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ABSTRACT: Like many widely cultivated crops, soybean [Glycine max (L.) Merr.] has a relatively narrow genetic base, while its perennial distant relatives in the subgenus Glycine Willd. are more genetically diverse and display desirable traits not present in cultivated soybean. To identify single-nucleotide polymorphisms (SNPs) between a pair of G. latifolia accessions that were resistant or susceptible to Sclerotinia sclerotiorum (Lib.) de Bary, reduced-representations of DNAs from each accession were sequenced. Approximately 30 % of the 36 million 100-nt reads produced from each of the two G. latifolia accessions aligned primarily to gene-rich euchromatic regions on the distal arms of G. max chromosomes. Because a genome sequence was not available for G. latifolia, the G. max genome sequence was used as a reference to identify 9,303 G. latifolia SNPs that aligned to unique positions in the G. max genome with at least 98 % identity and no insertions and deletions. To validate a subset of the SNPs, nine TaqMan and 384 GoldenGate allele-specific G. latifolia SNP assays were designed and analyzed in F2 G. latifolia populations derived from G. latifolia plant introductions (PI) 559298 and 559300. All nine TaqMan markers and 91 % of the 291 polymorphic GoldenGate markers segregated in a 1:2:1 ratio. Genetic linkage maps were assembled for G. latifolia, nine of which were uninterrupted and nearly collinear with the homoeologous G. max chromosomes. These results made use of a heterologous reference genome sequence to identify more than 9,000 informative high-quality SNPs for G. latifolia, a subset of which was used to generate the first genetic maps for any perennial Glycine species.
Theoretical and Applied Genetics 03/2013; · 3.30 Impact Factor
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ABSTRACT: Heterodera glycines is a nematode that is highly adapted to manipulate and parasitize plant hosts. The molecular players involved in these interactions have only recently begun to be identified. Here, the sequencing of the second stage juvenile transcriptome, followed by a bioinformatic screen for novel genes, identified seven new genes involved in biosynthesis and salvage of vitamins B(1), B(5), and B(7). With no confirmed reports in the literature, each of these biosynthesis pathways is believed to have been lost in multicellular animals. However, eukaryotic-like introns in the genomic sequences of the genes confirmed eukaryotic origin and nematode-specific splice leaders found on five of the cDNAs confirmed their nematode origin. Two of the genes were found to be flanked by known nematode sequences and quantitative polymerase chain reactions on individual nematodes showed similar and consistent amplification between the vitamin B biosynthesis genes and other known H. glycines genes. This further confirmed their presence in the nematode genome. Similarity to bacterial sequences at the amino acid level suggested a prokaryotic ancestry and phylogenetic analysis of the genes supported a likely horizontal gene transfer event, suggesting H. glycines re-appropriated the genes from the prokaryotic kingdom. This finding complements the previous discovery of a vitamin B(6) biosynthesis pathway within the nematode. However, unlike the complete vitamin B(6) pathway, many of these vitamin B pathways appear to be missing the initial enzymes required for full de novo biosynthesis, suggesting that initial substrates in the pathways are obtained exogenously. These partial vitamin B biosynthesis enzymes have recently been identified in other single-celled eukaryotic parasites and on rhizobia symbiosis plasmids, indicating that they may play an important role in host-parasite interactions and survival within the plant environment.
Journal of nematology 12/2009; 41(4):281-90. · 0.52 Impact Factor
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ABSTRACT: The expression of a candidate auxin-responsive gene was evaluated for use in a diagnostic assay for plant growth regulator (PGR) herbicide injury in soybean leaves. Expression of GH3, a primary auxin-responsive gene, was evaluated in response to dicamba and clopyralid at the RNA and protein levels, and proteomic analysis evaluated global expression of proteins in response to dicamba. Expression of GH3 was also analyzed in response to heat, drought, salt stress, and infection by soybean mosaic virus (SMV) and bean pod mottle virus (BPMV) to determine the specificity of GH3 expression as a diagnostic marker for PGR herbicide injury. At the RNA level, GH3 was strongly induced by dicamba and clopyralid within 8 h after application. Expression peaked 1 to 3 d after treatment (DAT) in response to 10% and 1% of a labeled dose of dicamba and clopyralid, with higher expression levels detected at higher herbicide rates. At the protein level, GH3 expression was also strongly induced at 1, 2, and 3 DAT by 10% vs. 1% of a labeled dose of dicamba and clopyralid. Heat, drought, and salt stress and infection with SMV or BPMV had no effect on GH3 expression at either the RNA or protein level. Proteomic analysis identified three proteins that were up-regulated in response to dicamba. Two were induced for less than 7 DAT, and a third was identified as a stress-response enzyme (superoxide dismutase) that is likely not specific to PGR herbicide injury. Expression of GH3 was highly induced by PGR herbicides at the RNA and protein level and was not affected by environmental stresses or viral infection, indicating that GH3 expression has excellent potential for use in a diagnostic assay for PGR herbicide injury. Nomenclature: Clopyralid; dicamba; soybean, Glycine max L. Merr.
Weed Science 01/2009; · 1.73 Impact Factor
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ABSTRACT: Heterodera glycines is an obligate plant parasite capable of biochemically and developmentally altering its host's cells in order to create a specialized feeding cell. Although the exact mechanism of feeding cell morphogenesis remains a mystery, the nematode's ability to manipulate the plant is thought to be due in part to horizontal gene transfers (HGTs). A bioinformatic screen of the nematode genome has revealed homologues of the genes SNZ and SNO, which comprise a metabolic pathway for the de novo biosynthesis of pyridoxal 5'-phosphate, the active form of vitamin B(6) (VB(6)). Analysis of the 2 genes, HgSNZ and HgSNO, show that they contain nematode-like introns, generate polyadenylated mRNAs, and map to the soybean cyst nematode genetic linkage map, indicating that they are part of the nematode genome. However, gene synteny, protein homology, and phylogenetic evidence suggest prokaryotic origin. This would represent the first case of the HGT of a complete pathway into a nematode or terrestrial animal. VB(6) acts as a cofactor in over 140 different enzymes, and recent studies point toward an important role as a potent quencher of reactive oxygen species. With H. glycines' penchant for acquiring parasitism genes through HGT along with the absence of this pathway in other land-based animals suggests a specific need for VB(6) which may involve the parasite-host interaction.
Molecular Biology and Evolution 07/2008; 25(10):2085-98. · 5.55 Impact Factor
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ABSTRACT: Arabidopsis transcriptional factors LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3), ABSCISIC ACID3 (ABI3), and ABSCISIC ACID5 (ABI5) are known to regulate multiple aspects of seed development. In an attempt to understand the developmental control of storage product accumulation, we observed the expression time course of the five transcripts. The sequential expression of these factors during seed fill suggests differentiation of their normal responsibilities. By extending the expression periods of the two early genes LEC1 and LEC2 in transgenic seeds, we demonstrated that the subsequent timing of FUS3, ABI3, and ABI5 transcripts depends on LEC1 and LEC2. Because a delayed onset or reduced level of FUS3 mRNA coincided with reduction of seed oil content in the transgenic seeds, the role of FUS3 in oil deposition was further examined. Analysis of published seed transcriptome data indicated that FUS3 transcript increased together with nearly all the plastidial fatty acid biosynthetic transcripts during development. The ability of FUS3 to rapidly induce fatty acid biosynthetic gene expression was confirmed using transgenic Arabidopsis seedlings expressing a dexamethasone (DEX)-inducible FUS3 and Arabidopsis mesophyll protoplasts transiently expressing the FUS3 gene. By accommodating the current evidence, we propose a hierarchical architecture of the transcriptional network in Arabidopsis seeds in which the oil biosynthetic pathway is integrated through the master transcriptional factor FUS3.
Planta 09/2007; 226(3):773-83. · 3.00 Impact Factor
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ABSTRACT: Chemicals called safeners protect cereal crops from herbicide toxicity. Proteomic methods (2-D PAGE and LC-MS/MS) were utilized to identify safener- and/or herbicide-regulated proteins in three tissues (root, leaf, and coleoptile) of Triticum tauschii seedlings to better understand a safener's mechanism of action. Growth experiments showed that the safener cloquintocet-mexyl protected seedlings from injury by the herbicide dimethenamid. In total, 29 safener-induced and 10 herbicide-regulated proteins were identified by LC-MS/MS. These proteins were classified into two major categories based on their expression patterns, and were further classified into several functional groups. Surprisingly, mutually exclusive sets of proteins were identified following herbicide or safener treatment, suggesting that different signaling pathways may be recruited. Safener-responsive proteins, mostly involved in xenobiotic detoxification, also included several new proteins that had not been previously identified as safener-responsive, whereas herbicide-regulated proteins belonged to several classes involved in general stress responses. Quantitative RT-PCR revealed that multidrug resistance-associated protein (MRP) transcripts were highly induced by safeners and two MRP genes were differentially expressed. Our results indicate that safeners protect T. tauschii seedlings from herbicide toxicity by coordinately inducing proteins involved in an entire herbicide detoxification pathway mainly in the coleoptile and root, thereby protecting new leaves from herbicide injury.
PROTEOMICS 05/2007; 7(8):1261-78. · 4.51 Impact Factor
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ABSTRACT: ABSTRACT Transgenic soybean (Glycine max) plants expressing Soybean mosaic virus (SMV) helper component-protease (HC-Pro) showed altered vegetative and reproductive phenotypes and responses to SMV infection. When inoculated with SMV, transgenic plants expressing the lowest level of HC-Pro mRNA and those transformed with the vector alone initially showed mild SMV symptoms. Plants that accumulated the highest level of SMV HC-Pro mRNA showed very severe SMV symptoms initially, but after 2 weeks symptoms disappeared, and SMV titers were greatly reduced. Analysis of SMV RNA abundance over time with region-specific probes showed that the HC-Pro region of the SMV genome was degraded before the coat protein region. Transgenic soybean plants that expressed SMV HC-Pro showed dose-dependent alterations in unifoliate leaf morphologies and seed production where plants expressing the highest levels of HC-Pro had the most deformed leaves and the lowest seed production. Accumulation of microRNAs (miRNAs) and mRNAs putatively targeted by miRNAs was analyzed in leaves and flowers of healthy, HC-Pro-transgenic, and SMV-infected plants. Neither expression of SMV HC-Pro nor SMV infection produced greater than twofold changes in accumulation of six miRNAs. In contrast, SMV infection was associated with twofold or greater increases in the accumulation of four of seven miRNA-targeted mRNAs tested.
Phytopathology 04/2007; 97(3):366-72. · 2.80 Impact Factor
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ABSTRACT: The soybean cyst nematode Heterodera glycines is the most destructive pathogen of soybean in the Unites States. Diversity in the parasitic ability of the nematode allows it to reproduce on nematode-resistant soybean. H. glycines chorismate mutase-1 (Hg-CM-1) is a nematode enzyme with the potential to suppress host plant defense compounds; therefore, it has the potential to enhance the parasitic ability of nematodes expressing the gene. Hg-cm-1 is a member of a gene family where two alleles, Hg-cm-1A and Hg-cm-1B, have been identified. Analysis of the Hg-cm-1 gene copy number revealed that there are multiple copies of Hg-cm-1 alleles in the H. glycines genome. H. glycines inbred lines were crossed to ultimately generate three F2 populations of second-stage juveniles (J2s) segregating for Hg-cm-1A and Hg-cm-1B. Segregation of Hg-cm-1A and 1B approximated a 1:2:1 ratio, which suggested that Hg-cm-1 is organized in a cluster of genes that segregate roughly as a single locus. The F2 H. glycines J2 populations were used to infect nematode-resistant (Hartwig, PI88788, and PI90763) and susceptible (Lee 74) soybean plants. H. glycines grown on Hartwig, Lee 74, and PI90763 showed allelic frequencies similar to Hg-cm-1A/B, but nematodes grown on PI88788 contained predominately Hg-cm-1A allele as a result of a statistically significant drop of Hg-cm-1B in the population. This result suggests that specific Hg-cm-1 alleles, or a closely linked gene, may aid H. glycines in adapting to particular soybean hosts.
Molecular Plant-Microbe Interactions 07/2005; 18(6):593-601. · 4.43 Impact Factor
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ABSTRACT: Symptoms resembling off-target plant growth regulator (PGR) herbicide injury are frequently found in soybean fields, but the causal agent is often difficult to identify. The expression of GH3, an auxin-regulated soybean gene, was quantified from soybean leaves injured by PGR herbicides using real-time RT-PCR. Expression of GH3 was analyzed to ascertain its suitability for use in a diagnostic assay to determine whether PGR herbicides are the cause of injury. GH3 was highly induced by dicamba within 3 days after treatment (DAT) and remained high at 7 DAT, but induction was much lower at 17 DAT. GH3 was also highly induced at 7 DAT by dicamba + diflufenzopyr, and to a lesser extent by the other PGR herbicides clopyralid and 2,4-D. The non-PGR herbicides glyphosate, imazethapyr, and fomesafen did not significantly induce GH3 expression above a low constitutive level. These results indicate that a diagnostic assay for PGR herbicide injury based on overexpression of auxin-responsive genes is feasible, and that GH3 is a potential candidate from which a diagnostic assay could be developed. However, time course analysis of GH3 expression indicates the assay would be effective for a limited time after exposure to the herbicide.
Journal of Agricultural and Food Chemistry 03/2004; 52(3):474-8. · 2.82 Impact Factor
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ABSTRACT: Parasitism genes from phytoparasitic nematodes are thought to be essential for nematode invasion of the host plant, to help the nematode establish feeding sites, and to aid nematodes in the suppression of host plant defenses. One gene that may play several roles in nematode parasitism is chorismate mutase (CM). This secreted enzyme is produced in the nematode's esophageal glands and appears to function within the plant cell to manipulate the plant's shikimate pathway, which controls plant cell growth, development, structure, and pathogen defense. Using degenerate polymerase chain reaction primers, we amplified and cloned a chorismate mutase (Hg-cm-1) from Heterodera glycines, the soybean cyst nematode (SCN), and showed it had CM activity. RNA in situ hybridization of Hg-cm-1 cDNA to SCN sections confirms that it is specifically expressed in the nematodes' esophageal glands. DNA gel blots of genomic DNA isolated from SCN inbred lines that have differing virulence on SCN resistant soybean show Hg-cm-1 is a member of a polymorphic gene family. Some Hg-cm family members predominate in SCN inbred lines that are virulent on certain SCN resistant soybean cultivars. The same polymorphisms and correlation with virulence are seen in the Hg-cm-1 expressed in the SCN second-stage juveniles. Based on the enzymatic activity of Hg-cm-1 and the observation that different forms of the mutase are expressed in virulent nematodes, we hypothesize that the Hg-cm-1 is a virulence gene, some forms of which allow SCN to parasitize certain resistant soybean plants.
Molecular Plant-Microbe Interactions 06/2003; 16(5):439-46. · 4.43 Impact Factor
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ABSTRACT: A developmental expression profile of the Meloidodgyne javanica esophageal gland gene chorismate mutase-1 (Mj-cm-1) could suggest when in the lifecycle of the nematode the Mj-cm-1 product is functional. This study used real-time quantitative RT-PCR to examine the variation in Mj-cm-1 transcript levels over six timepoints in the nematode lifecycle: egg, infective second-stage juveniles (Inf-J2), 2-day post-inoculation (pi), 7-day pi, 14-day pi, and adult. The Mj-cm-1 mRNA levels peaked at 2-day pi, about 100-fold above levels expressed at the egg and Inf-J2 stages. Some expression of Mj-cm-1 remained during the 7-day pi, 14-day pi, and adult stages. High transcript levels of the beta-actin control gene M. javanica Beta-actin-1 (Mj-ba-1) demonstrated the presence of cDNA at all timepoints. The peak in Mj-cm-1 transcript expression at 2-day pi as well as the previously shown esophageal gland localization of Mj-cm-1 mRNA suggest that the product of this gene may be involved early in the establishment of parasitism.
Journal of nematology 04/2003; 35(1):82-7. · 0.52 Impact Factor
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ABSTRACT: Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant cells for feeding. Nematodes inject secretions from their esophageal glands through their stylet and into plant cells to induce giant cell formation. Meloidogyne javanica chorismate mutase 1 (MjCM-1) is one such esophageal gland protein likely to be secreted from the nematode as giant cells form. MjCM-1 has two domains, an N-terminal chorismate mutase (CM) domain and a C-terminal region of unknown function. It is the N-terminal CM domain of the protein that is the predominant form produced in root-knot nematodes. Transgenic expression of MjCM-1 in soybean hairy roots results in a phenotype of reduced and aborted lateral roots. Histological studies demonstrate the absence of vascular tissue in hairy roots expressing MjCM-1. The phenotype of MjCM-1 expressed at low levels can be rescued by the addition of indole-3-acetic acid (IAA), indicating MjCM-1 overexpression reduces IAA biosynthesis. We propose MjCM-1 lowers IAA by causing a competition for chorismate, resulting in an alteration of chorismate-derived metabolites and, ultimately, in plant cell development. Therefore, we hypothesize that MjCM-1 is involved in allowing nematodes to establish a parasitic relationship with the host plant.
Molecular Plant-Microbe Interactions 03/2003; 16(2):123-31. · 4.43 Impact Factor
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ABSTRACT: Root-knot nematodes (Meloidogynejavanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.
Molecular Plant-Microbe Interactions 07/2002; 15(6):549-56. · 4.43 Impact Factor
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ABSTRACT: Soybean mosaic virus (SMV) helper component protease (HC-Pro), a suppressor of post-transcriptional gene silencing, was evaluated for its ability to enhance production of soybean hygromycin-resistant somatic embryos (HR-SEs), and stabilize transgene expression. Immature soybean cotyledonary explants were co-cultured with Agrobacterium tumefaciens strain KYRT1 harboring either pCAMBIA1302, carrying a hygromycin phosphotransferase gene (hpt) and a gene encoding green fluorescent protein; pCAMBIA1305.1, carrying hpt and beta-glucuronidase (uidA) genes; pG2-HC-Pro, a derivative of pCAMBIA1305.1 containing SMV G2 HC-Pro; or pG5-HC-Pro, a derivative of pCAMBIA1305.1 containing SMV G5 HC-Pro, but lacking uidA. Significantly (rho<0.02) higher numbers of HR-SEs were obtained from explants transformed with Agrobacterium harboring either pG2-HC-Pro or pG5-HC-Pro than with either of the vector controls (pCAMBIA1302 or pCAMBIA1305.1). Beta-glucuronidase (GUS) expression was significantly (rho<0.003) higher in 50-day-old transgenic plants expressing GUS along with SMV-HC-Pro and in SMV-infected GUS transgenic plants than in transgenic plants expressing GUS alone. Together, these data suggest that SMV-HC-Pro enhanced recovery of HR-SEs by suppressing silencing of the hygromycin phosphotransferase gene.
Plant Physiology and Biochemistry 43(10-11):1014-21. · 2.84 Impact Factor
