (GenBank accession number AF352059); C1, 40% identity, 55% similarity (GenBank accession number AF036960)] suggesting GmSUB1 is a novel subtilisin-like protease in soybean.



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  		Fig. 3. Structure and sequence analysis of the GmSUB1 gene. (A) Schematic diagram of GmSUB1. Open boxes represent exon sequences. Shaded boxes represent repeated sequences in the promoter region. (B) Alignment of the deduced amino acid sequence of GmSUB1 with AIR3 of Arabidopsis. Identical residues are shaded in black. The predicted cleavage sites for the signal peptide (open triangle) and propeptide domain (shaded triangle) are indicated. Conserved residues forming the catalytic triad and substrate binding site are indicated with an asterisk (*). GenBank accession number AY277949.

 
Analysis of the deduced amino acid sequence of GmSUB1 using the SignalP-HMM prediction program (Nielsen and Krogh, 1998) showed that the amino-terminal portion of the protein contains a 26-amino acid signal peptide sequence (Fig. 3B). An 89-amino acid propeptide domain was also identified. Cleavage of this domain is believed to occur immediately in front of the two conserved threonine residues at positions 116 and 117. The amino acid residues Asp-152, His-202, and Ser-559 were identified as the residues making up the catalytic triad on the basis of sequence similarity with other plant subtilases. The distance between the stabilizing Asn-328 and the reactive Ser-559 is 231 amino acids, characteristic of other plant subtilases.

Analysis of the region upstream of the translation start site has identified potential promoter sequences and a number of regulatory elements, including sequences involved in regulating root gene expression (ATATT), a silencer element characteristic of pathogenesis-related genes (YTGTCWC), and binding sites for Dof proteins (AAAG), transcription factors that coordinately regulate expression of multiple genes in a tissue-specific or inducible manner. Additionally, the subtilase promoter region contains a large, nearly perfect direct repeat located 419 bp upstream of the translation start site. The repeated sequences are approximately 90 bp in length, show 82% sequence identity, and are separated by 557 bp (Fig. 3A). These repeated sequences show no significant similarity to known sequences in the available databases (the nearest match is human sequence from chromosome 5, E value 0.13, GenBank accession number AC008958). However, they do contain sequences that may function as promoter elements.

GmSUB1 Gene Copy Number
Using a GmSUB1 gene-specific probe for Southern hybridization analysis with genomic DNA, we detected two (or at most three) fragments with each of the restriction enzymes tested, suggesting the presence of a small gene family for GmSUB1 (Fig. 4). Intensity of the hybridization signal for one of the fragments was consistent between PI 437654 and Williams 82, suggesting no difference in gene copy number for one member of the GmSUB1 family. A locus for this gene has not been determined. A difference in hybridization intensity between PI 437654 and Williams 82 was observed for GmSUB1 localized near the Rhg4 locus (Fig. 4B). Using a 26S rRNA probe to normalize for variation in DNA content between lanes, we determined that the GmSUB1 gene-specific probe produces a 4-fold (±0.48) increase in hybridization signal with PI 437654 compared with Williams 82 (Fig. 4). This indicates an increased copy number of GmSUB1 gene sequences in PI 437654.



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  		Fig. 4. Southern hybridization analysis to determine GmSUB1 copy number in soybean. Genomic DNA from Williams 82 (1, 3, and 5) and PI 437654 (2, 4, and 6) digested with BamHI (1 and 2), DraI (3 and 4), and EcoRV (5 and 6) were hybridized with a GmSUB1 gene-specific probe (A). Differences in hybridization intensity with GmSUB1 probe were normalized to 26S rRNA content to determine differences in gene copy number (B).

 
Differential Expression of GmSUB1 in PI 437654 and Williams 82
Expression patterns of GmSUB1 were examined in soybean roots and leaves to determine if differences in expression exist between PI 437654 and Williams 82. GmSUB1 mRNA levels were determined by relative quantitative real-time PCR using 18S rRNA to normalize the data. Relative GmSUB1 mRNA levels of PI 437654 uninfected roots were set equal to 1 and data for all other tissues were scaled accordingly (Fig. 5). In PI 437654, relative GmSUB1 mRNA levels were 1.22 (±0.09) for infected roots and 1.83 (±0.68) for leaves. In Williams 82, relative GmSUB1 mRNA levels were 0.43 (±0.11) for uninfected roots, 0.42 (±0.18) for infected roots, and 0.86 (±0.01) for leaves. These results show that GmSUB1 expression is higher in PI 437654 than in Williams 82 in all organs examined. Higher levels of expression are also observed in leaves as compared with roots for both germplasms. In PI 437654, expression levels are increased slightly in infected roots as compared with uninfected roots. However, no significant difference in expression levels is observed between infected and uninfected roots for Williams 82.



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  		Fig. 5. GmSUB1 expression in soybean. (A) Relative GmSUB1 mRNA levels in uninfected roots, SCN infected roots, and leaves of PI 437654 (resistant) and Williams 82 (susceptible) were determined by quantitative real-time PCR using gene-specific primers and normalizing to 18S rRNA values. Quantitation is based on CT value as described in Materials and Methods. (B) Representative data set from quantitative real-time PCR analysis with GmSUB1 and 18S gene-specific primers. Inset panel shows standard curve graph for GmSUB1 (top) and 18S (bottom).

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Genomic Southern hybridization analyses identified two overlapping BAC clones from PI 437654 that produced an RFLP between resistant and susceptible soybean germplasms. Hybridization of these overlapping BAC clones, 15G19 and 11K09, which are localized near the Rhg4 locus (Lewers et al., 2001), to genomic DNA produced discrete bands with little background, indicating that they contain little repetitive DNA sequence and are therefore likely to be gene-rich. Our results support this in that sequence analysis of a 13-kb region of one of the BAC clones identified two different genes located 1.5 kb apart.

Our strategy utilizing a combination of detailed RFLP analysis, subcloning, and sequencing allowed identification of a specific 8-kb fragment from PI 437654 BAC clone 15G19 that produces the genomic polymorphism. This fragment contains the promoter region and partial coding sequence of a subtilisin-like protease, GmSUB1 (GenBank accession number AY277949). Our results suggest that resistant germplasm PI 437654 contains only full-length copies of this DNA sequence, whereas the susceptible germplasm Williams 82 contains both full-length and truncated copies of the sequence. A comparison of GmSUB1 with genomic sequence surrounding the Rhg4 locus in susceptible germplasm A3244 (GenBank accession numbers AX196297 and AX197417, Hauge et al., 2001) identified both full-length and truncated copies of the gene, corroborating our results. Additionally, a 4-fold increase in GmSUB1 gene copy number was observed in PI 437654 compared with Williams 82. This difference in gene copy number and the occurrence of deletions could have pronounced effects on the overall level of GmSUB1 expression in the susceptible germplasm. In fact, stringent analysis using relative quantitative real-time PCR shows that steady state GmSUB1 mRNA levels are 2- to 3-fold higher in PI 437654 than in Williams 82.

Sequence analysis of GmSUB1 shows it to be a member of the subtilase class of serine proteases. The predicted protein sequence contains the three domains characteristic of subtilisin-like proteases: a signal peptide domain targeting the protein to the endoplasmic reticulum; a propeptide domain, which acts as an intramolecular chaperone to regulate folding and activity of the enzyme; and a catalytic domain that makes up the mature protein. The amino acid sequences surrounding Asp-152, His-202, and Ser-559 of the catalytic triad and the stabilizing Asn-328, as well as their relative positions in the protein, are similar to other plant subtilases. The GmSUB1 gene isolated in this study shows little sequence similarity to previously identified soybean subtilisin-like proteases (Batchelor et al., 2000; Beilinson et al., 2002; Boyd et al., 2002), and therefore represents a distinct subtilase in soybean.

GmSUB1 shows the highest degree of sequence similarity with the subtilisin-like protease AIR3 of Arabidopsis. AIR3 mRNA is highly expressed in leaves and stems and to a lower level in roots (Neuteboom et al., 1999a). Accumulation of AIR3 in roots is localized to the outer layers of the parental root at the site of lateral root development, and it has been proposed that AIR3 may act to facilitate lateral root emergence by digesting structural proteins of the extracellular matrix and weakening connections between cells (Neuteboom et al., 1999b).

Our results show that GmSUB1 mRNA is expressed in both roots and leaves of soybean. As with AIR3, higher levels of expression are observed in leaves as compared with roots. In leaves and uninfected roots, GmSUB1 steady state mRNA levels are approximately 2-fold higher in the resistant germplasm PI 437654 than the susceptible germplasm Williams 82. This increased level of gene expression corresponds to the increased gene copy number in PI 437654. After SCN infection an increase in GmSUB1 expression is observed in PI 437654, resulting in mRNA levels that are nearly 3-fold higher than in Williams 82, indicating that the gene may be upregulated in response to pathogen attack. Altered expression patterns in response to soybean cyst nematode infection have been observed for a number of genes involved in stress responses, including signal transduction, transcription regulation, and cell wall metabolism (Mahalingam et al., 1999; Vaghchhipawala et al., 2001; Mazarei et al., 2002; Puthoff et al., 2003).

The actual function of GmSUB1 is not known. However, because of its expression patterns and similarity to other proteases, it is possible to speculate about its potential role in soybean. Like other plant subtilases, GmSUB1 contains a signal peptide domain and is believed to be secreted into the extracellular space where it may play a role in the reorganization of structural proteins within the cell wall or in the activation of signal transduction pathways (Taylor et al., 1997; Vera et al., 1989). Genomic polymorphism of GmSUB1 at a region involved in pathogen resistance, increased GmSUB1 gene copy number, and the increased level of gene expression in PI 437654 may support a potential functional role in the defense response.

Proteases have been implicated in a diverse array of plant defense responses leading to pathogen resistance. They may act directly through interactions with the pathogen, as in the case of chitinases or collagenases, or through indirect pathways, such as signal transduction and activation of other functional proteins. The P69 family of subtilisin-like proteases was initially identified as pathogenesis-related proteins induced by viroid infection and treatment with defense signaling molecules such as ethylene and salicylic acid (Vera and Conejero, 1989; Tornero et al., 1996a). One possible role of the P69 proteases is in processing of a leucine-rich repeat protein LRP, potentially regulating signal transduction in response to pathogen attack. Activation of systemin in tomato has also been shown to involve processing by a subtilisin-like protease (Schaller and Ryan, 1994). Plant subtilases may also play a role in pathogen defense responses by mediating a reorganization of the extracellular matrix, resulting in reinforcement of the plant cell wall, and providing a physical barrier to pathogen movement (Dixon and Lamb, 1990). GmSUB1 protease may be involved in this aspect of the defense response. In PI 437654, an increase in deposition of cell wall materials in cells surrounding the forming syncytium precedes the collapse of the syncytium, resulting in an incompatibility response (Mahalingam and Skorupska, 1996).

The steady state expression level of GmSUB1 mRNA in PI 437654 is 2- to 3-fold higher than in Williams 82. This increased level of expression may play a critical role in determining the resistance level of the plant. Studies have shown that overexpression of extracellular proteases such as prb1 of Trichoderma harzianum and a serine protease produced by Stenotrophomonas maltophilia strain W81 results in enhance biological control activity against plant pathogenic fungi (Flores et al., 1997; Dunne et al., 2000).

Further experiments will need to be performed to determine the specific functional role of GmSUB1 in soybean, including an analysis of its potential substrates. Analysis of sequences within the GmSUB1 promoter region has identified the presence of regulatory elements that would point to a potential role in pathogen defense response. Most notable are GmSUB1 silencing element characteristic of pathogenesis-related genes (Boyle and Brisson, 2001) and Dof protein binding sites, which may play a role in coordinated expression of multiple genes (Yanagisawa and Schmidt, 1999; Yanagisawa, 2002). Expression of GmSUB1 in PI 437654 appears to be regulated, in part, in response to SCN infection. However, because GmSUB1 appears to be constitutively expressed in soybean before infection, it may play a role in the early defense response, as is reported for a number of constitutively expressed pathogenesis-related genes (Samac and Shah, 1991; Tornero et al., 1997b).


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This material is based on work supported by the CSREES/USDA, under project number SC-1700102. Clemson University Exp. Stn. Tech. Contribution No. 4899. This work was supported by the United Soybean Board through the "Application of Biotechnology to Control of the Soybean Cyst Nematode" project.

Received for publication April 28, 2003.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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