Response of Fe-S cluster assembly machinery of Escherichia coli to mechanical stress in a model of amino-acid crystal fermentation.

During amino-acid crystal fermentation, mechanical stress on bacterial cells caused by crystal collision often impacts negatively on bacterial growth and amino-acid production. When Escherichia coli cells were cultivated under mechanical stress of polyvinyl chloride particles as a model of the crystal fermentation, activities of iron-sulfur (Fe-S) cluster-containing enzymes were apparently decreased. Based on an assumption that function of Fe-S cluster assembly machinery would be elevated to recover the enzyme activities in such stressed cells, we analyzed levels of various components of Fe-S cluster assembly machinery by western blotting. It was found that the expression of HscA, a chaperon component of the machinery, was up-regulated and that shorter forms of HscA with the N-terminal region truncated were accumulated, suggesting an important role of HscA against the mechanical stress. An overexpression of HscA gene in E. coli cells gave a positive effect on rescue of the stress-induced decrease of the activity of Fe-S cluster-containing enzyme. These results may provide a new strategy to alleviate the mechanical stress during the amino-acid crystal fermentation.

Analysis on blast fungus-responsive characters of a flavonoid phytoalexin sakuranetin; accumulation in infected rice leaves, antifungal activity and detoxification by fungus.

To understand the role of the rice flavonoid phytoalexin (PA) sakuranetin for blast resistance, the fungus-responsive characteristics were studied. Young rice leaves in a resistant line exhibited hypersensitive reaction (HR) within 3 days post inoculation (dpi) of a spore suspension, and an increase in sakuranetin was detected at 3 dpi, increasing to 4-fold at 4 dpi. In the susceptible line, increased sakuranetin was detected at 4 dpi, but not at 3 dpi, by which a large fungus mass has accumulated without HR. Induced expression of a PA biosynthesis gene OsNOMT for naringenin 7-O-methyltransferase was found before accumulation of sakuranetin in both cultivars. The antifungal activity of sakuranetin was considerably higher than that of the major rice diterpenoid PA momilactone A in vitro and in vivo under similar experimental conditions. The decrease and detoxification of sakuranetin were detected in both solid and liquid mycelium cultures, and they took place slower than those of momilactone A. Estimated local concentration of sakuranetin at HR lesions was thought to be effective for fungus restriction, while that at enlarged lesions in susceptible rice was insufficient. These results indicate possible involvement of sakuranetin in blast resistance and its specific relation to blast fungus.

Mechanical damage to Escherichia coli cells in a model of amino-acid crystal fermentation.

We investigated the mechanical damage to the Escherichia coli cell caused by polyvinyl chloride particles as a model of amino-acid crystal fermentation. Our results indicated that the glucose-consumption rate and the intracellular ATP concentration temporarily increased by the mechanical damage, and decreased after considerable damage had occurred on cell membrane.

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.

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 Arabidopsis PR1 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.

Involvement of EIN3 homologues in basic PR gene expression and flower development in tobacco plants.

The TEIL (Tobacco EIN3-Like) gene is a tobacco homologue of arabidopsis Ethylene Insensitive 3 (EIN3), and the gene product binds an 8 bp sequence in the tobacco PR1a promoter in a sequence specific manner. It was found here that accumulation of TEIL transcript was induced by wounding and preceded basic PR gene expression. To study the downstream signalling pathway of TEIL, TEIL was overexpressed under the control of the constitutive 35S promoter in tobacco plants. In 35S::TEIL lines, basic PR genes, which are wound-, jasmonate-, and ethylene-inducible, were expressed constitutively. Next, the conserved 781 bp sequence among tobacco EIN3-like (EIL) protein genes was introduced as an inverted-repeat (IR) into tobacco to suppress expression of these genes. In two independent IRTEIL lines, the TEIL transcript was not found and transcripts of other tobacco EILs, NtEIL3, and NtEIL5, were reduced. In IRTEIL plants, wound-, jasmonate-, and ACC-induced accumulation of basic PR gene transcripts was significantly inhibited. These results indicate that TEIL functions upstream of tobacco basic PR genes in wound signalling via not only ethylene but also jasmonate. In 35S::TEIL plants, the pistil length of the flower was longer with a slight protrusion of the stigma compared with the control. In IRTEIL plants, the length of the stamens was shorter than the control with significant protrusion of the stigma in the flower. These observations indicate the involvement of tobacco EILs in flower development.

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants.

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.

Chemically induced expression of rice OSB2 under the control of the OsPR1.1 promoter confers increased anthocyanin accumulation in transgenic rice.

Anthocyanin pigmentation provides an excellent system with which to study the regulation of gene expression in higher plants. In this study, OsPR1.1 promoter was isolated and the promoter activity was monitored using a reporter gene OSB2, which encodes a transcription factor for anthocyanin synthesis in rice plants. We introduced PR::OSB2 plasmid into an isogenic Taichung 65, no. 99-962 T-65 CBA B9F5 (T65 CBA), rice line (Oryza sativa L.) and found that the transgenic rice plants exhibited anthocyanin accumulation by the induced expression of OSB2 after chemical treatments with methyl jasmonate (MeJA) and 2,6-dichloroisonicotinic acid (DCINA). The shoots of the PR::OSB2 transgenic rice plants changed color to red after application of the chemicals accompanying with the increased anthocyanin content to approximately 5-fold by MeJA and 2-fold by DCINA, respectively. The anthocyanin accumulation was consistent with the increase of the expression of OSB2 and anthocyanidin synthase (ANS). This color change system could provide a useful and easy way to produce transgenic plants for monitoring of chemicals in the environment.

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.

Ten rice peroxidases redundantly respond to multiple stresses including infection with rice blast fungus.

Class III plant peroxidases are believed to function in diverse physiological processes including disease resistance and wound response, but predicted low substrate specificities and the presence of 70 or more isoforms have made it difficult to define a specific physiological function(s) for each gene. To select pathogen-responsive POX genes, we analyzed the expression profiles of 22 rice POX genes after infection with rice blast fungus. The expression of 10 POX genes among the 22 genes was induced after fungal inoculation in both compatible and incompatible hosts. Seven of the 10 POX genes were expressed at higher levels in the incompatible host than in the compatible host 6-24 h after inoculation by which time no fungus-induced lesions have appeared. Organ-specific expression and stress-induced expression by wounding and treatment with probenazole, an agrichemical against blast fungus, jasmonic acid, salicylic acid and 1-aminocyclopropane-1-carboxylate, a precursor of ethylene, indicated that rice POXs have individual characteristics and can be classified into several types. A comparison of the amino acid sequences of POXs showed that multiple isoforms with a high sequence similarity respond to stress in different or similar ways. Such redundant responses of POX genes may guarantee POX activities that are necessary for self-defense in plant tissues against environmental stresses including pathogen infection.

The Nudix hydrolase Ndx1 from Thermus thermophilus HB8 is a diadenosine hexaphosphate hydrolase with a novel activity.

The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins. Ndx1 was overexpressed in Escherichia coli and purified. Ndx1 was stable up to 95 degrees C and at extreme pH. Size exclusion chromatography indicated that Ndx1 was monomeric in solution. Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always generated ATP as the product. Diadenosine hexaphosphate (Ap(6)A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap(6)A, with a K(m) of 1.4 microm and a k(cat) of 4.1 s(-1). These results indicate that Ndx1 is a (di)adenosine polyphosphate hydrolase. Ndx1 activity required the presence of the divalent cations Mn(2+), Mg(2+), Zn(2+), and Co(2+), whereas Ca(2+), Ni(2+), and Cu(2+) were not able to activate Ndx1. Fluoride ion inhibited Ndx1 activity via a non-competitive mechanism. Optimal activity for Ap(6)A was observed at around pH 8.0 and about 70 degrees C. We found two important residues with pK(a) values of 6.1 and 9.6 in the free enzyme and pK(a) values of 7.9 and 10.0 in the substrate-enzyme complex. Kinetic studies of proteins with amino acid substitutions suggested that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis. Trp-26 was likely involved in enzyme-substrate interactions based on fluorescence measurements. Based on these results, the mechanism of substrate recognition and catalysis are discussed.

Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants overproducing an oat cell-wall-bound thionin.

Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field. The thionins purified from seed and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens; however, we found that no endogenous rice thionin genes alone are enough for resistance to two major seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and B. glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria. Thus, we isolated thionin genes from oat, one of which was overexpressed in rice. When wild-type rice seed were germinated with these bacteria, all seedlings were wilted with severe blight. In the seedling infected with B. plantarii, bacterial staining was intensively marked around stomata and intercellular spaces. However, transgenic rice seedlings accumulating a high level of oat thionin in cell walls grew almost normally with bacterial staining only on the surface of stomata. These results indicate that the oat thionin effectively works in rice plants against bacterial attack.

Release from post-transcriptional gene silencing by cell proliferation in transgenic tobacco plants: possible mechanism for noninheritance of the silencing.

Transgenic tobacco plants that overproduce luciferase (Luc) frequently exhibit post-transcriptional gene silencing (PTGS) of luc. The silencing was observed over five generations and found not to be inherited but acquired by the next generation at a certain frequency. Luc imaging analysis of silenced plants revealed Luc activity only in proliferating tissues such as shoot meristem and developing flower. The luc gene expression has been recovered from silencing before development of germ cells, excluding a possible recovery from the PTGS at meiosis. A systemic silencing signal transferred from older tissue likely induces gene silencing of younger tissues in which cell proliferation has been completed. Only seeds maintained Luc activity, probably because of isolation from the silencing signal by a possible partition from the parent placenta. Calli newly induced from the leaf pieces of silenced plants recovered from the silencing and exhibited strong Luc activity similar to nonsilenced leaves, further indicating that the silencing cannot be maintained in proliferating cells. Thus release from PTGS in proliferating cells is a possible mechanism for noninheritance of silencing.