Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata.
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.
Gherbi
Hassen
H
Equipe Rhizogenèse, UMR DIA PC, IRD (Institut de Recherche pour le Développement), Montpellier Cedex 5, France.
Nambiar-Veetil
Mathish
M
Zhong
Chonglu
C
Félix
Jessy
J
Autran
Daphné
D
Girardin
Raphaël
R
Vaissayre
Virginie
V
Auguy
Florence
F
Bogusz
Didier
D
Franche
Claudine
C
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
D049452:Green Fluorescent Proteins; D005966:Glucuronidase