Journal Article

Nitrate Reductase is Responsible for Elicitin-induced Nitric Oxide Production in <i>Nicotiana benthamiana</i>

Ayako Yamamoto-Katou, Shinpei Katou, Hirofumi Yoshioka, Noriyuki Doke and Kazuhito Kawakita

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 47, issue 6, pages 726-735
Published in print June 2006 | ISSN: 0032-0781
Published online June 2006 | e-ISSN: 1471-9053 | DOI:
Nitrate Reductase is Responsible for Elicitin-induced Nitric Oxide Production in Nicotiana benthamiana

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry


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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.

Keywords: Agroinfiltration; Elicitor; Gene silencing; Nicotiana benthamiana; Nitrate reductase; Nitric oxide; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide; DAF, diaminofluorescein-2; GUS, β-glucuronidase; HA, hemagglutinin; HR, hypersensitive response; l-NAME, NG-nitro-l-Arg-methyl ester; LPS, lipopolysaccharides; NO, nitric oxide; NOS, nitric oxide synthase; NR, nitrate reductase; NED, N-(1-naphthyl)-ethylenediamine; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; TRV, tobacco rattle virus; VIGS, virus-induced gene silencing

Journal Article.  7145 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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