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Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid–liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared to wild type. In summary, (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active gibberellin A4 (GA4 ) to GA58 (12-OH-GA4 ) in vitro. MdDOX-Co also metabolized its precursors GA12 and GA9 to GA111 (12-OH-GA12 ) and GA70 (12-OH-GA9 ), respectively. (ii) Of the three 12-OH-GAs, GA58 was still active physiologically, but not GA70 or GA111 . (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d2 )-GA12 to d2 -GA111 but not to d2 -GA58 , whereas transformants converted applied d2 -GA9 to d2 -GA58 . (iv) GA111 is converted poorly to GA70 by GA 20-oxidases in vitro when GA12 is efficiently metabolized to GA9 . (v) No GA58 was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA12 by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.

The apple cultivar McIntosh Wijcik, which is a mutant of ‘McIntosh’, exhibits a columnar growth phenotype (short internodes, few lateral branches, many spurs, etc.) that is controlled by a dominant Co gene. The candidate gene (MdDOX-Co), encoding a 2-oxoglutarate-dependent dioxygenase, is located adjacent to an insertion mutation. Non-columnar apples express MdDOX-Co in the roots, whereas columnar apples express MdDOX-Co in the aerial parts as well as in the roots. However, the function of MdDOX-Co remains unknown. Here, we characterized tobacco plants overexpressing MdDOX-Co. The tobacco plants showed the typical dwarf phenotype, which was restored by application of gibberellin A3 (GA3). Moreover, the dwarf tobacco plants had low concentrations of endogenous bioactive gibberellin A1 (GA1) and gibberellin A4 (GA4). Similarly, ‘McIntosh Wijcik’ contained low endogenous GA4 concentration and its dwarf traits (short main shoot and internodes) were partially reversed by GA3 application. These results indicate that MdDOX-Co is associated with bioactive GA deficiency. Interestingly, GA3 application to apple trees also resulted in an increased number of lateral branches and a decrease in flower bud number, indicating that gibberellin (GA) plays important roles in regulating apple tree architecture by affecting both lateral branch formation (vegetative growth) and flower bud formation (reproductive growth). We propose that a deficiency of bioactive GA by ectopic expression of MdDOX-Co in the aerial parts of columnar apples not only induces dwarf phenotypes but also inhibits lateral branch development and promotes flower bud formation, and assembly of these multiple phenotypes constructs the columnar tree form.

Adhesion bonding of metals and polymers is attracting attention as an innovative bonding technology to realize high functionality and weight reduction of various mechanical parts and structural materials. This technology has been significantly demanded with the rapid development of multi-materials in recent years. However, it has been reported that natural oxide film and hydroxide film are formed on the metal surface, resulting in the change in adhesion, especially degradation of adhesion is sometimes accelerated by water molecules at the interface. The mechanism of adhesion degradation is still unclear. In this study, the interface between metal and resin was modeled using the molecular dynamics (MD) simulation to investigate how the moisture (water molecules) on the interfacial surface affects the adhesion. This study varied the amount of water molecules at the interface and investigated how water molecules affect the adhesive strength subjected to uni-axial tensile loading. In addition, the potential energy around the interface was calculated, and the adhesion mechanism with respected to water molecules was carefully discussed.

Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15–13 and 12–22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15–13 and 12–22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm–baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15–13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12–22 Fab has a weaker binding affinity (KD ~ μM) with the RABVG compared to the 15–13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15–13 and 12–22 antibodies as another potential biopharmaceutical for targeting rabies. The 15–13 and 12–22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the μM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.

We previously identified the FLOWERING LOCUS C (FLC)-like gene, a MADS-box transcription factor gene that belongs to Arabidopsis thaliana L. FLC clade, in apple (Malus $\times$ domestica Borkh.), and its expression in dormant flower buds is positively correlated with cumulative cold exposure. To elucidate the role of the MdFLC-like in the dormancy process and flower development, we first characterized the phenotypes of MdFLC-like overexpressing lines with the Arabidopsis Columbia-0 background. The overexpression of MdFLC-like significantly delayed the bolting date and reduced the plant size, but it did not significantly affect the number of rosette leaves or flower organ formation. Thus, MdFLC-like may affect vegetative growth and development rather than flowering when expressed in Arabidopsis, which is not like Arabidopsis FLC that affects development of flowering. We compared seasonal expression patterns of MdFLC-like in low-chill ‘Anna’ and high-chill ‘Fuji’ and ‘Tsugaru’ apples collected from trees grown in a cold winter region in temperate zone and found an earlier upregulation in ‘Anna’ compared with ‘Fuji’ and ‘Tsugaru’. Expression patterns were also compared in relation to developmental changes in the flower primordia during the chilling accumulation period. Overall, MdFLC-like was progressively upregulated during flower primordia differentiation and development in autumn to early winter and reached a maximum expression level at around the same time as the genotype-dependent chilling requirements were fulfilled in high-chill cultivars. Thus, we hypothesize MdFLC-like may be upregulated in response to cold exposure and flower primordia development during the progress of endodormancy. Our study also suggests MdFLC-like may have a growth-inhibiting function during the end of endodormancy and ecodormancy when the temperature is low and unfavorable for rapid bud outgrowth.

The columnar apple cultivar ‘McIntosh Wijcik’ was discovered as a spontaneous mutant from the top of a ‘McIntosh’ tree in the early 1960s. ‘McIntosh Wijcik’ exhibits the columnar growth phenotype: compact and sturdy growth, short internodes, and very few lateral shoots. Classical genetic analysis revealed that the columnar growth phenotype of ‘McIntosh Wijcik’ is controlled by a single dominant gene, Co. This review focuses on the advances made toward understanding the molecular mechanisms of columnar growth in the last decade. Molecular studies have shown that an 8.2 kb insertion in the intergenic region of the Co locus is responsible for the columnar growth phenotype of ‘McIntosh Wijcik’, implying that the insertion affects the expression patterns of adjacent genes. Among the candidate genes in the Co region, the expression pattern of MdDOX-Co, putatively encoding 2-oxoglutarate-dependent dioxygenase (DOX), was found to vary between columnar and non-columnar apples. Recent studies have found three functions of MdDOX-Co: facilitating bioactive gibberellin deficiency, increasing strigolactone levels, and positively regulating abscisic acid levels. Consequently, changes in these plant hormone levels caused by the ectopic expression of MdDOX-Co in the aerial organs of ‘McIntosh Wijcik’ can lead to dwarf trees with fewer lateral branches. These findings will contribute to the breeding and cultivation of new columnar apple cultivars with improved fruit quality.

In apple (Malus spp.), crown gall disease, caused by the bacterial pathogens Rhizobium radiobacter (Ti) and R. rhizogenes (Ti), can be severe. To control the disease, breeding of apple rootstocks that exhibit crown gall resistance is a promising approach. In this study, we used a full-sib F1 population derived from a ‘JM7’ (susceptible) × Sanashi 63 (resistant) cross to identify quantitative trait loci (QTLs) that control resistance to tumour-inducing Rhizobium isolates found in apple production areas in Japan. Three stable QTLs, associated with a wide range of crown gall resistances, were identified in three linkage groups (LGs). QTLs for resistance to isolates Peach CG8331, Nagano 1 and Nagano 2 co-localised at the middle of LG 2 in Sanashi 63, where the crown gall resistance gene Cg (renamed as Rrr1 in this study) was previously identified. Similarly, in ‘JM7’, QTLs were identified on LG 11 for resistance to isolates ARAT-001, ARAT-002 and Kazuno 2, and on LG 15 for resistance to isolate ARAT-001. Fine-mapping of Rrr1 and nucleotide sequencing of a contig obtained from two bacterial artificial chromosome (BAC) clones delimited the Rrr1 gene to a region spanning 217 kb. In silico gene prediction from the region identified four genes encoding the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class plant resistance genes.

To improve the safety of genetically modified live rabies vaccine strains, most studies have utilized an attenuating Arg-to-Glu mutation at position 333 in the glycoprotein (G333), which is responsible for attenuation of the live vaccine strain SAG2. The Glu residue requires two nucleotide substitutions to revert to pathogenic Arg, thus significantly lowering the probability of pathogenic reversion caused by the Glu-to-Arg mutation at G333. However, only one nucleotide substitution is sufficient to convert the Glu residue to another pathogenic residue, Lys, and thereby to cause pathogenic reversion. This indicates a potential safety problem of SAG2 and the live vaccine candidates attenuated by Glu at G333. In this study, aiming to solve this problem, we examined the utility of a Leu residue, which requires two nucleotide substitutions to be both Arg and Lys, as an attenuating mutation at G333. Using a reverse genetics system of the live vaccine strain ERA, we generated ERA-G333Leu by introducing an Arg-to-Leu mutation at G333. Similar to ERA-G333Glu, which is attenuated by an Arg-to-Glu mutation at G333, ERA-G333Leu did not cause obvious clinical signs in 6-week-old mice after intracerebral inoculation. Importantly, after 10 passages in suckling mouse brains, ERA-G333Glu acquired a pathogenic Lys or Arg at G333 and a high level of lethality in mice, whereas ERA-G333Leu retained the attenuating Leu at G333 and only showed a modest level of virulence probably caused by a mutation at G194. In addition, ERA-G333Leu and ERA-G333Glu induced neutralizing antibody response and protective immunity in mice with similar efficiencies. The results demonstrate that, compared to ERA-G333Glu, ERA-G333Leu is more stably attenuated, also indicating the high utility of a Leu residue as an attenuating mutation at G333 in the development of live rabies vaccine strains with a high level of safety.

The columnar apple ‘McIntosh Wijcik’, which is a mutation of ‘McIntosh’ shoots, has short internodes, thick stems, upright growth, poor lateral branches, and increased spur density. These columnar traits are controlled by a single dominant gene known as Co. We previously identified a putative dioxygenase gene (designated as 91071) as a promising Co candidate (expressed in the shoot apices of ‘McIntosh Wijcik’). However, tissue expression and function of the 91071 gene in noncolumnar apples is still not clear. In this study, we used reverse transcription polymerase chain reaction to demonstrate that the 91071 gene is mainly expressed in the roots of noncolumnar apples, whereas it is also expressed in shoot apices and leaves of columnar apples. In situ hybridization revealed that the 91071 gene is expressed at the primordium of lateral roots and root tips of both noncolumnar and columnar apple trees and in the shoot meristem and leaf primordium in the columnar apple ‘McIntosh Wijcik’. Grafting experiments of noncolumnar scion onto columnar rootstocks revealed that the columnar growth phenotype is not transmissible from rootstock to scion. These results indicated that ectopic expression of the 91071 gene in aerial parts causes columnar growth, whereas the expression of the 91071 gene in roots does not produce columnar growth. Furthermore, transgenic apples overexpressing the 91071 gene showed larger median adventitious root length and higher median number of lateral roots than control apples. Our result suggests that the 91071 gene may be related to root development.

Juiciness and freshness play a critical role in consumers’ choice of apples, and objective measurements of the traits are essential for adding a premium to fruits with high consumer demand. Juiciness and freshness were sensory evaluated by four apple-breeding experts. The score of sensory juiciness decreased with decreased instrumental measurements of water content (WC), firmness, and apoplastic solution (AS). The influence of firmness on sensory juiciness was not linear, and perception of juiciness rapidly decreased when firmness decreased below 60 N. The sensory freshness score was related to titratable acidity (TA), firmness, and the AS. A nonlinear regression model for freshness indicated that freshness was less likely to be perceived as the AS in flesh tissue decreased, and the perception of freshness rapidly decreased when firmness decreased below 60 N or TA decreased below 0.25 g 100 ml−1. The firmness of fruit treated with 1-MCP hardly decreased during storage. The titratable acidity of fruit treated with 1-MCP decreased; however, the degree of the decrease was significantly lower than that of non-treated fruit. The AS immediately decreased just after harvest and was not significantly different in most cases between 1-MCP-treated and non-treated fruit during storage. Therefore, measurements of firmness and TA were useful for distinguishing 1-MCP-treated fruit from non-treated fruit and the AS is necessary to distinguish between stored fruit and that just after harvest. The performance of calibration models developed by NIR spectroscopy for TA, firmness, and AS was lower than that for soluble solids content (SSC), which is used to grade apples by NIR spectroscopy in Japanese packing houses. Therefore, it is impractical to grade fruit by freshness using calibration models. However, high correlation coefficients were obtained in the calibration model for WC. The correlation coefficient of WC was about as high as that of SSC. Therefore, apples could be graded by juiciness using the calibration model for WC with the auxiliary use of calibration models for firmness and AS.

Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism underlying the less efficient replication of Ni-CE, we compared the activities of Ni-CE and Nishigahara phosphoproteins, viral interferon (IFN) antagonists, to suppress IFN-β promoter activity in muscle cells and we demonstrated a defect of Ni-CE phosphoprotein in this ability. Treatment with an IFN-β-neutralizing antibody improved the replication efficiency of Ni-CE in muscle cells, indicating that produced IFN inhibits Ni-CE replication. The results indicate the importance of IFN antagonism of rabies virus phosphoprotein for viral replication in muscle cells.

Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level of Ifn-β gene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-β promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves. IMPORTANCE Despite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.

To estimate the risk of interspecies transmission of rotavirus species A (RVA) from exotic pets to other mammalian species, the prevalence of RVA in sugar gliders (Petaurus breviceps) was investigated. RVAs were detected in 10 of 44 sugar gliders by reverse transcription (RT)-semi-nested PCR. These viruses were classified as G27P[3] and G27P[36] genotypes, with G27 and P[36] being new genotypes as assigned by the Rotavirus Classification Working Group. To characterize sugar glider RVA in detail, one strain, RVA/SugarGlider-tc/JPN/SG385/2012/G27P[36] (SG385-tc), was isolated. All of the genes of the strain were classified as new genotypes (G27-P[36]-I19-R10-C10-M9-A20-N11-T13-E17-H12). The enterotoxin domain in NSP4, which is important for the induction of diarrhoea, was conserved between SG385-tc and previously reported mammalian strains, suggesting the potential of sugar glider RVA to cause diarrhoea in mammalian species. In fact, seven out of nine suckling mice inoculated orally with 3.9 × 104 f.f.u. of strain SG385-tc had diarrhoea and the 50 % diarrhoea-inducing dose (DD50) of strain SG385-tc in suckling mice was 1.2 × 104 f.f.u. Our findings suggest that sugar glider RVA is infective to and possibly pathogenic in other mammalian species.

We isolated and confirmed two S-RNases, denoted as mpS1 and mpS2, from apple rootstock ``Marubakaido`` (Malus prunifolia Borkh. Var. ringo Asami). These S-RNases contained and conserved five cysteine residues and two histidine residues, which are essential for RNase activity. The mpS1 showed high similarity to S5 (99.1%) of Malus spectabilis, whereas the mpS2 showed 99.5% nucleotide sequence similarity to S26 of (Malus × domestica) and 99.6% to S35 of (Malus sieversii) when compared with reported S-RNases. In amino acid sequences, the mpS1-RNase was almost similar to the S5-RNase of Malus spectabilis, and the mpS2-RNase was similar to the S35 of Malus sieversii, with only one bp being different from the S26-RNase of Malus × domestica. The 57 S-RNases of Malus species were renamed and rearranged containing the new S-RNases, as mprpS35 (mpS2) and mprpS57 (mpS1), for determining S-genotypes and identifying new alleles from apple species (Malus spp.).

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.

In apple, two MYB transcription factors MdMYB10 (R6:MdMYB10) and MdMYB110a have been shown to be responsible for the type 1 and type 2 red flesh traits, respectively. While type 1 red-fleshed apples are characterized by a red coloration not only in fruit flesh but also in vegetative tissues such as leaves and flowers, red pigmentation in type 2 red-fleshed apples is limited at the fruit flesh. We have searched cultivars containing both functional MdMYB10 and MdMYB110a and then tried to breed new cultivars containing both functional genes by cross-pollination of ‘Geneva’ (type 1) and ‘Pink Pearl’ (type 2). The cultivar having both genes should exhibit superior characteristics, such as a stable red flesh trait throughout fruit maturity, as type 1 reduces its colouring until maturity, whereas type 2 increases until maturity. We could not identify red-fleshed cultivars having both genes; moreover, only one plant of 80 F1 progeny having both genes died in its juvenile stage. From the results, it was suggested that some sort of breeding depression must have occurred. We analysed the expression patterns of the genes within two F1 plants having either MdMYB10 or MdMYB110a gene and found that the expression pattern of MdMYB110a was different from that observed in ‘JPP35’ (‘Jonathan’ × ‘Pink Pearl’). The MdMYB110a gene in the No. 2804 F1 plant derived from ‘Pink Pearl’ × ‘Geneva’ was expressed in the flesh from the beginning of the red coloration through maturity, and seemed to cause upregulation, not only the latter half, but also the first half of the gene in the anthocyanin pathway.

A total of 568 normal feces from calves on a beef farm in Fukui Prefecture, Japan, in 2011-2012 were examined by RT-semi-nested PCR for rotavirus A (RVA) VP4 genes. Through partial sequencing and BLAST analyses of 84 VP4-positive specimens, we identified an avian-like RVA strain, N2342, which shares highest nucleotide identity (80.0%) with known avian-like bovine strain 993/83, in one specimen. Phylogenetic analysis also revealed a close genetic relationship between N2342 and avian RVAs, suggesting bird-to-cattle transmission. We observed frequent contact of wild birds with calves in the farm, suggesting that these birds were the source of the virus.

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars 'Orin' and 'Akane' using F1 seedlings derived from a cross between these cultivars. The 'Orin' map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the 'Akane' map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by 'Akane' are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.

Background Rabies virus (RABV) causes rabies disease resulting in >55,000 human deaths/year. The multifunctional RABV P-protein has essential roles in genome replication, and forms interactions with cellular STAT proteins that are thought to underlie viral antagonism of interferon-dependent immunity. However, the molecular details of P-protein-STAT interaction, and its importance to disease are unresolved. Methods Studies were performed using sequence/structure analysis, mutagenesis, immunoprecipitation, luciferase and qRT-PCR-based signaling assays, confocal microscopy and reverse genetics/in vivo infection. Results We identified a hydrophobic pocket of the P-protein C-terminal domain as critical to STAT-binding/antagonism. This interface was found to be functionally and spatially independent of the region responsible for N-protein interaction, which is critical to genome replication. Based on these findings, we generated the first mutant RABV lacking STAT-association. Growth of the virus in vitro was unimpaired, but it lacked STAT-antagonist function and was highly sensitive to interferon. Importantly, growth of the virus was strongly attenuated in brains of infected mice, producing no major neurological symptoms, compared with the invariably lethal wild-type virus. Conclusions These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication.

Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene ( Ifn -β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar 'Spartan' (S(9)S(10)). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S(9) -RNase and S(10-)RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1-11). All types included F-box genes derived from S(3-) and S(9-)haplotypes, and seven types included F-box genes derived from S(3-), S(9-), and S(10-)haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S(3-), S(9-), and S(10-)haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.

SummaryMost cultivars of European pear (Pyrus communis L.) exhibit S-RNase-based self-incompatibility. Their S-genotypes have mainly been assigned by analysing PCR-amplified S-RNase alleles from the genomic DNA of each cultivar. In this study, we identified eight European pear cultivars that had only one S-RNase allele amplified by conventional genomic PCR using a consensus primer pair. Rapid amplification of cDNA ends (RACE) on stylar RNAs of the cultivars was used to obtain the full-length sequence of the putative S 25-RNase. Long-PCR successfully amplified the S25-RNase allele, including a long 3,131 bp intron, the longest intron reported so far among S-RNase alleles in fruit tree species in the sub-tribe Pyrinae. Conventional PCR using a consensus primer and a primer designed from the intron sequence amplified a 385 bp fragment of the S25-RNase allele from the genomic DNA of all eight cultivars. An S-RNase-based, cleaved amplified polymorphic sequence (CAPS) marker system and S25-RNase allele-specific...

SummarySeveral cultivars of European pear (Pyrus communis L.) are triploid (2n = 51), produce little viable pollen, and exhibit S-RNase-based cross-incompatibility. In orchards where triploids are grown, two diploids (one to pollinate the triploids, and the other to pollinate the other diploid) must be inter-planted. In this study, seven European pear cultivars were confirmed to be triploid by flow cytometry analysis. An S-RNase-based, cleaved amplified polymorphic sequence (CAPS) marker system and S-RNase allele-specific PCR were used to genotype these triploids. A comparison of S-genotypes between the triploids and diploids of European pear identified those diploid(s) that could not be used to pollinate the triploids. Pollination tests confirmed the cross-incompatibility between a triploid and a diploid. The S-genotypes of the seven triploids will be useful for pollination management in orchards, and for breeding new cultivars using the triploids as seed parents. In addition, rapid amplification of cDNA...

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

The current live rabies vaccine SAG2 is attenuated by only one mutation (Arg-to-Glu) at position 333 in the glycoprotein (G333). This fact generates a potential risk of the emergence of a pathogenic revertant by a back mutation at this position during viral propagation in the body. To circumvent this risk, it is desirable to generate a live vaccine strain highly and stably attenuated by multiple mutations. However, the information on attenuating mutations other than that at G333 is very limited. We previously reported that amino acids at positions 273 and 394 in the nucleoprotein (N273/394) (Leu and His, respectively) of fixed rabies virus Ni-CE are responsible for the attenuated phenotype by enhancing interferon (IFN)/chemokine gene expressions in infected neural cells. In this study, we found that amino acid substitutions at N273/394 (Phe-to-Leu and Tyr-to-His, respectively) attenuated the pathogenicity of the oral live vaccine ERA, which has a virulent-type Arg at G333. Then we generated ERA-N273/394-G333 attenuated by the combination of the above attenuating mutations at G333 and N273/394, and checked its safety. Similar to the ERA-G333, which is attenuated by only the mutation at G333, ERA-N273/394-G333 did not cause any symptoms in adult mice after intracerebral inoculation, indicating a low level of residual pathogenicity of ERA-N273/394-G333. Further examination revealed that infection with ERA-N273/394-G333 induces IFN-β and CXCL10 mRNA expressions more strongly than ERA-G333 infection in a neuroblastoma cell line. Importantly, we found that the ERA-N273/394-G333 stain has a lower risk for emergence of a pathogenic revertant than does the ERA-G333. These results indicate that ERA-N273/394-G333 has a potential to be a promising candidate for a live rabies vaccine strain with a high level of safety.

To obtain complete genome sequences of turkey rotavirus A strains Ty-1 and Ty-3, we sequenced the gene segments that had not been decoded previously. The genotype constellations of the respective strains were determined to be G17-P[38]-I4-R4-C4-M4-A16-N4-T4-E4-H4 and G7-P[35]-I4-R4-C4-M4-A16-N4-T4-E11-H14. Notably, their VP4 and NSP5 genes were classified into novel genotypes.

In situ observation of the formation process of mesophase of isotactic polypropylene (iPP) is reported in structural point of view. Combining a rapid temperature jump and a high-flux synchrotron radiation X-ray scattering techniques, very rapid transformation from the molten amorphous state to the mesophase has been observed. The transformation proceeded very quickly in a narrow temperature range accompanied by instantaneous fluctuations in micrometer scale, suggesting the mesophase formation proceeds similarly to spinodal decomposition.

SummaryTo elucidate the influence of temperature on water flow from soil to stem in a dwarfing rootstock, sap flow was measured under different temperature conditions in 1-year-old trees of apple rootstocks of different vigour. Sap flow was largely determined by solar radiation, and increased linearly as solar radiation increased when the temperatures above and below ground were held constant. The adjusted means of sap flow in the trees tested were not significantly influenced by the total leaf area, but were significantly influenced by the root mass. The adjusted means of sap flow at 30oC were two- to three-times greater than those at 20oC in all rootstocks tested, except one super-dwarfing rootstock, during the period of shoot extension. As the degree of rootstock vigour increased, the adjusted means of sap flow increased. The difference in sap flow between an invigorating rootstock and a dwarfing one increased under high temperature conditions. Because the degree of dwarfing was clearly expressed as th...

SummaryWe have determined the S-RNase genotypes of 33 new apple cultivars and lineages produced in Japan, 44 unknown cultivars and two lineages, and 22 triploid progenies. We have speculated on the putative parentage of new cultivars and lineages based on their S-RNase genotypes and also identified mistaken parents. In the case of the triploid progenies, the breeding of new cultivars using a triploid paternal parent may pose problems due to its low pollen viability. Nevertheless, diploid and triploid progenies were obtained using a triploid maternal parent. We have compiled a database of 516 apple S-RNase genotypes, including those previously investigated, which included a survey system for cultivar combinations, showing those that were fully-incompatible, semi-compatible, or fully-compatible, together with information on the PCR-RFLP method used for the identification of S-RNase genotypes and S-RNase allele designation (available at http://www.agr.nagoya-u.ac.jp/~0hort/apple/).

Differentiation of cultivars with simple sequence repeat (SSR) markers is a very useful technique for the true-to-type characterization of cultivars and clarification of parent-offspring relationships. We developed an SSR marker set for cultivar identification comprising 15 markers that were screened from 46 previously published SSRs. This marker set could be used for apple varieties including Malus × domestica and/or other Malus species. These SSRs successfully characterized 95 apples, including the leading and major founding cultivars used worldwide for modern apple breeding. Therefore, this marker set could be applied to almost all apple cultivars. We also analyzed the parent-offspring relationships of 69 cultivars by considering allele transmissions. This analysis revealed the true parentage of the following seven cultivars: ‘Kizashi’, ‘Chinatsu’, ‘Honey Queen’, ‘Haruka’, ‘Seirin’, ‘Ozenokurenai’, and Morioka #48. This analysis also revealed a parentage discrepancy for ‘Hacnine’. From the parent-offspring analysis, two microsatellite mutation events at alleles inherited from pollen parents were observed.

Japanese pear exhibits gametophytic self-incompatibility controlled by a single S-locus with multiple alleles. The S-locus encodes an S-RNase as a stylar product, and 10 S-RNase alleles (S1 to S9, and Sk) have been cloned from major cultivars. To investigate the diversity of S-alleles in Japanese pear, we analyzed the S-genotypes of three local cultivars (‘Senryo’, ‘Kuroki’, and ‘Hogyoku’). Two S-RNase fragments were amplified from each cultivar by genomic PCR with S-RNase-specific primers. A cleaved amplified polymorphic sequence (CAPS) marker system to distinguish S1- to S9-RNases assigned S3- and S2-RNase alleles to ‘Senryo’ and ‘Kuroki’, respectively. Cloning and sequencing of the other S-RNase genes identified Se-RNase of European pear in ‘Senryo’, S12-RNase of Chinese pear in ‘Kuroki’, and S30-RNase of Chinese pear and Sk-RNase in ‘Hogyoku’. Therefore, S-genotypes of ‘Senryo’, ‘Kuroki’, and ‘Hogyoku’ were assigned as S3Se, S2S12, and SkS30, respectively. These results revealed that Japanese pear has some of the same S-alleles as European and Chinese pears as well as S1- to S9- and Sk-alleles.

SummaryMost cultivars of European pear (Pyrus communis L.) exhibit S-RNase-based gametophytic self-incompatibility (SI), but the cultivar, ‘Grand Champion’, is partially self-compatible (SC). We used pollination and molecular genetic approaches to study the cause of the partial SC, and its effects on fruit set and quality. ‘Grand Champion’ was genotyped to SbSe by an S-RNase-based cleaved amplified polymorphic sequence marker system. Crossing ‘Grand Champion’ with pollen from an SI cultivar, ‘California’ (SbSe), showed that partial SC was caused by the disruption of pistil function. Of the Sb- and Se-RNase alleles cloned from ‘Grand Champion’, the Sb-RNase allele had two nonsynonymous nucleotide substitutions compared with the Sb-RNase allele previously cloned from ‘Doyenne du Comice’, but it retained the typical primary structure of S-RNases of the Maloideae. The sequence of the Sb-RNase allele from ‘Grand Champion’ was also obtained from two SI cultivars: ‘Josephine de Malines’ and ‘Urbaniste’. Similar ...

Vegetative propagation enables the multiplication of plants that are genetically identical to the original. Hardwood cuttings have a low cost advantage and are used widely for the propagation of JM series apple rootstocks, which root well from hardwood cuttings. To breed new apple rootstocks with similarly high rooting capabilities, the development of a marker-assisted selection method is desirable. Therefore, it is important to understand the genetic mechanisms behind the rooting capabilities of hardwood cuttings. We identified quantitative trait loci (QTLs) for the rooting capability of ‘JM7’ using a QTL analysis with phenotypic data obtained under two environmental conditions. The QTLs for rooting rate and root quantity co-located. The QTL for rooting rate explained 66 % of the genetic variance and 57 % of the phenotypic variance. Graphical genotyping and database searching revealed candidate genes, including three related to auxin response, one related to ethylene response, and a gene encoding a WRKY transcription factor.

Nine full-length cDNAs of S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars by RT-PCR and 3' and 5' RACE. Comparison of the nucleotide sequences between the nine S-RNases cloned and 13 putative S alleles previously amplified by genomic PCRs revealed that seven corresponded to Sa, Sb, Sd, Se, Sh, Sk and Sl alleles, and the other two were new S alleles (designated as Sq and Sr alleles). Genomic PCR with a set of a8FTQQYQa9 and a8EP-anti-IIWPNVa9 primers was used to amplify nine S alleles; 1,414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb) and ca. 350 bp (Sa, Sd, Sh and Sr). Among these, S alleles of similar size were discriminated by digestion with BaeI, BglII, BssHII, HindIII, EcoO109I and SphI. The PCR amplification of S allele following digestion with the restriction enzymes provided a PCR-RFLP system for rapid S-genotyping European pear cultivars harboring nine S alleles. The PCR-RFLP system assigned a total of 63 European pear cultivars to 25 genotypes. Among these, 14 genotypes were shared by two or more cultivars, which were cross-incompatible. These results suggested that the genes cloned represented the S-RNases from European pear, and that there were many cross-incompatible combinations among European pear varieties.

A new S9-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S9-RNase, a stylar product of S9-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S9-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S9-RNase had a hypervariable (HV) region different from S1- to S8-RNase and shared higher similarity (95.2%) with apple S3-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S1- to S9-amplicon (product), but could not discriminate the S2 from the S9 of ca. 1.3 kb. The S2 and S9 were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S1 to S9, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S1- to S9-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S4S9 were determined to be cross incompatible.

A Japanese pear 'Kumoi' was previously determined as S3S4 by pollination tests, but its S-genotype was reconsidered following our PCR-RFLP (S1 to S9) analyses and pollination tests. Based on its compatibility with 'Seigyoku' (S3S4), and PCR-RFLP analysis, 'Kumoi' was classified as S1S3 for the first time. Additional pollination tests were necessary to prove our contention, but 'Kumoi' did not supply sufficient flowers. 'Sekaiichi' was also assigned as S1S3 by PCR-RFLP analysis, and incompatibility with 'Kumoi'. Instead of 'Kumoi', 'Sekaiichi' was pollinated with the pollen from an 55-homozygote and that from an Ssm4-homozygote. The lack of fruit set revealed that 'Sekaiichi' was incompatible with the S3 and Ssm4 pollen, leading us to predict that the 5-genotype of 'Sekaiichi' was S1S3 or 8384. Two S-genotypes with S1S3 and S2S3 segregated in hybrid progenies between 'Doitsu' (S1S2) and 'Sekaiichi', indicating that S1 was present in 'Sekaiichi'. These results of pollination tests with 'Sekaiichi' indicated the S-genotype of 'Kumoi' was S1S3.

On the basis of computer simulations of the time-dependent Ginzburg - Landau model including thermal noise effects, we evaluate the surface barrier against magnetic flux penetration in type-II superconductors. We discuss several parameter dependencies of the first field for flux penetration, which is estimated from the first peak position of the magnetization curve in the presence of an external field slowly increasing over time. It is shown that the first penetration field is a decreasing function of temperature and of the degree of sample shape deformation. The surface barrier is also found to be suppressed by the surface irregularity on the scale of the magnetic penetration depth.

The ac response of type-II superconductors to an alternating magnetic field is numerically studied on the basis of the time-dependent Ginzburg - Landau equations. We examine the temperature dependence of the ac susceptibility associated with a small ac magnetic field in the absence of a bias dc field. It is shown that with increasing temperature the in-phase component of the fundamental susceptibility exhibits a step-like change from a negative constant value to zero, while the out-of-phase component of the fundamental susceptibility and the third-harmonic component have a peak at a certain temperature near the superconducting transition temperature. These results are in qualitative agreement with those of recent experiments on high- superconductors.

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB(-α-γ)) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S(4)-RNase and 378 kb around S(2)-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB(4-u1-u4, 4-d1-d2) and PpSFBB(2-u1-u5,) (2-d1-d5), respectively) were found, but PpSFBB(4-α-γ) and PpSFBB(2-γ) were absent. The PpSFBB(4) genes showed 66.2-93.1% amino acid identity with the PpSFBB(2) genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3-94.9% identity, while group II consisted of gene pairs with higher identities (92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.