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Post-Transcriptional Gene Silencing in the Root System of the Actinorhizal Tree Allocasuarina verticillata

May 2008 , Volume 21 , Number  5
Pages  518 - 524

Hassen Gherbi,1 Mathish Nambiar-Veetil,1,2 Chonglu Zhong,1,3 Jessy Félix,1 Daphné Autran,1 Raphaël Girardin,1 Virginie Vaissayre,1 Florence Auguy,1 Didier Bogusz,1 and Claudine Franche1

1Equipe Rhizogenèse, UMR DIA PC, IRD (Institut de Recherche pour le Développement), 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France; 2Plant Biotechnology Division, Institute of Forest Genetics and Tree Breeding, Forest Campus, R. S. Puram, Coimbatore 641 002 India; 3Research Institute of Tropical Forestry, Chinese Academy of Forestry, Longdong, Guangzhou 510520, China

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Accepted 27 January 2008.

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 β-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.

Additional keywords:actinorhizal symbiosis, Frankia.

The American Phytopathological Society, 2008