Virus-induced gene silencing of 14-3-3 genes abrogates dark repression of nitrate reductase activity in Nicotiana benthamiana
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In order to study the effect of repression of 14-3-3 genes on actual activity of the nitrate reductase (NR) in Nicotiana benthamiana leaves, Nb14-3-3a gene was silenced by virus-induced gene silencing (VIGS) method using potato virus X (PVX). Expression of Nb14-3-3a as well as Nb14-3-3b genes was altogether repressed in the leaves of PVX-14-3a-infected plants. Furthermore, two-dimensional gel electrophoresis and immunoblot analysis with anti-14-3-3 antiserum suggested that the expressions of Nb14-3-3a and Nb14-3-3b proteins are accordingly repressed in PVX-14-3a-infected plants. It is well known that binding of 14-3-3 proteins to phosphorylated NR leads to substantial decrease in NR activity of leaves under darkness. Therefore, we studied the changes in NR activity in response to light/dark transitions in the leaves of PVX-14-3a-infected plants. NR activation state was kept at a high level under darkness in PVX-14-3a-infected plants, but not in PVX-green fluorescent protein (GFP)-infected and control plants. This result suggests that Nb14-3-3a and/or Nb14-3-3b proteins are indeed involved in the inactivation of NR activity under darkness in N. benthamiana.
Keywords14-3-3 proteins Nicotiana benthamiana Nitrate reductase Virus-induced gene silencing
We thank Dr David C. Baulcombe (Sainsbury Laboratory, John Innes Centre, Norwich, UK) for the kind gift of plasmid pPC2S and pTXS.GFP. We are grateful to Dr Carol MacKintosh and Dr Jean Harthill (University of Dundee, Dundee, UK) for the generous gift of antiserum to spinach 14-3-3 protein. AI, TB, RT and HS thank a support from “Program for Promotion of Basic Research Activities for Innovative Biosciences” (Japan).
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