Shigemi Seo

Jasmonate-Based Wound Signal Transduction Requires Activation of WIPK, a Tobacco Mitogen-Activated Protein Kinase

A gene encoding a tobacco mitogen-activated protein kinase (WIPK) is transcriptionally activated in response to wounding. Transgenic tobacco plants, in which expression of endogenous wipk was suppressed, did not accumulate jasmonic acid or its methyl ester when wounded, suggesting that WIPK is involved in jasmonate-mediated wound signal transduction. Here, we demonstrate that activation of WIPK is required for triggering the jasmonate-mediated signal transduction cascade that occurs when wild-type tobacco plants are wounded. We also show that when plants are wounded, WIPK is rapidly and transiently activated, whereas the quantity of WIPK protein is maintained at a constant level. A transgenic tobacco plant in which the wipk gene was constitutively expressed at a high level showed constitutive enzymatic activation of WIPK and exhibited three- to fourfold higher levels of jasmonate than did its wild-type counterpart. This plant also showed constitutive accumulation of jasmonate-inducible proteinase inhibitor II transcripts. These results show that WIPK is activated in response to wounding, which subsequently causes an increase in jasmonate synthesis.

Reduced Levels of Chloroplast FtsH Protein in Tobacco Mosaic Virus–Infected Tobacco Leaves Accelerate the Hypersensitive Reaction

In tobacco cultivars resistant to tobacco mosaic virus (TMV), infection results in the death of the infected cells accompanying the formation of necrotic lesions. To identify the genes involved in this hypersensitive reaction, we isolated the cDNA of tobacco DS9, the transcript of which decreases before the appearance of necrotic lesions. The DS9 gene encodes a chloroplastic homolog of bacterial FtsH protein, which serves to maintain quality control of some cytoplasmic and membrane proteins. A large quantity of DS9 protein was found in healthy leaves, whereas the quantity of DS9 protein in infected leaves decreased before the lesions appeared. In transgenic tobacco plants containing less and more DS9 protein than wild-type plants, the necrotic lesions induced by TMV were smaller and larger, respectively, than those on wild-type plants. These results suggest that a decrease in the level of DS9 protein in TMV-infected cells, resulting in a subsequent loss of function of the chloroplasts, accelerates the hypersensitive reaction.

Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180)

Pathogenesis-related (PR) proteins have been used as markers of plant defense responses, and are classified into 17 families. However, precise information on the majority members in specific PR families is still limited. We were interested in the individual characteristics of rice PR1 family genes, and selected 12 putatively active genes using rice genome databases for expressed genes. All were upregulated upon compatible and/or incompatible rice-blast fungus interactions; three were upregulated in the early infection period and four in the late infection period. Upon compatible rice–bacterial blight interaction, four genes were upregulated, six were not affected, and one was downregulated. These results are in striking contrast to those among 22 ArabidopsisPR1 genes where only one gene was pathogen-inducible. The responses of individual genes to salicylic acid, jasmonic acid, and ethylene induced defense signaling pathways in rice are likely to be different from those in dicot plants. Transcript levels in healthy leaves, roots, and flowers varied according to each gene. Analysis of the partially overlapping expression patterns of rice PR1 genes in healthy tissues and in response to pathogens and other stresses would be useful to understand their possible functions and for use as characteristic markers for defense-related studies in rice.

Contribution of Ethylene Biosynthesis for Resistance to Blast Fungus Infection in Young Rice Plants

The role of ethylene (ET) in resistance to infection with blast fungus (Magnaporthe grisea) in rice (Oryza sativa) is poorly understood. To study it, we quantified ET levels after inoculation, using young rice plants at the four-leaf stage of rice cv Nipponbare (wild type) and its isogenic plant (IL7), which contains the Pi-i resistance gene to blast fungus race 003. Small necrotic lesions by hypersensitive reaction (HR) were formed at 42 to 72 h postinoculation (hpi) in resistant IL7 leaves, and whitish expanding lesions at 96 hpi in susceptible wild-type leaves. Notable was the enhanced ET emission at 48 hpi accompanied by increased 1-aminocyclopropane-1-carboxylic acid (ACC) levels and highly elevated ACC oxidase (ACO) activity in IL7 leaves, whereas only an enhanced ACC increase at 96 hpi in wild-type leaves. Among six ACC synthase (ACS) and seven ACO genes found in the rice genome, OsACS2 was transiently expressed at 48 hpi in IL7 and at 96 hpi in wild type, and OsACO7 was expressed at 48 hpi in IL7. Treatment with an inhibitor for ACS, aminooxyacetic acid, suppressed enhanced ET emission at 48 hpi in IL7, resulting in expanding lesions instead of HR lesions. Exogenously supplied ACC compromised the aminooxyacetic acid-induced breakdown of resistance in IL7, and treatment with 1-methylcyclopropene and silver thiosulfate, inhibitors of ET action, did not suppress resistance. These findings suggest the importance of ET biosynthesis and, consequently, the coproduct, cyanide, for HR-accompanied resistance to blast fungus in young rice plants and the contribution of induced OsACS2 and OsACO7 gene expression to it.

Phytoalexin Accumulation in the Interaction Between Rice and the Blast Fungus

Blast fungus-induced accumulations of major rice diterpene phytoalexins (PA), momilactones A and B, and phytocassanes A through E were studied, focusing on their biosynthesis and detoxification. In resistant rice, all PA started to accumulate at 2 days postinoculation (dpi), at which hypersensitive reaction (HR)-specific small lesions became visible and increased 500- to 1,000-fold at 4 dpi, while the accumulation was delayed and several times lower in susceptible rice. Expression of PA biosynthetic genes was transiently induced at 2 dpi only in resistant plants, while it was highly induced in both plants at 4 dpi. Fungal growth was severely suppressed in resistant plants by 2 dpi but considerably increased at 3 to 4 dpi in susceptible plants. Momilactone A treatment suppressed fungal growth in planta and in vitro, and the fungus detoxified the PA in vitro. These results indicate that HR-associated rapid PA biosynthesis induces severe restriction of fungus, allowing higher PA accumulation in resistant rice, while in susceptible rice, failure of PA accumulation at the early infection stage allows fungal growth. Detoxification of PA would be a tactic of fungus to invade the host plant, and prompt induction of PA biosynthesis upon HR would be a trait of resistant rice to restrict blast fungus.

Circadian and senescence-enhanced expression of a tobacco cysteine protease gene

A cDNA clone encoding a cysteine protease was isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from degenerated primers based on the conserved sequences of plant cysteine protease genes. A putative protein encoded by the clone NTCP-23 had an amino acid sequence with significant similarities to those of plant senescence-associated cysteine proteases and mammalian cathepsin H. Northern blot analysis showed that NTCP-23 mRNA is expressed in all organs and the mRNA and protein expression is enhanced during natural senescence. We propose that NTCP-23 is responsible for amino acid remobilization especially in senescencing leaves. Furthermore, it was found that the mRNA expression follows a circadian rhythm and is reduced by continuous darkness, wounding and hypersensitive reaction (HR). NTCP-23 is the first cysteine protease whose mRNA expression has been shown to be temporarily reduced by wounding.

An HR-Induced Tobacco Peroxidase Gene Is Responsive to Spermine, but Not to Salicylate, Methyl Jasmonate, and Ethephon

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.

Antagonistic Plant Defense System Regulated by Phytohormones Assists Interactions Among Vector Insect, Thrips and a Tospovirus

The western flower thrips (Frankliniella occidentalis) is a polyphagous herbivore that causes serious damage to many agricultural plants. In addition to causing feeding damage, it is also a vector insect that transmits tospoviruses such as Tomato spotted wilt virus (TSWV). We previously reported that thrips feeding on plants induces a jasmonate (JA)-regulated plant defense, which negatively affects both the performance and preference (i.e. host plant attractiveness) of the thrips. The antagonistic interaction between a JA-regulated plant defense and a salicylic acid (SA)-regulated plant defense is well known. Here we report that TSWV infection allows thrips to feed heavily and multiply on Arabidopsis plants. TSWV infection elevated SA contents and induced SA-regulated gene expression in the plants. On the other hand, TSWV infection decreased the level of JA-regulated gene expression induced by thrips feeding. Importantly, we also demonstrated that thrips significantly preferred TSWV-infected plants to uninfected plants. In JA-insensitive coi1-1 mutants, however, thrips did not show a preference for TSWV-infected plants. In addition, SA application to wild-type plants increased their attractiveness to thrips. Our results suggest the following mechanism: TSWV infection suppresses the anti-herbivore response in plants and attracts its vector, thrips, to virus-infected plants by exploiting the antagonistic SA-JA plant defense systems.

Cyanide, a Coproduct of Plant Hormone Ethylene Biosynthesis, Contributes to the Resistance of Rice to Blast Fungus

Rice (Oryza sativa) plants carrying the Pi-i resistance gene to blast fungus Magnaporthe oryzae restrict invaded fungus in infected tissue via hypersensitive reaction or response (HR), which is accompanied by rapid ethylene production and formation of small HR lesions. Ethylene biosynthesis has been implicated to be important for blast resistance; however, the individual roles of ethylene and cyanide, which are produced from the precursor 1-aminocyclopropane-1-carboxylic acid, remain unevaluated. In this study, we found that Pi-i-mediated resistance was compromised in transgenic rice lines, in which ethylene biosynthetic enzyme genes were silenced and then ethylene production was inhibited. The compromised resistance in transgenic lines was recovered by exogenously applying cyanide but not ethephon, an ethylene-releasing chemical in plant tissue. In a susceptible rice cultivar, treatment with cyanide or 1-aminocyclopropane-1-carboxylic acid induced the resistance to blast fungus in a dose-dependent manner, while ethephon did not have the effect. Cyanide inhibited the growth of blast fungus in vitro and in planta, and application of flavonoids, secondary metabolites that exist ubiquitously in the plant kingdom, enhanced the cyanide-induced inhibition of fungal growth. These results suggested that cyanide, whose production is triggered by HR in infected tissue, contributes to the resistance in rice plants via restriction of fungal growth.

Silencing of NtMPK4 impairs CO2-induced stomatal closure, activation of anion channels and cytosolic Ca2+signals in Nicotiana tabacum guard cells

SUMMARY Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of NtMPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of NtMPK4-silenced plants do not close in elevated atmospheric CO(2), and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO(2) activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in NtMPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca(2+) concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca(2+) than in those with steady Ca(2+) levels. Both the darkness-induced anion channel activation and Ca(2+) signals were repressed in NtMPK4-silenced guard cells. These data show that CO(2) and darkness can activate anion channels in a Ca(2+)-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca(2+) concentration. NtMPK4 plays an essential role in CO(2)- and darkness-induced activation of guard-cell anion channels, through Ca(2+)-independent as well as Ca(2+)-dependent signalling pathways.