Kazuhito Kawakita

Nicotiana benthamiana gp91phox Homologs NbrbohA and NbrbohB Participate in H2O2 Accumulation and Resistance to Phytophthora infestans

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 H2O2 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, MEKDD, is compromised by VIGS of NbrbohB. We found that MEKDD 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.

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 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.

The oxidative burst protects plants against pathogen attack: Mechanism and role as an emergency signal for plant bio-defence — a review ☆

Various aspects, mechanisms and functions of the oxidative burst with generation of O2- superoxide anions in plant cells, which is stimulated by active defence-inducing agents such as fungal infection or elicitor treatment, were reviewed mainly on the basis of experimental evidence obtained in a system of Solanaceae plants and Phytophthora spp. The oxidative burst may be due to an O(2-)generating NADPH oxidase in the plasma membrane, which is activated with combinations of cytosolic proteins, Ca2+, calmodulin and protein kinase, following stimulation by elicitor molecules. The oxidative burst may play the role of an internal emergency signal for induction of the metabolic cascade for active defence.

Induction of Plant gp91 phox Homolog by Fungal cell wall, arachidonic acid, and salicylic acid in potato

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.

Rewiring Mitogen-Activated Protein Kinase Cascade by Positive Feedback Confers Potato Blight Resistance

Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.

Nitrate reductase, a nitric oxide-producing enzyme: induction by pathogen signals

Nitric oxide (NO) is believed to act as an effector for defense signaling in plant cells. Nitrate reductase (NR) is one of the NO-producing enzymes in plants. Here, we report that infection of Phytophthora infestans, the fungal pathogen of potato late blight, into potato tubers caused a transient increase in the NR transcript in an incompatible, but not a compatible, interaction. Treatment of potato tubers with the fungal elicitor hyphal wall components (HWC) from the fungus induced NR gene at the transcriptional and protein level. Soluble proteins from HWC-treated tubers exhibited enhanced nitrite-dependent NO production. The inhibitor experiments for protein kinase and extracellular Ca2+ on the HWC-induced accumulation of the NR transcript suggested involvement of a calcium-independent protein kinase in regulation of the NR gene. Additionally, we isolated cDNA clones encoding NR from a potato tuber cDNA library; two isogenes were designated StNR5 and StNR6, respectively. Reverse transcription-polymerase chain reaction analyses using gene-specific primers showed that transcripts for both StNR5 and StNR6 were induced by HWC in potato tubers. This is the first report that pathogen signals induce expression of the NR gene.

Proteomic analysis of S-nitrosylated proteins in potato plant.

Nitric oxide (NO) has various functions in physiological responses in plants, such as development, hormone signaling and defense. The mechanism of how NO regulates physiological responses has not been well understood. Protein S-nitrosylation, a redox-related modification of cysteine thiol by NO, is known to be one of the important post-translational modifications to regulate activity and interactions of proteins. To elucidate NO function in plants, proteomic analysis of S-nitrosylated proteins in potato (Solanum tuberosum) was performed. Detection and functional analysis of internal S-nitrosylated proteins is technically demanding because of the instability and reversibility of the protein S-nitrosylation. By using a modified biotin switch assay optimized for potato tissues, and nano liquid chromatography combined with mass spectrometry, approximately 80 S-nitrosylated candidate proteins were identified in S-nitrosoglutathione-treated potato leaves and tuber extracts. Identified proteins included redox-related enzymes, defense-related proteins and metabolic enzymes. Some of identified proteins were synthesized in Escherichia coli, and S-nitrosylation of recombinant proteins was confirmed in vitro. Dehydroascorbate reductase 1 (DHAR1, EC 1.8.5.1), one of the identified S-nitrosylated target proteins, showed glutathione-dependent dehydroascorbate-reducing activity. Either point mutation in a target cysteine of S-nitrosylation or treatment with an NO donor, S-nitroso-L-cysteine, significantly reduced the activity of DHAR1, indicating that DHAR1 is negatively regulated by S-nitrosylation of the cysteine residue essential for the enzymatic activity. These results show that the modified method developed in this study can be used to identify proteins regulated by S-nitrosylation in potato tissues.

Involvement of PPS3 Phosphorylated by Elicitor-Responsive Mitogen-Activated Protein Kinases in the Regulation of Plant Cell Death

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant innate immunity. Overexpression of StMEK1DD, a constitutively active MAPK kinase that activates salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), provokes hypersensitive response-like cell death in Nicotiana benthamiana. Here we purified a 51-kD MAPK, which was activated in potato (Solanum tuberosum) tubers treated with hyphal wall elicitor of a plant pathogen, and isolated the cDNA designated StMPK1. The deduced amino acid sequence of the StMPK1 showed strong similarity to stress-responsive MAPKs, such as tobacco (Nicotiana tabacum) SIPK and Arabidopsis (Arabidopsis thaliana) AtMPK6. To investigate the downstream signaling of StMPK1, we identified several proteins phosphorylated by StMPK1 (PPSs) using an in vitro expression cloning method. To dissect the biological function of PPSs in the plant defense, we employed virus-induced gene silencing (VIGS) in N. benthamiana. VIGS of NbPPS3 significantly delayed cell death induced by the transient expression of StMEK1DD and treatment with hyphal wall elicitor. Furthermore, the mobility shift of NbPPS3 on SDS-polyacrylamide gel was induced by transient expression of StMEK1DD. The mobility shift of NbPPS3 induced by StMEK1DD was not compromised by VIGS of WIPK or SIPK alone, but drastically reduced by the silencing of both WIPK and SIPK. This work strongly supports the idea that PPS3 is a physiological substrate of StMPK1 and is involved in cell death activated by a MAPK cascade.

Age-Related Resistance of Nicotiana benthamiana Against Hemibiotrophic Pathogen Phytophthora infestans Requires Both Ethylene- and Salicylic Acid–Mediated Signaling Pathways

Phytophthora infestans, the agent of late blight disease of potato, is a hemibiotrophic pathogen with biotrophic action during early infection and necrotrophic in the later stage of colonization. Mature Nicotiana benthamiana was resistant to P. infestans, whereas relatively young plants were susceptible to this pathogen. Young plants became resistant following a pretreatment with acibenzolar-S-methyl, a functional analog of salicylic acid (SA), indicating that susceptibility of young plants is due to a lack of induction of SA signaling. Further analysis with virus-induced gene silencing indicated that NbICS1 and NbEIN2, the genes for SA biosynthesis and ethylene (ET) signaling, respectively, are required for the resistance of mature N. benthamiana against P. infestans. Furthermore, these genes are required for the production of reactive oxygen species (ROS) induced by treatment of the INF1 elicitor. In NbICS1-silenced plants, cell death induced by either INF1 or necrosis-inducing protein NPP1.1 was significantly accelerated. Expression of genes for phytoalexin (capsidiol) biosynthesis, NbEAS and NbEAH, were regulated by ET, and gene silencing of either of them compromised resistance of N. benthamiana to P. infestans. Together, these results suggest that resistance of N. benthamiana against hemibiotrophic P. infestans requires both SA-regulated appropriate induction of cell death and ET-induced production of phytoalexin.

Elicitation of primary and secondary metabolism during defense in the potato

Exposure of potato tuber tissues to Phytophthora infestans, the late blight pathogen, elicits multiple defense responses, including an oxidative burst, accumulation of phenylpropanoid compounds, and sesquiterpenoid phytoalexins (lubimin and rishitin), which are accompanied by de novo synthesis of the enzymes that produce them. These reactions are also induced in potato tubers treated with a crude elicitor (hyphal wall components, or HWC) prepared from the mycelia of the pathogen. Differential hybridization was used to identify elicitor-responsive cDNAs from potato tubers. Database searches revealed that these genes appear to encode tyramine hydroxycinnamoyl transferase, arginine decarboxylase, glucose 6-phosphate dehydrogenase, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, NADP-dependent malic enzyme, pyruvate decarboxylase, and other potential defense components. RNA gel blot analyses indicated that the accumulation of transcripts related to both secondary and primary metabolism was induced by the HWC elicitor, arachidonic acid or salicylic acid. This suggests that the signals from the pathogen activate not only secondary metabolism but also the primary metabolism that supports the rapid expression of defense responses in potato tubers.