1. Introduction
Soybean,
Gene duplication is a major source of evolutionary novelties and can occur through duplication of individual genes, chromosomal segments, or entire genomes (polyploidization). Under the classic model of duplicate gene evolution, one of the duplicated genes is free to accumulate mutations, which results in either the inactivation of transcription and/or a function (pseudogenization or nonfunctionalization) or the gain of a new function (neofunctionalization) as long as another copy retains the requisite physiological functions [10; and references therein]. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classic model [11].
Recent advances in genome study have led to the formulation of several evolutionary models: a model proposed by Hughes [12] suggests that gene sharing, whereby a single gene encodes a protein with two distinct functions, precedes the evolution of two functionally distinct proteins; the duplication–degeneration–complementation model suggests that duplicate genes acquire debilitating yet complementary mutations that alter one or more subfunctions of the single gene progenitor, an evolutionary consequence for duplicated loci referred to as subfunctionalization [4, 11, 13]. In addition to this notion, models involving epigenetic silencing of duplicate genes [14] or purifying selection for gene balance [15, 16] have also been proposed. In soybean, differential patterns of expression have often been detected between homoeologous genes [17, 18], which indicates that subfunctionalization has occurred in these genes.
When the extent of subfunctionalization is limited, mutations in only one of multiple cognate gene copies do not often result in phenotypic changes. Therefore, methods that allow suppression of all copies of the duplicated gene are required for analyzing gene function or engineering novel traits. RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression, either at the posttranscriptional or transcriptional level, which provides a powerful tool to downregulate a gene or a gene family [19, 20]. Suppression of gene expression through RNA silencing is particularly useful for analyzing the function(s) of duplicated genes or engineering novel traits because it allows silencing of multiple cognate genes having nucleotide sequence identity. In fact, to produce soybean lines that have a novel trait, researchers have frequently used RNA silencing induced by a transgene.
In this review, we describe application of RNA silencing to understand the roles of genes or engineering novel traits in soybean. We describe methods to induce simultaneous silencing of duplicated genes and selective silencing of each copy of duplicated genes through RNA silencing. In addition to intentionally induced RNA silencing, we also refer to naturally occurring RNA silencing. Based on our knowledge of RNA silencing in soybean, we propose a hypothesis that plants may have used subfunctionalization of duplicated genes as a means to avoid the occurrence of simultaneous silencing of duplicated genes, which could be deleterious to the organism.
2. Mechanisms and diverse pathways of RNA silencing
Gene silencing is one of the regulatory mechanisms of gene expression in eukaryotes, which refers to diverse RNA-guided sequence-specific inhibition of gene expression, either at the posttranscriptional or transcriptional level [19, 20]. Post-transcriptional gene silencing (PTGS) was first discovered in transgenic petunia plants whose flower color pattern was changed as a consequence of overexpression of a gene that encodes the key enzyme for anthocyanin biosynthesis in 1990 [21, 22]. Similar phenomena have also been reported for plants transformed with various genes, which include virus resistance of plants that have gene or gene segments derived from the viral genome [23, 24]. Because of these findings, gene silencing is thought to have developed to defend against viruses. Several lines of research in plants indicated that double-stranded RNA (dsRNA) is crucial for RNA degradation [25, 26]. The potency of dsRNA to induce gene silencing was demonstrated in
Subsequent genetic and biochemical analyses in several organisms revealed that PTGS and RNAi share the same pathway and consist of two main processes: (i) processing of dsRNA into 20–26-nt small RNA molecules (short interfering RNA; siRNA) by an enzyme called Dicer that has RNaseIII-like endonuclease activity; (ii) cleavage of RNA guided by siRNA at a complementary nucleotide sequence in the RNA-induced silencing complex (RISC) containing the Argonaute (AGO) protein (Figure 1) [28]. The formation of dsRNA from single-stranded sense RNA was explained by the synthesis of its complementary strand by RNA-dependent RNA polymerase (RdRP). This process provides templates for Dicer cleavage that produces siRNAs and consequently allows amplification of silencing [29]. siRNA is responsible for not only induction of sequence-specific RNA degradation but also epigenetic changes involving DNA methylation and histone modification in the nucleus, which leads to transcriptional gene silencing (TGS) [30]. It has become evident that siRNA plays a role in systemic silencing as a mobile signal [31, 32]. In addition to siRNA, small RNA molecules called micro RNAs (miRNAs) are also involved in negative regulation of gene expression [33]. These gene silencing phenomena that are induced by sequence-specific RNA interaction are collectively called RNA silencing [34, 35].
RNA silencing plays an important role in many biological processes including development, stability of the genome, and defense against invading nucleic acids such as transgenes and viruses [20, 29, 30]. It can also be used as a tool for analyzing specific gene functions and producing new features in organisms including plants [36-38].
3. Methods of the induction of RNA silencing in soybean
3.1. Transgene-induced RNA silencing
Engineering novel traits through RNA silencing in soybean has been done using transgenes or virus vectors (Figure 1). RNA silencing in some transgenic soybean lines was induced by introducing a transgene that transcribes sense RNA homologous to a gene present in the plant genome, a phenomenon termed co-suppression [21]. This type of silencing was first discovered in transgenic petunia plants that had silencing of
An interesting finding reported in soybean is that RNA silencing is induced by a transgene that transcribes inverted repeats of a fatty acid desaturase
Transcribing a transgene with a strong promoter tends to induce RNA silencing more frequently than that with a weak promoter [47]. For obtaining a higher level of transcription in soybean plants, the
A gene construct that induces RNA silencing has been introduced to the soybean genome using either
3.2. Virus-induced gene silencing (VIGS)
RNA silencing has also been induced using a virus vector in soybean. Plants intrinsically have the ability to cope with viruses through the mechanisms of RNA silencing. When plants are infected with an RNA virus, dsRNA of the viral genome is degraded by the infected plants [49, 50]. The dsRNA in the virus-infected cells is thought to be the replication intermediate of the viral RNA [51] or a duplex structure formed within single-stranded viral RNA [52]. The viral genomic RNA can be processed into siRNAs, then targeted by the siRNA/RNase complex. In this scenario, if a nonviral segment is inserted in the viral genome, siRNAs would also be produced from the segment. Therefore, if the insert corresponds to a sequence of the gene encoded in the host plant, infection by the virus results in the production of siRNAs corresponding to the plant gene and subsequently induces loss of function of the gene product (Figure 2). This fact led to the use of a virus vector as a source to induce silencing of a specific gene in the plant genome, which is referred to as virus-induced gene silencing (VIGS) [42, 53, 54]. So far, at least 11 RNA viruses and five DNA viruses were developed as a plant virus vector for gene silencing, as listed previously [37]. Three vectors are now available in soybean: those based on
4. Examples of RNA silencing reported in soybean
4.1. Metabolic engineering by transgene-induced RNA silencing
To the authors’ knowledge, 28 scientific papers that describe metabolic engineering by transgene-induced RNA silencing in soybean have been published up to 2011 [58]. Because soybean seeds are valued economically for food and oil production, most modifications to transgenic soybean plants using RNA silencing are focused on seed components. Metabolic pathways in developing seeds have been targeted in terms of altering nutritional value for human or animals, e.g., changing seed storage protein composition [59, 60], reducing phytic acids [61, 62], saponin [63] or allergens [64], and increasing isoflavone [65]. Metabolic engineering has also targeted oil production [66-72]. These modifications were done by inhibiting a step in a metabolic pathway to decrease a product or by blocking a competing branch pathway to increase a product.
RNA silencing can be induced efficiently in soybean roots using
Transgene-induced RNA silencing has also been induced in leaf tissues for the β-glucuronidase gene [80] or the senescence-associated receptor-like kinase gene [81] and in calli for the amino aldehyde dehydrogenase gene to induce the biosynthesis of 2-acetyl-1-pyrroline [82].
4.2. Disease resistance acquired by transgene-induced RNA silencing
Another focus of modifying soybean plants through RNA silencing is resistance against diseases, particularly to those caused by viruses. Resistance to viruses was achieved by transforming plants with genes or segments of genes derived from viruses and was referred to as pathogen-derived resistance [23, 24, 83, 84]. The resistance did not need protein translated from the transgene [85-87], which led to the understanding that RNA is the factor that conferred resistance to the plants and that the enhanced resistance is acquired via a mechanism analogous to that involved in co-suppression. Using this strategy, soybean plants resistant to
In addition to resistance against a virus, transgenic soybean plants resistant to cyst nematode (
4.3. Gene functional analysis by VIGS
An advantage of VIGS is its ease for making a gene construct and introducing nucleic acids to cells. In addition, the effect of silencing can be monitored within a short time after inoculating plants with the virus. Because of these features, VIGS is suitable for gene function analysis [51, 100, 101] and has been used for gene identification via downregulating a candidate gene(s) responsible for a specific phenomenon in soybean. VIGS was used to demonstrate that genes present in the genetically identified loci actually encode the genes responsible for the phenotype: VIGS of the putative
4.4. Naturally occurring RNA silencing
In addition to artificially induced RNA silencing, naturally occurring RNA silencing has also been known in soybean. Naturally occurring RNA silencing, involving mRNA degradation induced as a consequence of certain genetic changes, has been detected based on phenotypic changes. Most commercial varieties of soybean produce yellow seeds due to loss of pigmentation in seed coats, and this phenotype has been shown to be due to PTGS of the
The occurrence of RNA silencing that leads to changes in pigmentation of plant tissues has also been reported for the
5. Diagnosis of an RNA silencing-induced phenotype using viral infection
In the course of the analysis of
6. What do phenotypic changes induced by RNA silencing in soybean indicate?
Soybean is thought to be derived from an ancestral plant(s) with a tetraploid genome, and as a consequence, large portions of the soybean genome are duplicated [7], with nearly 75% of the genes present in multiple copies [117]. In addition, genes in the soybean genome are sometimes duplicated in tandem [118-121]. Our recent studies have indeed shown functional redundancy of duplicated genes in soybean [122, 123]. Such gene duplication can be an obstacle to producing mutants by conventional methods of mutagenesis. In this regard, the gene silencing technique is particularly useful because it allows silencing of multiple cognate genes having nucleotide sequence identity.
Changes in phenotypes as a consequence of inducing RNA silencing have been successful for many genes in soybean as mentioned above. Considering that many genes are duplicated in soybean genome, this fact indicates either that RNA silencing worked on all duplicated genes that have the same function or that the genes were subfunctionalized after duplication, so that RNA silencing of even a single gene of the duplicated genes resulted in the phenotypic changes.
It is of interest to understand whether duplicated genes have identical or diversified functions, which may depend on the time after duplication event and/or the selection pressure on the genes. To analyze the functions of each copy of the duplicated genes, we need to silence a specific copy of the duplicated genes. If the duplicated genes are expressed in different tissues, RNA silencing of both genes can lead to understanding the function of each gene. PTGS by transcribing inverted repeat with a constitutive promoter or VIGS will be suitable for this analysis. An example of such an approach is the VIGS of duplicated
7. Methods to induce selective RNA silencing of duplicated genes
When duplicated genes are subfunctionalized with only limited nucleotide changes and are expressed in overlapping tissues, specific silencing of each gene will be necessary for understanding their function(s). Silencing a specific copy of duplicated genes can be achieved by targeting a gene portion whose nucleotide sequence is differentiated between the duplicated genes. A condition that allows this type of silencing involves a lack of silencing of the other copy of duplicated genes even when they have the same sequence in the other portions.
In plants, miRNAs or siRNAs promote production of secondary siRNAs from the 5′ upstream region and/or the 3′ downstream region of the initially targeted region via production of dsRNA by RdRP. These secondary siRNAs can lead to silencing of a secondary target that is not directly targeted by the primary silencing trigger [124]. Studies so far have indicated that such a spread of RNA silencing, called transitive RNA silencing, does not occur with the majority of endogenous genes, although it can happen to a transgene [45; and references therein]. Assuming the lack of transitive RNA silencing, it is possible to induce silencing of a specific copy of a duplicated gene. Targeting a region specific for each copy, e.g., the 3′ untanslated region (UTR), can induce silencing of the gene copy only, whereas targeting a region conserved in duplicated gene copies can induce silencing of the multiple gene copies simultaneously (Figure 3). Such selective RNA silencing was successful in a gene family of rice [125] and this strategy may work for analyzing functional diversification of duplicated genes in any plant species.
An alternative approach to suppress gene expression in plants is the use of artificial miRNAs (Figure 4) (amiRNAs; also called synthetic miRNAs) [38, 126]. This approach involves modification of plant miRNA sequence to target specific transcripts, originally not under miRNA control, and downregulation of gene expression via specific cleavage of the target RNA. Melito
Induction of TGS by targeting dsRNA to a gene promoter can also be the method of choice. Gene silencing through transcriptional repression can be induced by dsRNA targeted to a gene promoter (Figure 4). However, until recently, no plant has been produced that harbors an endogenous gene that remains silenced in the absence of promoter-targeting dsRNA. We have reported for the first time that TGS can be induced by targeting dsRNA to the endogenous gene promoters in petunia and tomato plants, using a
8. Differentiation of duplicated genes and induction of RNA silencing
How much sequence difference will be necessary to induce selective RNA silencing? A factor that affects induction of RNA silencing is the extent of sequence identity between the dsRNA that triggers RNA silencing and its target gene. IR-PTGS could be induced by IR-transcripts that can form 98-nt or longer dsRNAs [39]. In VIGS, the lower size limit of the inserted fragments required for inducing PTGS is 23-nt, a size almost corresponding to that of siRNAs [134], and that for inducing TGS is 81-91 nt [135]. Silencing a gene probably requires sequence identity longer than the size of siRNAs between dsRNA and its target, although the efficiency of silencing may depend on the system of silencing induction.
We previously induced
The naturally occurring RNA silencing of the
9. Conclusion and perspectives
RNA silencing has been used as a powerful tool to engineer novel traits or analyze gene function in soybean. Soybean plants that have engineered a metabolic pathway or acquired resistance to diseases have been produced by transgene-induced gene silencing. VIGS has been used as a tool to analyze gene function in soybean. In addition to RNA silencing, site-directed mutagenesis using zinc-finger nucleases has been applied to mutagenizing duplicated genes in soybean [138]. Such reverse genetic approaches may be supplemented by forward genetic approaches such as high linear energy transfer radiation-based mutagenesis, e.g., irradiation of ion beam [139] and fast neutron [140]. Similarly, gene tagging systems using maize Ds transposon [141] and rice
AGO, Argonaute; ALSV,
Acknowledgments
Our work is supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References
- 1.
Lackey J. Chromosome-numbers in the Phaseoleae (Fabaceae, Faboideae) and their relation to taxonomy 1980 67 595 602 - 2.
Hymowitz T. 2004 Speciation and cytogenetics. In: Boerma HR, Specht JE. (eds) Soybeans: improvement, production, and uses, Ed 3, Agronomy Monograph No. 16. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc. Madison, Wisconsin, USA; 2004. p97-136. - 3.
Shoemaker R. C. Schlueter J. Doyle J. J. Paleopolyploidy and gene duplication in soybean and other legumes 2006 9 104 9 - 4.
Moore R. C. Purugganan M. D. 2005 The evolutionary dynamics of plant duplicate genes 8 122 8 - 5.
Lohnes D. Specht J. Cregan P. Evidence for homoeologous linkage groups in the soybean 1997 37 254 7 - 6.
Zhu T. Schupp J. M. Oliphant A. Keim P. 1994 Hypomethylated sequences: characterization of the duplicate soybean genome 244 638 45 - 7.
Shoemaker R. C. Polzin K. Labate J. Specht J. Brummer E. C. Olson T. et al. Genome duplication in soybean ( Glycine subgenussoja )1996 144 329 38 - 8.
Lee J. Bush A. Specht J. Shoemaker R. Mapping of duplicate genes in soybean 1999 42 829 36 - 9.
Schlueter J. A. Dixon P. Granger C. Grant D. Clark L. Doyle J. J. et al. 2004 Mining EST databases to resolve evolutionary events in major crop species 47 868 76 - 10.
Lynch M. Conery J. S. The evolutionary fate and consequences of duplicate genes 2000 290 1151 5 - 11.
Force A. Lynch M. Pickett F. B. Amores A. Yan Y. L. Postlethwait J. Preservation of duplicate genes by complementary, degenerative mutations 1999 151 1531 45 - 12.
Hughes A. L. 1994 The evolution of functionally novel proteins after gene duplication 256 119 24 - 13.
Lynch M. Force A. The probability of duplicate gene preservation by subfunctionalization 2000 154 459 73 - 14.
Rodin S. N. Riggs A. D. 2003 Epigenetic silencing may aid evolution by gene duplication 56 718 29 - 15.
Freeling M. Thomas B. C. 2006 Gene-balanced duplications, like tetraploidy, provide predictable drive to increase morphological complexity 16 805 14 - 16.
Birchler J. A. Veitia R. A. 2007 The gene balance hypothesis: from classical genetics to modern genomics 19 395 402 - 17.
Schlueter J. A. Scheffler B. E. Schlueter S. D. Shoemaker R. C. 2006 Sequence conservation of homeologous bacterial artificial chromosomes and transcription of homeologous genes in soybean ( Glycine max L. Merr.)174 1017 28 - 18.
Schlueter J. Vaslenko-Sanders I. Deshpande S. Yi J. Siegfried M. Roe B. et al. 2007 The FAD2 gene family of soybean: insights into the structural and functional divergence of a paleopolyploid genome 47 S14 S26 - 19.
Brodersen P. Voinnet O. The diversity of RNA silencing pathways in plants 2006 22 268 80 - 20.
Vaucheret H. 2006 Post-transcriptional small RNA pathways in plants: mechanisms and regulations 20 759 71 - 21.
Napoli C. Lemieux C. Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans 1990 2 279 89 - 22.
van der Krol A. R. Mur L. A. Beld M. Mol J. N. Stuitje A. R. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression 1990 2 291 9 - 23.
Wilson T. M. 1993 Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms 90 3134 41 - 24.
Baulcombe D. C. 1996 Mechanisms of pathogen-derived resistance to viruses in transgenic plants 8 1833 44 - 25.
Metzlaff M. O’Dell M. Cluster P. D. Flavell R. B. 1997 RNA-mediated RNA degradation and chalcone synthase A silencing in petunia 88 845 54 - 26.
Waterhouse P. Graham H. Wang M. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA 1998 95 13959 64 - 27.
Fire A. Xu S. Montgomery M. K. Kostas S. A. Driver S. E. Mello C. C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans 1998 391 806 11 - 28.
Matzke M. Matzke A. J. Kooter J. M. 2001 RNA: guiding gene silencing 293 1080 3 - 29.
Baulcombe D. 2004 RNA silencing in plants 431 356 63 - 30.
Matzke M. Kanno T. Daxinger L. Huettel B. Matzke A. J. RNA-mediated chromatin-based silencing in plants 2009 21 367 76 - 31.
Dunoyer P. Schott G. Himber C. Meyer D. Takeda A. Carrington J. C. et al. 2010 Small RNA duplexes function as mobile silencing signals between plant cells 328 912 6 - 32.
Molnár A. Melnyk C. W. Bassett A. Hardcastle T. J. Dunn R. Baulcombe D. C. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells 2010 328 872 5 - 33.
Mallory A. C. Vaucheret H. Functions of microRNAs and related small RNAs in plants 2006 38 S31-6 - 34.
Voinnet O. 2002 RNA silencing: small RNAs as ubiquitous regulators of gene expression 5 444 51 - 35.
Matzke M. Aufsatz W. Kanno T. Daxinger L. Papp I. Mette M. F. et al. Genetic analysis of RNA-mediated transcriptional gene silencing 2004 1677 129 41 - 36.
Mansoor S. Amin I. Hussain M. Zafar Y. Briddon R. W. Engineering novel traits in plants through RNA interference 2006 11 559 65 - 37.
Kanazawa A. 2008 RNA silencing manifested as visibly altered phenotypes in plants 25 423 35 - 38.
Frizzi A. Huang S. 2010 Tapping RNA silencing pathways for plant biotechnology 8 655 77 - 39.
Wesley S. V. Helliwell C. A. Smith N. A. Wang M. B. Rouse D. T. Liu Q. et al. Construct design for efficient, effective and high-throughput gene silencing in plants 2001 27 581 90 - 40.
Helliwell C. A. Waterhouse P. M. 2005 Constructs and methods for hairpin RNA-mediated gene silencing in plants 392 24 35 - 41.
Wagner N. Mroczka A. Roberts P. D. Schreckengost W. Voelker T. RNAi trigger fragment truncation attenuates soybean FAD2-1 transcript suppression and yields intermediate oil phenotypes2011 9 723 8 - 42.
Ruiz M. T. Voinnet O. Baulcombe D. C. Initiation and maintenance of virus-induced gene silencing 1998 10 937 46 - 43.
Metzlaff M. O’Dell M. Hellens R. Flavell R. B. Developmentally and transgene regulated nuclear processing of primary transcripts of chalcone synthase A in petunia2000 23 63 72 - 44.
Hoffer P. Ivashuta S. Pontes O. Vitins A. Pikaard C. Mroczka A. et al. Posttranscriptional gene silencing in nuclei 2011 108 409 14 - 45.
Vermeersch L. De Winne N. Depicker A. Introns reduce transitivity proportionally to their length, suggesting that silencing spreads along the pre-mRNA 2010 64 392 401 - 46.
Christie M. Croft L. J. Carroll B. J. Intron splicing suppresses RNA silencing in Arabidopsis 2011 68 159 67 - 47.
Que Q. Wang H. Y. English J. J. Jorgensen R. A. The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence 1997 9 1357 68 - 48.
Yamada T. Takagi K. Ishimoto M. Recent advances in soybean transformation and their application to molecular breeding and genomic analysis 2012 61 480 94 - 49.
Covey S. AlKaff N. Langara A. Turner D. Plants combat infection by gene silencing 1997 385 781 2 - 50.
Al-Kaff N. S. Covey S. N. Kreike M. M. Page A. M. Pinder R. Dale P. J. Transcriptional and posttranscriptional plant gene silencing in response to a pathogen 1998 279 2113 5 - 51.
Lu R. Martin-Hernandez A. M. Peart J. R. Malcuit I. Baulcombe D. C. Virus-induced gene silencing in plants 2003 30 296 303 - 52.
Molnár A. Csorba T. Lakatos L. Várallyay E. Lacomme C. Burgyán J. Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs 2005 79 7812 8 - 53.
Kumagai M. H. Donson J. della -Cioppa G. Harvey D. Hanley K. Grill L. K. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA 1995 92 1679 83 - 54.
Purkayastha A. Dasgupta I. 2009 Virus-induced gene silencing: a versatile tool for discovery of gene functions in plants 47 967 76 - 55.
Zhang C. Ghabrial S. A. Development of Bean pod mottle virus -based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean2006 344 401 11 - 56.
Nagamatsu A. Masuta C. Senda M. Matsuura H. Kasai A. Hong J. S. et al. Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing 2007 5 778 90 - 57.
Yamagishi N. Yoshikawa N. Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors2009 71 15 24 - 58.
Kasai M. Kanazawa A. 2012 RNA silencing as a tool to uncover gene function and engineer novel traits in soybean 61 468 79 - 59.
Kinney A. J. Jung R. Herman E. M. Cosuppression of the α subunits of β-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies 2001 13 1165 78 - 60.
Schmidt M. A. Barbazuk W. B. Sandford M. May G. Song Z. Zhou W. et al. Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome 2011 156 330 45 - 61.
Nunes A. C. Vianna G. R. Cuneo F. Amaya-Farfán J. de Capdeville G. Rech E. L. et al. RNAi-mediated silencing of the myo -inositol-1-phosphate synthase gene (GmMIPS1 ) in transgenic soybean inhibited seed development and reduced phytate content2006 224 125 32 - 62.
Shi J. Wang H. Schellin K. Li B. Faller M. Stoop J. M. et al. Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds 2007 25 930 7 - 63.
Takagi K. Nishizawa K. Hirose A. Kita A. Ishimoto M. Manipulation of saponin biosynthesis by RNA interference-mediated silencing of β-amyrin synthase gene expression in soybean 2011 30 1835 46 - 64.
Herman E. M. Helm R. M. Jung R. Kinney A. J. Genetic modification removes an immunodominant allergen from soybean 2003 132 36 43 - 65.
Yu O. Shi J. Hession A. O. Maxwell C. A. McGonigle B. Odell J. T. Metabolic engineering to increase isoflavone biosynthesis in soybean seed 2003 63 753 63 - 66.
Kinney A. Development of genetically engineered soybean oils for food applications 1996 3 273 92 - 67.
Chen R. Matsui K. Ogawa M. Oe M. Ochiai M. Kawashima H. et al. 2006 Expression of Δ6, Δ5 desaturase and GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [Glycine max (L.) Merrill] seeds170 399 406 - 68.
Flores T. Karpova O. Su X. Zeng P. Bilyeu K. Sleper D. A. et al. 2008 Silencing of GmFAD3 gene by siRNA leads to low alpha-linolenic acids (18:3) of fad3-mutant phenotype in soybean [Glycine max (Merr.)]17 839 50 - 69.
Schmidt M. A. Herman E. M. 2008 Suppression of soybean oleosin produces micro-oil bodies that aggregate into oil body/ER complexes 1 910 24 - 70.
Wang G. Xu Y. Hypocotyl-based Agrobacterium-mediated transformation of soybean ( Glycine max ) and application for RNA interference2008 27 1177 84 - 71.
Lee J. Welti R. Schapaugh W. T. Trick H. N. Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed 2011 9 359 72 - 72.
Wagner N. Mroczka A. Roberts P. D. Schreckengost W. Voelker T. RNAi trigger fragment truncation attenuates soybean FAD2-1 transcript suppression and yields intermediate oil phenotypes2011 9 723 8 - 73.
Lee M. Y. Shin K. H. Kim Y. K. Suh J. Y. Gu Y. Y. Kim M. R. et al. 2005 Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots 139 1881 9 - 74.
Subramanian S. Stacey G. Yu O. Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum 2006 48 261 73 - 75.
Hayashi S. Gresshoff P. M. Kinkema M. Molecular analysis of lipoxygenases associated with nodule development in soybean 2008 21 843 53 - 76.
Dalton D. A. Boniface C. Turner Z. Lindahl A. Kim H. J. Jelinek L. et al. Physiological roles of glutathione S-transferases in soybean root nodules 2009 150 521 30 - 77.
Govindarajulu M. Kim S. Y. Libault M. Berg R. H. Tanaka K. Stacey G. et al. 2009 GS52 ecto-apyrase plays a critical role during soybean nodulation 149 994 1004 - 78.
Libault M. Zhang X. C. Govindarajulu M. Qiu J. Ong Y. T. Brechenmacher L. et al. 2010 A member of the highly conserved FWL (tomatoFW2.2-like ) gene family is essential for soybean nodule organogenesis62 852 64 - 79.
Yi J. Derynck M. R. Li X. Telmer P. Marsolais F. Dhaubhadel S. 2010 A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean62 1019 34 - 80.
Reddy M. S. Dinkins R. D. Collins G. B. 2003 Gene silencing in transgenic soybean plants transformed via particle bombardment 21 676 83 - 81.
Li X. P. Gan R. Li P. L. Ma Y. Y. Zhang L. W. Zhang R. et al. Identification and functional characterization of a leucine-rich repeat receptor-like kinase gene that is involved in regulation of soybean leaf senescence 2006 61 829 44 - 82.
Arikit S. Yoshihashi T. Wanchana S. Uyen T. T. Huong N. T. Wongpornchai S. et al. Deficiency in the amino aldehyde dehydrogenase encoded by GmAMADH2 , the homologue of riceOs2AP , enhances 2-acetyl-1-pyrroline biosynthesis in soybeans (Glycine max L.)2011 9 75 87 - 83.
Prins M. Goldbach R. RNA-mediated virus resistance in transgenic plants 1996 141 2259 76 - 84.
Goldbach R. Bucher E. Prins M. Resistance mechanisms to plant viruses: an overview 2003 92 207 12 - 85.
Smith H. A. Swaney S. L. Parks T. D. Wernsman E. A. Dougherty W. G. 1994 Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs 6 1441 53 - 86.
Mueller E. Gilbert J. Davenport G. Brigneti G. Baulcombe D. 1995 Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing 7 1001 13 - 87.
Sijen T. Wellink J. Hiriart J. B. Van Kammen A. 1996 RNA-mediated virus resistance: role of repeated transgenes and delineation of targeted regions 8 2277 94 - 88.
Wang X. Eggenberger A. Nutter F. Hill J. Pathogen-derived transgenic resistance to soybean mosaic virus in soybean 2001 8 119 27 - 89.
Furutani N. Hidaka S. Kosaka Y. Shizukawa Y. Kanematsu S. Coat protein gene-mediated resistance to soybean mosaic virus in transgenic soybean 2006 56 119 24 - 90.
Furutani N. Yamagishi N. Hidaka S. Shizukawa Y. Kanematsu S. Kosaka Y. Soybean mosaic virus resistance in transgenic soybean caused by posttranscriptional gene silencing 2007 57 123 8 - 91.
Tougou M. Furutani N. Yamagishi N. Shizukawa Y. Takahata Y. Hidaka S. Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus 2006 25 1213 8 - 92.
Tougou M. Yamagishi N. Furutani N. Shizukawa Y. Takahata Y. Hidaka S. Soybean dwarf virus-resistant transgenic soybeans with the sense coat protein gene 2007 26 1967 75 - 93.
Steeves R. Todd T. Essig J. Trick H. Transgenic soybeans expressing siRNAs specific to a major sperm protein gene suppress Heterodera glycines reproduction2006 33 991 9 - 94.
Li J. Todd T. C. Oakley T. R. Lee J. Trick H. N. Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe2010 232 775 85 - 95.
Ibrahim H. M. Alkharouf N. W. Meyer S. L. Aly M. A. Gamal El-Din AlK. Hussein E. H. et al. 2011 Post-transcriptional gene silencing of root-knot nematode in transformed soybean roots 127 90 9 - 96.
Subramanian S. Graham M. Y. Yu O. Graham T. L. 2005 RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and to enhanced susceptibility to Phytophthora sojae 137 1345 53 - 97.
Graham T. L. Graham M. Y. Subramanian S. Yu O. RNAi silencing of genes for elicitation or biosynthesis of 5-deoxyisoflavonoids suppresses race-specific resistance and hypersensitive cell death in Phytophthora sojae infected tissues2007 144 728 40 - 98.
Lozovaya V. V. Lygin A. V. Zernova O. V. Ulanov A. V. Li S. Hartman G. L. et al. Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase 2007 225 665 79 - 99.
Melito S. Heuberger A. L. Cook D. Diers B. W. MacGuidwin A. E. Bent A. F. 2010 A nematode demographics assay in transgenic roots reveals no significant impacts of the Rhg1 locus LRR-Kinase on soybean cyst nematode resistance10 104 - 100.
Metzlaff M. 2002 RNA-mediated RNA degradation in transgene- and virus-induced gene silencing 383 1483 9 - 101.
Burch-Smith T. M. Anderson J. C. Martin G. B. Dinesh-Kumar S. P. 2004 Applications and advantages of virus-induced gene silencing for gene function studies in plants 39 734 46 - 102.
Liu B. Watanabe S. Uchiyama T. Kong F. Kanazawa A. Xia Z. et al. The soybean stem growth habit gene Dt1 is an ortholog of ArabidopsisTERMINAL FLOWER1 2010 153 198 210 - 103.
Kachroo A. Fu D. Q. Havens W. Navarre D. Kachroo P. Ghabrial S. A. An oleic acid-mediated pathway induces constitutive defense signaling and enhanced resistance to multiple pathogens in soybean 2008 21 564 75 - 104.
Fu D. Q. Ghabrial S. Kachroo A. 2009 GmRAR1 andGmSGT1 are required for basal, R gene-mediated and systemic acquired resistance in soybean22 86 95 - 105.
Meyer J. D. Silva D. C. Yang C. Pedley K. F. Zhang C. van de Mortel M. et al. Identification and analyses of candidate genes for Rpp4 -mediated resistance to Asian soybean rust in soybean2009 150 295 307 - 106.
Pandey A. K. Yang C. Zhang C. Graham M. A. Horstman H. D. Lee Y. et al. Functional analysis of the Asian soybean rust resistance pathway mediated by Rpp2 2011 24 194 206 - 107.
Singh A. K. Fu D. Q. El-Habbak M. Navarre D. Ghabrial S. Kachroo A. Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean2011 24 506 15 - 108.
Senda M. Masuta C. Ohnishi S. Goto K. Kasai A. Sano T. et al. Patterning of virus-infected Glycine max seed coat is associated with suppression of endogenous silencing of chalcone synthase genes2004 16 807 18 - 109.
Senda M. Kurauchi T. Kasai A. Ohnishi S. Suppressive mechanism of seed coat pigmentation in yellow soybean 2012 61 523 30 - 110.
Kasai A. Kasai K. Yumoto S. Senda M. Structural features of GmIRCHS, candidate of the I gene inhibiting seed coat pigmentation in soybean: implications for inducing endogenous RNA silencing of chalcone synthase genes 2007 64 467 79 - 111.
Della Vedova C. B. Lorbiecke R. Kirsch H. Schulte M. B. Scheets K. Borchert L. M. et al. The dominant inhibitory chalcone synthase allele C2-Idf (Inhibitor diffuse ) fromZea mays (L.) acts via an endogenous RNA silencing mechanism2005 170 1989 2002 - 112.
Koseki M. Goto K. Masuta C. Kanazawa A. The star-type color pattern in Petunia hybrida ’Red Star’ flowers is induced by sequence-specific degradation of chalcone synthase RNA2005 46 1879 83 - 113.
Jorgensen R. A. 1995 Cosuppression, flower color patterns, and metastable gene expression States 268 686 91 - 114.
Kusaba M. Miyahara K. Iida S. Fukuoka H. Takano T. Sassa H. et al. 2003 Low glutelin content1 : a dominant mutation that suppresses theglutelin multigene family via RNA silencing in rice15 1455 67 - 115.
Kasai M. Koseki M. Goto K. Masuta C. Ishii S. Hellens R. P. et al. 2012 Coincident sequence-specific RNA degradation of linked transgenes in the plant genome 78 259 73 - 116.
Silhavy D. Burgyán J. Effects and side-effects of viral RNA silencing suppressors on short RNAs 2004 9 76 83 - 117.
Schmutz J. Cannon S. B. Schlueter J. Ma J. Mitros T. Nelson W. et al. Genome sequence of the palaeopolyploid soybean 2010 463 178 83 - 118.
Yoshino M. Kanazawa A. Tsutsumi K. Nakamura I. Takahashi K. Shimamoto Y. Structural variation around the gene encoding the α subunit of soybean β-conglycinin and correlation with the expression of the α subunit 2002 52 285 92 - 119.
Matsumura H. Watanabe S. Harada K. Senda M. Akada S. Kawasaki S. et al. Molecular linkage mapping and phylogeny of the chalcone synthase multigene family in soybean 2005 110 1203 9 - 120.
Schlueter J. A. Scheffler B. E. Jackson S. Shoemaker R. C. Fractionation of synteny in a genomic region containing tandemly duplicated genes across Glycine max ,Medicago truncatula , andArabidopsis thaliana 2008 99 390 5 - 121.
Kong F. Liu B. Xia Z. Sato S. Kim B. M. Watanabe S. et al. Two coordinately regulated homologs of FLOWERING LOCUS T are involved in the control of photoperiodic flowering in soybean2010 154 1220 31 - 122.
Liu B. Kanazawa A. Matsumura H. Takahashi R. Harada K. Abe J. Genetic redundancy in soybean photoresponses associated with duplication of the phytochrome A gene 2008 180 995 1007 - 123.
Kanazawa A. Liu B. Kong F. Arase S. Abe J. Adaptive evolution involving gene duplication and insertion of a novel Ty1 /copia -like retrotransposon in soybean2009 69 164 75 - 124.
Voinnet O. 2008 Use, tolerance and avoidance of amplified RNA silencing by plants 13 317 28 - 125.
Miki D. Itoh R. Shimamoto K. 2005 RNA silencing of single and multiple members in a gene family of rice 138 1903 13 - 126.
Ossowski S. Schwab R. Weigel D. Gene silencing in plants using artificial microRNAs and other small RNAs 2008 53 674 90 - 127.
Zhang B. Pan X. Stellwag E. J. Identification of soybean microRNAs and their targets 2008 229 161 82 - 128.
Chen R. Hu Z. Zhang H. Identification of microRNAs in wild soybean ( Glycine soja )2009 51 1071 9 - 129.
Song Q. X. Liu Y. F. Hu X. Y. Zhang W. K. Ma B. Chen S. Y. et al. Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing 2011 11 5 - 130.
Turner M. Yu O. Subramanian S. Genome organization and characteristics of soybean microRNAs 2012 13 169 - 131.
Kanazawa A. Inaba J. Shimura H. Otagaki S. Tsukahara S. Matsuzawa A. et al. Virus-mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants 2011 65 156 68 - 132.
Kanazawa A. Inaba J. Kasai M. Shimura H. Masuta C. R. N. RNA-mediated epigenetic modifications of an endogenous gene targeted by a viral vector: A potent gene silencing system to produce a plant that does not carry a transgene but has altered traits 2011 6 1090 3 - 133.
Arase S. Kasai M. Kanazawa A. In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein2012 8 10 - 134.
Thomas C. L. Jones L. Baulcombe D. C. Maule A. J. Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector2001 25 417 25 - 135.
Otagaki S. Kawai M. Masuta C. Kanazawa A. Size and positional effects of promoter RNA segments on virus-induced RNA-directed DNA methylation and transcriptional gene silencing 2011 6 681 91 - 136.
Kasai A. Watarai M. Yumoto S. Akada S. Ishikawa R. Harada T. et al. 2004 Influence of PTGS on chalcone synthase gene family in yellow soybean seed coat 54 355 60 - 137.
Tuteja J. H. Clough S. J. Chan W. C. Vodkin L. O. 2004 Tissue-specific gene silencing mediated by a naturally occurring chalcone synthase gene cluster in Glycine max 16 819 35 - 138.
Curtin S. J. Zhang F. Sander J. D. Haun W. J. Starker C. Baltes N. J. et al. Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases 2011 156 466 73 - 139.
Arase S. Hase Y. Abe J. Kasai M. Yamada T. Kitamura K. et al. Optimization of ion-beam irradiation for mutagenesis in soybean: effects on plant growth and production of visibly altered mutants 2011 28 323 9 - 140.
Bolon Y. T. Haun W. J. Xu W. W. Grant D. Stacey M. G. Nelson R. T. et al. Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean 2011 156 240 53 - 141.
Mathieu M. Winters E. K. Kong F. Wan J. Wang S. Eckert H. et al. Establishment of a soybean ( Glycine max Merr. L) transposon-based mutagenesis repository2009 229 279 89 - 142.
Hancock C. N. Zhang F. Floyd K. Richardson A. O. Lafayette P. Tucker D. et al. The rice miniature inverted repeat transposable element mPing is an effective insertional mutagen in soybean2011 157 552 62 - 143.
Nagamatsu A. Masuta C. Matsuura H. Kitamura K. Abe J. Kanazawa A. Down-regulation of flavonoid 3’-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence 2009 166 32 9