title: Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata.
abstract: In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.
authors.affiliation: Equipe Rhizogenèse, UMR DIA PC, IRD (Institut de Recherche pour le Développement), Montpellier Cedex 5, France.
authors.forename: Hassen
authors.initials: H
authors.lastname: Gherbi
authors.forename: Mathish
authors.initials: M
authors.lastname: Nambiar-Veetil
authors.forename: Chonglu
authors.initials: C
authors.lastname: Zhong
authors.forename: Jessy
authors.initials: J
authors.lastname: Félix
authors.forename: Daphné
authors.initials: D
authors.lastname: Autran
authors.forename: Raphaël
authors.initials: R
authors.lastname: Girardin
authors.forename: Virginie
authors.initials: V
authors.lastname: Vaissayre
authors.forename: Florence
authors.initials: F
authors.lastname: Auguy
authors.forename: Didier
authors.initials: D
authors.lastname: Bogusz
authors.forename: Claudine
authors.initials: C
authors.lastname: Franche
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D029624:Ferns / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D040161:Frankia / Q000254:growth & development; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018517:Plant Roots / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D018520:Plant Shoots / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D049452:Green Fluorescent Proteins; D005966:Glucuronidase