Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase
Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase
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DOI：
10.1105/tpc.106.048884
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发表时间：
2007-03-01
期刊：
影响因子：
11.6
通讯作者：
Yoshioka, Hirofumi
中科院分区：
文献类型：
--
作者：
Kobayashi, Michie;Ohura, Ikuko;Yoshioka, Hirofumi
活性氧（ROS）参与植物先天免疫。NADPH氧化酶（RBOH;呼吸爆发氧化酶同源物）在氧化爆发中起中心作用，并且该蛋白质的N末端的EF-手状基序表明可能受Ca 2+调节。然而，监管机制在很大程度上是未知的。我们在马铃薯（Solanum tuberosum）St RBOHB的N末端鉴定了Ser-82和Ser-97作为潜在的磷酸化位点。抗磷酸肽抗体（pSer-82）表明，植物体内的病原菌信号使Ser-82磷酸化。我们克隆了两个马铃薯钙依赖性蛋白激酶，圣CDPK 4和圣CDPK 5，和质谱分析表明，这些CDPKs磷酸化只有Ser-82和Ser-97在N端的圣RBOHB在钙依赖的方式。异位表达的组成型活性突变体的St CDPK 5，St CDPK 5VK，激发活性氧的生产烟草本塞姆氏叶片。在N.本萨米亚纳功能的丧失通过野生型马铃薯St RBOHB的异源表达而不是突变体（S82 A/S97 A）来补充。此外，St CDPK 5VK磷酸化的St RBOHB的Ser-82在N.本萨米亚纳这些结果表明，St CDPK 5诱导St RBOHB的磷酸化并调节氧化爆发。
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.