Plant and Cell Physiology Advance Access originally published online on April 11, 2006
Plant and Cell Physiology 2006 47(6):726-735; doi:10.1093/pcp/pcj044
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Nitrate Reductase is Responsible for Elicitin-induced Nitric Oxide Production in Nicotiana benthamiana
Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan
* Corresponding author: E-mail, kkawakit{at}agr.nagoya-u.ac.jp; Fax, +81-52-789-5525.
Recent works have established a key role for nitric oxide (NO) in activating disease resistance in plants. Nitrate reductase (NR) is one of the enzymes that are capable of producing NO in plants. In a previous study, we reported that pathogen signals induce expression of NR genes in potato, suggesting the involvement of NR in NO production induced by pathogen signals. In this study, we cloned NR genes from Nicotiana benthamiana and investigated their involvement in NO production induced by INF1, a major elicitin secreted by Phytophthora infestans. Treatment of protoplasts prepared from N. benthamiana leaves with INF1 elevated NO production to a maximum level 1–3 h after treatment. INF1-induced NO generation was suppressed completely by an NO-specific scavenger, but partially by a nitric oxide synthase inhibitor. To investigate the involvement of NR in INF1-induced NO production, NR genes were silenced by virus-induced gene silencing. The NR-silenced plants showed yellowish leaves which resemble the characteristic of Arabidopsis NR double mutants. Silencing of NR genes significantly decreased both NO2 –-producing activity and INF1-induced NO production, indicating that NR is involved in INF1-induced NO production. In contrast, overexpression of NbNR1 encoding N. benthamiana NR by Agrobacterium-mediated transient expression elevated NO2 –-producing activity nine times over the control; however, INF1-induced NO production in protoplasts overexpressing NbNR1 was comparable with that in control protoplasts. These results suggest that NR is involved in INF1-induced NO production, and post-translational modification of NR or availability of substrate NO2 – may be a rate-limiting step of NO production by NR.
The nucleotide sequences reported in this paper have been submited to EMBL/GenBank/DDBJ under accession numbers AB238224 and AB245431 for NbNR1 and NbNR2, respectively.
1 Present address: Plant Physiology Department, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, 305-8602 Japan.
(Received January 14, 2006; Accepted March 31, 2006)
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