Calcium-Dependent Protein Kinases Regulate the Production of Reactive Oxygen Species by Potato NADPH Oxidase

• Published in: The Plant cell 2007-03, Vol.19 (3), p.1065-1080
• Main Authors: Kobayashi, Michie, Ohura, Ikuko, Kawakita, Kazuhito, Yokota, Naohiko, Fujiwara, Masayuki, Shimamoto, Ko, Doke, Noriyuki, Yoshioka, Hirofumi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Antibodies; Calcium; Cell membranes; Chemical suspensions; Gels; Gene Expression Regulation, Plant; Gene Silencing; Genetic Complementation Test; Mass Spectrometry; Models, Biological; Molecular Sequence Data; NADPH Oxidases - metabolism; Nicotiana - enzymology; Nicotiana benthamiana; Oxidases; Phosphorylation; Plant cells; Plant Leaves - metabolism; Plant Proteins - metabolism; Potatoes; Protein Kinases - chemistry; Protein Kinases - genetics; Protein Kinases - metabolism; Proteins; Reactive Oxygen Species - metabolism; Recombinant Proteins - metabolism; Sequence Deletion; Serine - metabolism; Signal Transduction; Solanum tuberosum; Solanum tuberosum - enzymology; Solanum tuberosum - genetics; Solubility; Tubers; Vegetables
• ISSN: 1040-4651; 1532-298X; 1532-298X
• DOI: 10.1105/tpc.106.048884

Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca²⁺. However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.