Noriyuki Doke

Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components

Abstract Potato tuber tissue discs, which were aged after wounding in order to acquire hypersensitive reactivity, reduced extracellular cytochrome c and nitroblue tetrazolium (NBT) following inoculation with an incompatible, but not compatible, race of Phytophthora infestans . The cytochrome c -reducing activity rapidly increased from l to 4 h after inoculation along with an increase in the percentage of hypersensitively dead cells, and then decreased from the time when most of the penetrated cells had died. A localized activation of NBT reduction around invading hyphae of the incompatible, but not those of the compatible, race was observed at early stages of penetration before cell death. The reductive activity of the discs was also elicited by treatment with a hypersensitivity-eliciting substance, hyphal wall components of the fungus. Superoxide dismutase (SOD), an enzyme catalysing the conversion of the superoxide anion (O 2 − ) to H 2 O 2 and O 2 inhibited the enhanced reducing activity of the discs when added to the assay solution, indicating that cytochrome c and NBT may be reduced by O 2 − generated from the discs. Pre-infectional, vacuum infiltration of the discs with a solution containing SOD significantly delayed the occurrence of hypersensitive cell death caused by infection with the incompatible race as well as the accumulation of phytoalexin. Application of SH-binding reagents and NADP + , but not respiratory inhibitors, inhibited the elicitation of the reducing activity caused by infection with the incompatible race. These results indicate that an O 2 − -generating system may be activated in potato tissues during the incompatible interaction induced by invading fungi or fungal wall components, and also that the generation of O 2 − may be involved during hypersensitive cell death as a trigger of the sequence of resistance reactions.

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.

Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity

Abstract Generation of superoxide anion (O 2 − ) by potato tuber protoplasts following treatment with hyphal wall components (HWC) of Phytophthora infestans was demonstrated by the superoxide dismutase (SOD)-sensitive reduction of extracellular cytochrome c or nitroblue tetrazolium (NBT). In the presence of NADPH ability of protoplasts to reduce cytochrome c was enhanced within 2 min after adding HWC and then proceeded as a sigmoidal curve. Addition of SOD during the course of the reaction prevented further reduction of cytochrome c . In the reaction mixture without NADPH or HWC, little reducing activity was detected. A similar reduction of NBT was also found in the protoplasts treated with HWC. Water-soluble glucans from compatible races of P. infestans , which are known as suppressors of hypersensitivity, inhibited the activation of reducing activity when they were applied before elicitation of the reaction with HWC. The glucans from incompatible races caused little inhibition. Of the tested inhibitors of hypersensitive cell death, SH-binding reagent and NADP + inhibited the reducing activity whereas respiratory inhibitors, NaN 3 , antimycine A, and 2,4-dinitrophenol were ineffective. These results indicate that O 2 − generation and NADPH oxidation occur on the plasma-membrane upon incompatible recognition of host cells leading to hypersensitive cell death. Suppression of the NADPH-dependent OZ- generation may be involved in compatible relationships in the potato— P. infestans system.

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.

NADPH-dependent O2− generation in membrane fractions isolated from wounded potato tubers inoculated with Phytophthora infestans

A membrane-rich fraction from wounded potato tubers showed increasing activity of NADPH-dependent cytochrome c, but not of acetylated cytochrome c reducing activity, during ageing after slicing (wound-induced activity, WIA). In the fraction from aged tissues inoculated with an incompatible, but not a compatible, race of Phyiophthora infestans, as increase in native and acetylated cytochrome c reducing activities was closely associated with the occurrence of hypersensitive cell death and phytoalexin production (infection-induced activity, IIA). Treatment of aged tissues with hyphal wall components (HWC), a hypersensitivity-eliciting factor of the fungus, also activated both native and acetylated cytochrome c reducing activities similarly to infection. IIA and WIA were inhibited by NADP+, but only the former was appreciably inhibited by superoxide dismutase (SOD). Water-soluble glucans (WSG), a hypersensitivity-inhibiting factor from compatible, but not incompatible, races of P. infestans, significantly inhibited the IIA but not the WIA in vitro. These results suggest that a novel O2− generating NADPH oxidase system in the membrane of potato tissues may be activated following an incompatible cell reaction, which results in hypersensitive cell death and phytoalexin production. The lack of IIA activation in the compatible interaction may result from an inhibition of the reaction system by the water-soluble glucans from the fungus, thus resulting in the establishment of a compatible interaction.

Involvement of an O2− generating system in the induction of necrotic lesions on tobacco leaves infected with tobacco mosaic virus

Leaf discs of the tobacco cultivar Samsun NN [a cultivar of Nicotiana tabacum containing the N gene for resistance to tobacco mosaic virus (TMV)], inoculated with TMV and grown at 30 °C for 36 h to allow infection, formed ring-shaped necrotic lesions 6–7 h after transfer to 20–26 °C. The infected leaf discs showed a marked O2− generating activity almost immediately after transfer to 20 °C, which decreased rapidly and then underwent a cyclic oscillation three times within a further 4 h incubation at 20 °C. The net activity detected for the initial 10 min after transfer to 20 °C was significantly correlated with the number of necrotic lesions which appeared later. Leaves of non-infected Samsun NN, and those of TMV-infected and non-infected Samsun (a cultivar without the N gene) showed little O2− generating activity after transfer from 30 to 20 °C. The accumulation of formazan at the sites of subsequent lesion development in TMV-infected Samsun leaf discs, infiltrated with nitroblue tetrazolium (NBT) at the beginning of the 20 °C incubation, suggested that O2− generation may be activated in sites of TMV-multiplication. A membrane-rich fraction isolated from TMV-infected Samsun NN leaf discs incubated at 30 °C showed an enhanced O2− generating activity dependent on NADPH and Ca2+ at 20–26 °C, but not at temperatures higher than 28 °C. Infiltration of TMV-infected Samsun NN leaves with NADP+, SOD or catalase caused a reduction in necrotic lesion formation following transfer from 30 to 20 °C. These results indicate that the activation of an NADPH-dependent O2− generating reaction in leaves of TMV-infected tobacco cultivars carrying the N gene may be involved in the induction of the necrotic lesions caused by virus infection.

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.

Effect of hyphal wall components from Phytophthora infestans on protoplasts of potato tuber tissues

Abstract Effect of hyphal wall components of various races of Phytophthora infestans on protoplasts prepared from tuber tissues of potato cultivars having various resistance genes to the pathogen was investigated to know the interactions with surfaces of host cell membrane. Reaction of protoplasts to the components was characterized by rapid protoplasmic aggregation followed by exolysis of the aggregate leaving a ghost of spherical membrane behind. There was no significant difference in the physiological activities of the components from 7 races of the fungus. Significant differences in the reactivity of protoplasts from different cultivars used was found regardless of the resistance genes of the cultivars. There was a linear correlation between % of reacted protoplasts and concentration of fungal components (expressed as log) used for the treatments in each reaction system. The higher the field resistance of the cultivars used, the more reactive to components were the protoplasts prepared from them. Nearly 1 μg ml−1 of components was enough to initiate the reaction of protoplasts and more than 125 μg ml−1 elicited the reaction at maximum rate within 5 to 10 min after application. These phenomena suggested a possibility that the recognition sites of the protoplasts for the fungal components may reside in the protoplasmic membrane, resulting in occurrence of a hypersensitive reaction in potato cells. These results are discussed in connection with the host-parasite interaction in potato late blight.

Partial characterization and aspects of the mode of action of a hypersensitivity-inhibiting factor (HIF) isolated from Phytophthora infestans☆

Abstract Factors which inhibit the hypersensitive reaction of potato tuber tissue (Kennebec, R1) to Phytophthora infestans were isolated from mycelia and zoospores of race 1234 (compatible) and race 4 (incompatible) of the fungus. They were partially characterized as glucans containing β-1→3 and β-1→6 linkages and 17 to 23 glucose units. The glucans from both mycelia and zoospores included a non-anionic glucan and an anionic glucan; one or two residues of the latter were esterified with a phosphoryl monoester. Death of host cells, browning and the accumulation of rishitin (hypersensitive reaction) in tuber slices inoculated with race 4 or treated with an elicitor from the fungus were suppressed by pretreatment of slices with the glucans. The glucans from the compatible race were more active in suppressing the hypersensitive reaction than those from the incompatible race. The anionic glucan was more active than the non-anionic glucan. Crude elicitors from races 4 and 1234 lost terpenoid-eliciting activity when mixed with a microsomal fraction prepared from potato tuber tissue. The glucans from the compatible race, but not the incompatible race, markedly reduced the loss resulting from the reaction between the crude elicitor and the microsomal fraction. The data suggest that the compatible interaction between potato tissue and P. infestans may be caused by a suppression of the hypersensitive response of the host tissue by water-soluble glucans from the fungus.

Suppression of the hypersensitive response of potato tuber protoplasts to hyphal wall components by water soluble glucans isolated from Phytophthora infestans

Abstract The suppressive effect of water soluble glucans isolated from Phytophthora infestans on the hypersensitive response of potato tuber-protoplasts was investigated qualitatively and quantitatively in relation to host-parasite specificity in potato late blight. Protoplasts were prepared from nine cultivars having different resistance genes (r, R1, R3, R4 R1, R2, R1, R3, R1, R4, R2, R3 and R2, R4), and glucans were isolated from 7 races (0, 1, 3, 4, 1.2, 1.4, and 1.2.3.4). Each glucan itself did not cause any visible effect on the protoplasts. Pretreatment of protoplasts with glucans from each race for 15 min suppressed the hypersensitive reaction of protoplasts elicited by hyphal wall components isolated from P. infestans. Greater suppressive activity of the glucans was partly characteristic of the compatible relationships between protoplasts and races used as a source of glucans. There was a linear correlation between the suppressive effect of glucans and the log concentration of glucans applied. Each glucan showed some different activity in suppressing the hypersensitive response of protoplasts among compatible cultivars. These results suggested that glucans from P. infestans may be a determinant of host-parasite specificity in potato late blight as a suppressor of the effects of hyphal wall components on elicitation of the hypersensitive reaction.