Link to home

Virus-Induced Gene Silencing (VIGS) as a Reverse Genetic Tool to Study Development of Symbiotic Root Nodules

June 2008 , Volume 21 , Number  6
Pages  720 - 727

G. D. Constantin,1 M. Grønlund,1 I. E. Johansen,1 J. Stougaard,2 and O. S. Lund1,3

1Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, University of Aarhus, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; 2Laboratory of Gene Expression, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark; 3Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

Go to article:
Accepted 13 February 2008.

Virus-induced gene silencing (VIGS) can provide a shortcut to plants with altered expression of specific genes. Here, we report that VIGS of the Nodule inception gene (Nin) can alter the nodulation phenotype and Nin gene expression in Pisum sativum. PsNin was chosen as target because of the distinct non-nodulating phenotype of nin mutants in P. sativum, Lotus japonicus, and Medicago truncatula. The vector based on Pea early browning virus (PEBV) was engineered to carry one of three nonoverlapping fragments (PsNinA, PsNinB, and PsNinC) derived from the PsNin cDNA. Vector inoculation was mediated by agroinfiltration and, 2 weeks later, a Rhizobium leguminosarum bv. viceae culture was added in order to induce root nodulation. At this time point, it was estimated that systemic silencing was established because leaves of reference plants inoculated with PEBV carrying a fragment of Phytoene desaturase displayed photo bleaching. Three weeks after Rhizobium spp. application, plants inoculated with a control vector nodulated normally, whereas nodulation was almost eliminated in plants inoculated with a vector carrying PsNinA and PsNinC. For plants inoculated with a vector carrying PsNinB, nodulation was reduced by at least 45%. Down-regulation of PsNin transcripts in plants inoculated with vectors carrying PsNin cDNA fragments was confirmed and these plants displayed a relative increase in the root/shoot ratio, as expected if nitrogen fixation had been impaired.

Additional keywords:sym35, symbiosis, Tobravirus

© 2008 The American Phytopathological Society