Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase.
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 Ca(2+). 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.
Kobayashi
Michie
M
Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Ohura
Ikuko
I
Kawakita
Kazuhito
K
Yokota
Naohiko
N
Fujiwara
Masayuki
M
Shimamoto
Ko
K
Doke
Noriyuki
N
Yoshioka
Hirofumi
H
D000595:Amino Acid Sequence; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005816:Genetic Complementation Test; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics; D012995:Solubility; D014026:Nicotiana / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D019255:NADPH Oxidases; D010940:Plant Proteins; D011494:Protein Kinases; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases