title: Nitrate reductase is responsible for elicitin-induced nitric oxide production in Nicotiana benthamiana.
abstract: 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 NO(2) (-)-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 NO(2) (-)-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 NO(2) (-) may be a rate-limiting step of NO production by NR.
authors.affiliation: Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Yamamoto-Katou
authors.forename: Shinpei
authors.initials: S
authors.lastname: Katou
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yoshioka
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Doke
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Kawakita
mesh_terms: D020418:Algal Proteins; D000595:Amino Acid Sequence; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D005656:Fungal Proteins / Q000494:pharmacology*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects / Q000502:physiology; D017343:Genes, Plant / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D050901:Nitrate Reductase / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011506:Proteins; D011523:Protoplasts / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012231:Rhizobium / Q000502:physiology; D014026:Nicotiana / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*
chemical_list: D020418:Algal Proteins; D018744:DNA, Plant; D005656:Fungal Proteins; D011506:Proteins; C085101:elicitin, Phytophthora; D009569:Nitric Oxide; D050901:Nitrate Reductase