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 2þ . 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.

Active oxygen species (AOS) are responsible for triggering defense responses in plants. Respiratory burst oxidase homologs ( rboh genes) have been implicated in AOS generation. We have isolated two rboh cDNAs, NbrbohA and NbrbohB , from Nicotiana benthamiana leaves. NbrbohA was expressed constitutively at a low level and the transcripts were increased after mechanical stress of control leaf infiltration, whereas NbrbohB was induced specifically by the protein elicitor INF1 from the potato pathogen Phytophthora infestans . We examined the function of the Nbrboh genes in AOS generation and in the hypersensitive response (HR) using virus-induced gene silencing (VIGS). VIGS indicated that both genes are required for H 2 O 2 accumulation and for resistance to Phytophthora. VIGS of Nbrboh genes also led to a reduction and delay of HR cell death caused by INF1. We further demonstrate that the induction of HR-like cell death by overexpression of a constitutively active mutant of a mitogen-activated protein kinase kinase, MEK DD , is compromised by VIGS of NbrbohB . We found that MEK DD induced NbrbohB but not NbrbohA . This work provides genetic evidence for the involvement of a mitogen-activated protein kinase cascade in the regulation of rboh genes.

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The oxidative burst has been suggested to be a primary event responsible for triggering the cascade of defense responses in various plant species against infection with avirulent pathogens or pathogen-derived elicitors. The molecular mechanisms of rapid production of active oxygen species (AOS), however, are not well known. We isolated homologs of gp91 phox, a plasma membrane protein of the neutrophil NADPH oxidase, from a potato cDNA library. Molecular cloning of the cDNA showed that there are two isogenes, designated StrbohA and StrbohB, respectively. The RNA gel blot analyses showed that StrbohA was constitutively expressed at a low level, whereas StrbohB was induced by hyphal wall components (HWC elicitor) from Phytophthora infestans in potato tubers. Treatment of potato tubers with HWC elicitor caused a rapid but weak transient accumulation of H2O2 (phase I), followed by a massive oxidative burst 6 to 9 h after treatment (phase II). Diphenylene iodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, blocked both bursts, whereas pretreatment of the protein synthesis inhibitor cycloheximide with the tuber abolished only the second burst. These results suggest that the expression of StrbohA and StrbohB contributes to phase I and II bursts, respectively. The same is true for arachidonic acid, a lipid component of P. infestans-stimulated biphasic oxidative burst, whereas an endogenous signaling molecule, salicylic acid, only induced a weak phase II burst. Both molecules induced the StrbohB expression, which is in agreement with the second burst. To characterize the signal transduction pathway leading to the oxidative burst, we examined the role of protein phosphorylation in HWC-stimulated StrbohB gene expression. K252a and staurosporine, two protein kinase inhibitors, blocked the transcript accumulation. Two inhibitors of extracellular Ca2+ movement, however, did not abolish the transcript accumulation of StrbohB, suggesting that certain calcium-independent protein kinases are involved in the process of StrbohB gene expression. Additionally, we examined a causal relationship between the oxidative burst and expression of defense genes induced by the HWC elicitor. The transcript accumulation of genes related to sesquiterpenoid phytoalexin synthesis (lubimin and rishitin) and phenylpropanoid pathway was inhibited slightly by the DPI treatment, suggesting that the oxidative burst is not essential to activate these genes. Interestingly, the concomitant presence of DPI with the elicitor resulted in an increase in lubimin accumulation and a decrease in rishitin accumulation. Because it is known that lubimin is metabolized into rishitin via oxylubimin, we propose that AOS mediates the synthesis of rishitin from lubimin.

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