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Accepted: 2012-01-25, 資料番号: SA1004224000

We found that the whole plant-specific resistance (WPSR), which was expressed in the intact leaf sheaths of susceptible rice plants, was suppressed by low temperature or abscisic acid (ABA) treatment. In this study, quantitative changes in protein expression characteristic to WPSR reaction were analyzed in susceptible rice plants, ZTS, with or without ABA treatment, against the challenge of a M. oryzae isolate Hoku1. In addition, these results were compared with those of the R gene-mediated hypersensitive response (HR) in a resistant cultivar, ZTR. Proteins extracted from leaf sheaths were separated with two-dimensional polyacrylamide gel electrophoresis, and 13 proteins were changed by fungal inoculation in WPSR and R-gene-mediated resistance, but not in plants in which WPSR was suppressed by ABA. Partial amino acid sequencing of these proteins revealed that they contained - 1,3-glucanase, a thaumatin-like protein precursor (a member of the pathogenesis-related protein [PR-] group 5), and the root-specific PR-10. In transgenic ZTS plants expressing anti-sense RNA of the thaumatin-like protein, WPSR was significantly suppressed, suggesting that the induction of the thaumatin-like protein after the fungal inoculation is associated with the expression of WPSR in the susceptible rice plant.

A large number of cometary dust particles were captured with low-density silica aerogels by NASA’s Stardust Mission. Knowledge of the details of the capture mechanism of hypervelocity particles in silica aerogel is needed in order to correctly derive the original particle features from impact tracks. However, the mechanism has not been fully understood yet. We shot hard spherical projectiles of several different materials into silica aerogel of density 60 mg cm −3 and observed their penetration processes using an image converter or a high-speed video camera. In order to observe the deceleration of projectiles clearly, we carried out impact experiments at two velocity ranges; ∼4 km s −1 and ∼200 m s −1 . From the movies we took, it was indicated that the projectiles were decelerated by hydrodynamic force which was proportional to v 2 ( v : projectile velocity) during the faster penetration process (∼4 km s −1 ) and they were merely overcoming the aerogel crushing strength during the slower penetration process (∼200 m s −1 ). We applied these deceleration mechanisms for whole capture process to calculate the track length. Our model well explains the track length in the experimental data set by Burchell et al. (Burchell, M.J., Creighton, J.A., Cole, M.J., Mann, J., Kearsley, A.T. [2001]. Meteorit. Planet. Sci. 36, 209–221).

Since preliminary studies demonstrated unsuitability of the existing Japanese standardized methods for evaluating the termite-resistance of plastics, a new laboratory method was tested for its applicability. Assembled units, each consisting of a plastic material and a wood attachment surrounding a plastic sample, were placed around a laboratory nest of Coptotermes formosanus for 6 weeks at 28 ± 2 °C and over 80% relative humidity, and the termite attack was assessed visually. Amorphous polyamide performed best, and low-density polyethylene did worst. This new laboratory method succeeded in demonstrating termite damage to materials that are susceptible under actual service conditions, although it is not applicable to evaluating insecticide-incorporated plastics. A modified JWPA Standard No. 17 test method was employed to determine the minimum number of termites required to attack high-density polyethylene film so that it was possible to standardize the method to compare termite-resistance of non-woody materials with or without chemical treatment. It was concluded that 300 workers of C. formosanus in 12.6 cm 2 of foraging area, which was equivalent to the termite density (pressure) of 24 workers cm −2 foraging area, were required for termites to attack from a straight scratched surface.

The C-terminal catalytic domain of tobacco N-acetylglucosaminyltransferase I fused to maltose-binding protein was produced in Escherichia coli as a soluble form with significant activity. The protein was affinity-purified using amylose resin, and its enzymatic properties were investigated, including its divalent cation requirements, optimal temperature, optimal pH, and substrate specificity.

We established a novel strategy for preparing uniformly stable isotope-labeled proteins by using suspension-cultured plant cells and an inducible virus vector encoding the research target. By using this new method, we demonstrated the expression of three proteins, namely, Escherichia coli dihydrofolate reductase (DHFR), chicken calmodulin (CaM), and porcine protein kinase C-dependent protein phosphatase-1 inhibitor with a molecular mass of 17-kDa (CPI-17). In addition, we successfully expressed bovine pancreatic trypsin inhibitor (BPTI), which contains three pairs of disulfide bonds, as the soluble form. In the most efficient case, as little as 50 ml culture yielded 3-4 mg ^N-labeled protein suitable for NMR experiments. The ^1H-^N HSQC spectra of all of these proteins clearly indicated that their structures were identical to those of their counterparts reported previously. Thus, the present results suggest that our novel protocol is a potential method for NMR sample preparation.

Day length perceived by a leaf is a major environmental factor that controls the timing of flowering. It has been believed that a mobile, long-distance signal called florigen is produced in the leaf under inductive day length conditions, and is transported to the shoot apex where it triggers floral morphogenesis. Grafting experiments have shown that florigen is transmissible from a donor plant that has been subjected to inductive day length to an uninduced recipient plant. However, the nature of florigen has long remained elusive. Arabidopsis FLOWERING LOCUS T (FT) is expressed in cotyledons and leaves in response to inductive long days (LDs). FT protein, with a basic region/leucine zipper (bZIP) transcription factor FD, acts in the shoot apex to induce target meristem identity genes such as APETALA1 (AP1) and initiates floral morphogenesis. Recent studies have provided evidence that the FT protein in Arabidopsis and corresponding proteins in other species are an important part of florigen. Our work shows that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a day or two. The sequence and structure of mRNA are not of critical importance for the long-distance action of the FT gene. These observations led to the conclusion that the FT protein, but not mRNA, is the essential component of florigen.

Resistance in the leaf blades of rice plants against a virulent race of the rice blast fungus Magnaporthe oryzae was quantitatively examined using a modified spot inoculation method. Numbers of conidia produced in the lesions were affected by plant age and paralleled the frequency of resistance infection types, which is indicative of whole-plant-specific resistance (WPSR), in the inoculated leaf sheaths of the corresponding plants. Exogenous abscisic acid treatment, which suppresses WPSR, also increased the susceptibility of the leaf blades. These results indicate a correlation between the resistance of the leaf blades and the WPSR in the leaf sheaths.

Summary Fungal plant pathogens have evolved diverse strate- gies to overcome the multilayered plant defence responses that confront them upon host invasion. Here we show that pathogenicity of the cucumber anthracnose fungus, Colletotrichum lagenarium, and the rice blast fungus, Magnaporthe grisea, requires a gene orthologous to Saccharomyces cerevisiae SSD1, a regulator of cell wall assembly. Screening for C. lagenarium insertional mutants deficient in patho- genicity led to the identification of ClaSSD1. Follow- ing targeted gene replacement, appressoria of classd1 mutants retained the potential for penetration but were unable to penetrate into host epidermal cells. Transmission electron microscopy suggested that appressorial penetration by classd1 mutants was restricted by plant cell wall-associated defence responses, which were observed less frequently with the wild-type strain. Interestingly, on non-host onion epidermis classd1 mutants induced papilla formation faster and more abundantly than the wild type. Simi- larly, colonization of rice leaves by M. grisea was severely reduced after deletion of the orthologous MgSSD1 gene and attempted infection by the mutants was accompanied by the accumulation of reactive oxygen species within the host cell. These results suggest that appropriate assembly of the fungal cell wall as regulated by SSD1 allows these pathogens to establish infection by avoiding the induction of host defence responses.

Previously, we reported on an inducible viral vector system for foreign protein production in cultured plant cells, in which the transcription of the recombinant viral vector RNA encoding a foreign protein is controlled by an estradiol-inducible promoter. In this study, we used the inducible virus vector system to test the efficiency of a modified tomato mosaic virus (ToMV) encoding the movement protein. The virus inducibly produced a foreign jellyfish green fluorescent protein encoded in the virus in transgenic tobacco BY-2 suspension-cultured cells as efficiently as modified ToMV without the movement protein. We then produced transgenic BY-2 cell lines ER–ToMV–MP–DHFR and ER–ToMV–MP–GUS, which encoded modified ToMV with Escherichia coli dihydrofolate reductase (DHFR) and β-glucuronidase (GUS), respectively. After estradiol was added to the cell culture, DHFR and GUS activity was detected in the ER–ToMV–MP–DHFR and ER–ToMV–MP–GUS cells, respectively. In contrast, no DHFR or GUS activity was detected in untreated transgenic lines. Three days after induction, DHFR accumulation accounted for up to 15% of the total soluble proteins extracted from the cells, indicating that an inducible viral vector is an effective option for efficiently producing active enzymes in cultured plant cells.

Although suspension-cultured plant cells have many potential merits as sources of useful proteins, the lack of an efficient expression system has prevented using this approach. In this study, we established an inducible tomato mosaic virus (ToMV) infection system in tobacco BY-2 suspension-cultured cells to inducibly and efficiently produce a foreign protein. In this system, a modified ToMV encoding a foreign protein as replacement of the coat protein is expressed from stably transformed cDNA under the control of an estrogen-inducible promoter in transgenic BY-2 cells. Estrogen added to the culture activates an estrogen-inducible transactivator expressed constitutively from the transgene and induces transcription and replication of viral RNA. In our experiments, accumulation of viral RNA and expression of green fluorescent protein (GFP) encoded in the virus were observed within 24 h after induction. The amount of GFP reached approximately 10% of total soluble protein 4 d after induction. In contrast, neither viral RNA nor GFP were detected in uninduced cells. The inducible virus infection system established here should be utilized not only for the expression of foreign proteins, but also for investigations into the viral replication process in cultured plant cells.

Transgenic plants that produce anti-insect substances are vital in improving crop yields and in reducing the environmental risks of chemical insecticides. Enhancin is a metalloprotease produced in occlusion bodies of the Trichoplusia ni granulovirus (TnGV). It is a key substance that enhances infection of the nucleopolyhedrovirus in lepidopteran insects. Rice (Oryza sativa L. cv. Nipponbare) protoplasts were cotransformed with pREXΦVEF and pLTHyg, which respectively bear the chimeric enhancin gene and the hygromycin-resistance gene. Hygromycin-resistant regeneration plants were examined by genomic polymerase chain reaction and genomic Southern and northern blotting analyses to confirm the presence and expression of the enhancin gene. Fourteen transgenic plant lines harboring the enhancin gene were obtained, and stable inheritance and expression of the enhancin gene were confirmed in the second, third, and fourth plant generations. Feeding Spodoptera exigua larvae leaves of enhancin-expressing rice plants in the presence of S. exigua nucleopolyhedrovirus occlusion bodies enhanced infection of the virus. Further, the development of Pseudaletia separata, S. exigua, and S. litura, none of which are host insects of TnGV, was inhibited when these larvae were fed enhancin-expressing rice leaves. This indicates that expression of the enhancin gene confers resistance to lepidopteran insect pests in rice.

We describe a new method designated “the resurrection method” by which a modified protein is expressed in higher plants in place of the original protein. The modified gene constructed by introducing synonymous codon substitutions throughout the original gene to prevent the sequence-specific degradation of its mRNA during RNA silencing is expressed while the expression of the original gene is suppressed. Here, we report the successful alteration of the biochemical properties of green fluorescent protein expressed in transgenic Nicotiana benthamiana, suggesting that this method could be useful for gene control in living plants.

The susceptibility of rice plants to the rice blast fungus Magnaporthe grisea is increased by cold stress. However, the mechanism of this phenomenon is unknown. In this study, we found that the whole plant-specific resistance (WPSR), which was expressed in the intact leaf sheaths of susceptible rice plants, was suppressed by low temperature or abscisic acid (ABA) treatment. An inhibitor of ABA biosynthesis prevented the suppression of WPSR by low temperature treatment. These results indicate that ABA synthesized de novo under low temperature condition is a key factor for suppressing WPSR and making rice plants susceptible to M. grisea.

A novel inoculation method of the rice blast fungus Magnaporthe grisea using intact leaf sheaths of rice plants, designated as the intact method, was devised to observe the cytological events in living tissues during infection. In leaf sheaths inoculated by using the intact method, the symptoms closely resembled those occurring in inoculated plants, and progressive steps of fungal invasion and cellular changes could be observed by microscopy. Significantly, a cellular resistant reaction was observed in a susceptible line, with cell death preventing fungal development. This inoculation procedure provides a useful technique for cytological investigations of rice-pathogen interactions.

Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.

Subcellular localization of the Brome mosaic virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.

Golgi α1,2-mannosidase I is involved in the N-linked oligosaccharide processing pathway. In this study, two truncated genes encoding for human Golgi α1,2-mannosidase I (hManIA2: amino acids 127–626 and hManIC: amino acids 118–617) were expressed in Escherichia coli to characterize the enzymes. These genes were fused to a T7 protein tag and a histidine tag at the N- and C-terminal ends, respectively, and purified using Co2+ affinity chromatography. The properties including optimal temperature, optimal pH, and substrate specificity of the purified enzymes were investigated by HPLC using pyridylamino (PA)-labeled oligosaccharides as substrates. The stability of hManIA2 was dependent on the presence of Ca2+, which was also required for its activity. On the other hand, hManIC was stable in the absence of Ca2+, even though Ca2+ was also effective for the activity of hManIC. While the similarity of the amino acid sequences is over 60%, hManIA2 and hManIC showed different substrate specificities particularly toward M9A and M8C.

An inducible virus infection system was demonstrated to be an efficient protein expression system for inducing synchronous virus vector multiplication in suspension-cultured plant cells. A GFP-tagged tomato mosaic virus (ToMV-GFP) derivative that has a defect in its 130 K protein, a silencing suppressor of ToMV, was synchronously infected to tobacco BY2 cultured cells using this system. In the infection-induced cells, viral RNA was degraded rapidly, and a cytosol extract prepared from the infected cells showed RNA degradation activity specific for ToMV- or GFP-related sequences. In lysate prepared from cells infected by ToMV-GFP carrying the wild-type 130 K protein, sequence-specific RNA degradation activity was suppressed, although siRNA derived from the virus was generated. Furthermore, the 130 K protein interfered with 3′-end methylation of siRNA. The inducible virus infection system may provide a method for biochemical analysis of antiviral RNA silencing and silencing suppression by ToMV.

A major obstacle in the genetic manipulation of tomato mosaic virus (ToMV) is the instability of the plasmid containing the infectious full-length cDNA of the ToMV vector, which often prevents the subcloning of a foreign gene of interest into the vector. We found that an insertion of a 0.3-1.6-kbp DNA fragment in the movement protein (MP) coding region effectively attenuated bacterial toxicity of the plasmid and greatly increased plasmid yield. Accumulation of a modified ToMV containing a 0.3-kb insertion in the MP coding region was comparable to that of a modified ToMV without an insertion in tobacco BY-2 protoplasts, while an insertion more than 0.6 kb significantly reduced accumulation of the viral RNA. The modified ToMV vector containing a 0.3-kb insertion was easily manipulated to introduce a coding sequence for human interferon-gamma (HuIFN-gamma) and successfully utilized to produce HuIFN-gamma in both BY-2 protoplasts and transgenic BY-2 cells.

Fungal plant pathogens have evolved diverse strategies to overcome the multilayered plant defence responses that confront them upon host invasion. Here we show that pathogenicity of the cucumber anthracnose fungus, Colletotrichum lagenarium, and the rice blast fungus, Magnaporthe grisea, requires a gene orthologous to Saccharomyces cerevisiae SSD1, a regulator of cell wall assembly. Screening for C. lagenarium insertional mutants deficient in pathogenicity led to the identification of ClaSSD1. Following targeted gene replacement, appressoria of classd1 mutants retained the potential for penetration but were unable to penetrate into host epidermal cells. Transmission electron microscopy suggested that appressorial penetration by classd1 mutants was restricted by plant cell wall-associated defence responses, which were observed less frequently with the wild-type strain. Interestingly, on non-host onion epidermis classd1 mutants induced papilla formation faster and more abundantly than the wild type. Similarly, colonization of rice leaves by M. grisea was severely reduced after deletion of the orthologous MgSSD1 gene and attempted infection by the mutants was accompanied by the accumulation of reactive oxygen species within the host cell. These results suggest that appropriate assembly of the fungal cell wall as regulated by SSD1 allows these pathogens to establish infection by avoiding the induction of host defence responses.

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.

Brome mosaic bromovirus (BMV) 3a protein is required for cell-to-cell movement of the virus in host plants. The BMV 3a protein (B3a) was produced in Escherichia coli using an expression vector. Gel retardation analysis and UV cross-linking experiments demonstrated that B3a bound single-stranded RNA cooperatively without sequence specificity. Binding competition analysis showed that B3a bound to single-stranded nucleic acids more strongly than to double-stranded nucleic acids. Deletion mutagenesis located a nucleic acid-binding domain to amino acids 189-242. Western blot analysis of fractionated proteins of BMV-infected barley using monoclonal antibodies against B3a indicated that B3a may interact with membrane materials and form complexes in the cytoplasm. Immunogold labelling of thin sections of infected barley tissues revealed that B3a was associated with plasmodesmata and cytoplasmic inclusions.

Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5′-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions., Author Summary Many important pathogens of plants, animals, and humans are positive-strand RNA viruses. They replicate via complementary RNA in replication complexes formed on host intracellular membranes. In the replication process, not only viral replication proteins but also host factors play important roles. Although many host factors whose knockdown affects the multiplication of positive-strand RNA viruses have been identified, the function of each host factor in virus multiplication is only poorly understood in most instances. In this paper, we show that a host small GTP-binding protein ARL8 is required for the multiplication of Tomato mosaic virus (ToMV), and that it forms a complex with ToMV replication proteins and another essential host factor TOM1 that is a seven-pass transmembrane protein. We further demonstrate that the replication proteins acquire the ability to synthesize negative-strand ToMV RNA and RNA 5′ cap only in the presence of both TOM1 and ARL8. The replication proteins of ToMV are multifunctional proteins that participate in RNA replication on membranes and RNA silencing suppression in the cytosol. Our results suggest that ToMV replication proteins are programmed to express their replication-related activities only on membranes through interactions with these host membrane proteins.

Fragments generated by high-velocity collisions between solid planetary bodies are one of the main sources of new interplanetary dust particles. However, only limited ranges of collision velocity, ejecta size, and target materials have been studied in previous laboratory experiments, and the collision condition that enables the production of dust-sized particles remains unclear. We conducted hypervelocity impact experiments on silicate rocks at relative velocities of 9 to 61 km s{sup -1}, which is beyond the upper limit of previous laboratory studies. Sub-millimeter-diameter aluminum and gold spheres were accelerated by laser ablation and were shot into dunite and basalt targets. We analyzed the surfaces of aerogel blocks deployed near the targets using an electron probe micro analyzer and counted the number of particles that contained the target material. The size distributions of ejecta ranged from five to tens of microns in diameter. The total cross-sectional area of dust-sized ejecta monotonically increased with the projectile kinetic energy, independent of impact velocity, projectile diameter, and projectile and target material compositions. The slopes of the cumulative ejecta-size distributions ranged from -2 to -5. Most of the slopes were steeper than the -2.5 or -2.7 that is expected for a collisional equilibrium distribution in a collisionmore » cascade with mass-independent or mass-dependent catastrophic disruption thresholds, respectively. This suggests that the steep dust size-distribution proposed for the debris disk around HD172555 (an A5V star) could be due to a hypervelocity collision.« less