Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase (CHS) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase (F3'H) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.

[1]  Paul M Porter,et al.  Phenolic acids and flavonoids in soybean root and leaf extracts , 1986 .

[2]  R. Koes,et al.  Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. , 2005, Trends in plant science.

[3]  K. Osakabe,et al.  Molecular characterization of true and ectopic gene targeting events at the acetolactate synthase gene in Arabidopsis. , 2006, Plant & cell physiology.

[4]  V. Sundaresan,et al.  Analysis of Flanking Sequences from Dissociation Insertion Lines: A Database for Reverse Genetics in Arabidopsis , 1999, Plant Cell.

[5]  D. Baulcombe,et al.  Gene Silencing without DNA: RNA-Mediated Cross-Protection between Viruses , 1999, Plant Cell.

[6]  H. Tanaka,et al.  Broad resistance to plant viruses in transgenic plants conferred by antisense inhibition of a host gene essential in S-adenosylmethionine-dependent transmethylation reactions. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A. Kinney,et al.  Genetic Modification Removes an Immunodominant Allergen from Soybean1,212 , 2003, Plant Physiology.

[8]  Yuichiro Watanabe,et al.  Use of an Attenuated Strain of Tobamovirus for Early Detection of Virus-Induced Gene Silencing , 2004 .

[9]  D. Choi,et al.  A method of high frequency virus-induced gene silencing in chili pepper (Capsicum annuum L. cv. Bukang). , 2004, Molecules and cells.

[10]  A. Kikuchi,et al.  Low Isoflavone Content in Some Early Maturing Cultivars, So-called “Summer-type Soybeans” (Glycine max (L) MERRILL). , 1991 .

[11]  Kazuo Tsugane,et al.  Efficient gene targeting by homologous recombination in rice , 2002, Nature Biotechnology.

[12]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[13]  M. Senda,et al.  Patterning of Virus-Infected Glycine max Seed Coat Is Associated with Suppression of Endogenous Silencing of Chalcone Synthase Genes , 2004, The Plant Cell Online.

[14]  A. Kinney,et al.  Cosuppression of the α Subunits of β-Conglycinin in Transgenic Soybean Seeds Induces the Formation of Endoplasmic Reticulum–Derived Protein Bodies , 2001, Plant Cell.

[15]  Akira Kanazawa,et al.  The star-type color pattern in Petunia hybrida 'red Star' flowers is induced by sequence-specific degradation of chalcone synthase RNA. , 2005, Plant & cell physiology.

[16]  J. Amaya-Farfan,et al.  RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content , 2006, Planta.

[17]  C. M. Woodworth.  Inheritance of Cotyledon, Seed-Coat, Hilum and Pubescence Colors in Soy-Beans. , 1921, Genetics.

[18]  K. Oparka,et al.  Efficient Virus-Induced Gene Silencing in Roots Using a Modified Tobacco Rattle Virus Vector1[w] , 2004, Plant Physiology.

[19]  M. Sussman,et al.  T-DNA as an Insertional Mutagen in Arabidopsis , 1999, Plant Cell.

[20]  M. Gijzen,et al.  Isoflavonoid biosynthesis and accumulation in developing soybean seeds , 2003, Plant Molecular Biology.

[21]  S. Volrath,et al.  Gene targeting in Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[22]  D. Robertson,et al.  Geminivirus-based vectors for gene silencing in Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[23]  Concetta Vazzana,et al.  Polyphenolic content in different plant parts of soy cultivars grown under natural conditions. , 2003, Journal of agricultural and food chemistry.

[24]  S. Ghabrial,et al.  Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean. , 2006, Virology.

[25]  S. Clough,et al.  Tissue-Specific Gene Silencing Mediated by a Naturally Occurring Chalcone Synthase Gene Cluster in Glycine max , 2004, The Plant Cell Online.

[26]  O. Lund,et al.  Virus-induced gene silencing as a tool for functional genomics in a legume species. , 2004, The Plant journal : for cell and molecular biology.

[27]  M. Senda,et al.  Influence of PTGS on Chalcone Synthase Gene Family in Yellow Soybean Seed Coat , 2004 .

[28]  Shuichi Asanuma,et al.  Association of T Gene with Chilling Tolerance in Soybean , 1996 .

[29]  D. Baulcombe,et al.  High throughput virus‐induced gene silencing implicates heat shock protein 90 in plant disease resistance , 2003, The EMBO journal.

[30]  Zhong Zheng,et al.  Development of a Virus-Induced Gene-Silencing System for Functional Analysis of the RPS2-Dependent Resistance Signalling Pathways in Arabidopsis , 2006, Plant Molecular Biology.

[31]  J. Abe,et al.  Identification and Characterization of Wild Soybean (Glycine soja Sieb. et Zecc.) Strains with High Lutein Content , 2006 .

[32]  Y. Takahata,et al.  Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus , 2006, Plant Cell Reports.

[33]  David C Baulcombe,et al.  Virus-induced gene silencing in plants. , 2003, Methods.

[34]  Makoto Kusaba,et al.  A simple and rapid method to detect plant siRNAs using nonradioactive probes , 2003, Plant Molecular Biology Reporter.

[35]  C. Maxwell,et al.  Metabolic engineering to increase isoflavone biosynthesis in soybean seed. , 2003, Phytochemistry.

[36]  C. Masuta,et al.  Infection of soybean by cucumber mosaic virus as determined by viral movement protein , 2007, Archives of Virology.

[37]  G. Hartman,et al.  Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase , 2007, Planta.

[38]  W. Stiekema,et al.  A Two-Component Enhancer-Inhibitor Transposon Mutagenesis System for Functional Analysis of the Arabidopsis Genome , 1999, Plant Cell.

[39]  C. Masuta,et al.  Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system. , 1991, Virology.

[40]  T. Nishio,et al.  Low glutelin content1: A Dominant Mutation That Suppresses the Glutelin Multigene Family via RNA Silencing in Rice Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.011452. , 2003, The Plant Cell Online.

[41]  O. Yu,et al.  RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae1 , 2005, Plant Physiology.

[42]  Ralf Reski,et al.  Development, Genetics and Molecular Biology of Mosses , 1998 .

[43]  M. Kumagai,et al.  Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[44]  S. Dinesh-Kumar,et al.  Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. , 2002, The Plant journal : for cell and molecular biology.

[45]  M. Senda,et al.  Sequence divergence at chalcone synthase gene in pigmented seed coat soybean mutants of the Inhibitor locus. , 2002, Genes & genetic systems.

[46]  Peter M. Waterhouse,et al.  Exploring plant genomes by RNA-induced gene silencing , 2003, Nature Reviews Genetics.

[47]  Kyuya Harada,et al.  A single-base deletion in soybean flavonoid 3′-hydroxylase gene is associated with gray pubescence color , 2002, Plant Molecular Biology.

[48]  M. Metzlaff,et al.  SVISS - a novel transient gene silencing system for gene function discovery and validation in tobacco plants. , 2002, The Plant journal : for cell and molecular biology.

[49]  Jonathan D. G. Jones,et al.  Virus-induced gene silencing in Solanum species. , 2004, The Plant journal : for cell and molecular biology.

[50]  S. Holzberg,et al.  Barley stripe mosaic virus-induced gene silencing in a monocot plant. , 2002, The Plant journal : for cell and molecular biology.

[51]  Yuan-yuan Ma,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, Plant Molecular Biology.

[52]  L. Hileman,et al.  Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy). , 2005, The Plant journal : for cell and molecular biology.

[53]  K. Mysore,et al.  Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species. , 2004, The Plant journal : for cell and molecular biology.

[54]  L. Vodkin,et al.  Cloning of the pleiotropic T locus in soybean and two recessive alleles that differentially affect structure and expression of the encoded flavonoid 3' hydroxylase. , 2003, Genetics.

[55]  C. Masuta,et al.  Development of Cucumber mosaic virus as a vector modifiable for different host species to produce therapeutic proteins , 2006, Planta.

[56]  S. Dinesh-Kumar,et al.  Virus-induced gene silencing in tomato. , 2002, The Plant journal : for cell and molecular biology.

[57]  D. Treutter.  Significance of Flavonoids in Plant Resistance and Enhancement of Their Biosynthesis , 2005, Plant biology.

[58]  C. Napoli,et al.  Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. , 1990, The Plant cell.

[59]  G. Martin,et al.  Applications and advantages of virus-induced gene silencing for gene function studies in plants. , 2004, The Plant journal : for cell and molecular biology.

[60]  L. Maquat.  Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics , 2004, Nature Reviews Molecular Cell Biology.

[61]  Peter M Waterhouse,et al.  RNA silencing platforms in plants , 2005, FEBS letters.

[62]  Makoto Arai,et al.  Rapid induction of transcriptional and post-transcriptional gene silencing using a novel Cucumber mosaic virus vector , 2006 .

[63]  J. Ross,et al.  Dietary flavonoids: bioavailability, metabolic effects, and safety. , 2002, Annual review of nutrition.

[64]  D. Baulcombe.  RNA silencing in plants , 2004, Nature.

[65]  L. Hanley-Bowdoin,et al.  Silencing of a meristematic gene using geminivirus-derived vectors. , 2001, The Plant journal : for cell and molecular biology.

[66]  W. F. Thompson,et al.  Gene silencing from plant DNA carried by a Geminivirus. , 1998, The Plant journal : for cell and molecular biology.

[67]  Olivier Voinnet,et al.  Initiation and Maintenance of Virus-Induced Gene Silencing , 1998, Plant Cell.

[68]  Randy C. Shoemaker,et al.  National Science Foundation-Sponsored Workshop Report. Draft Plan for Soybean Genomics1 , 2004, Plant Physiology.

[69]  T. Mengiste,et al.  Prospects for the Precise Engineering of Plant Genomes by Homologous Recombination , 1999, Biological chemistry.