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Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 (TAW1) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1. We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.

Antibody-dependent cellular cytotoxicity (ADCC) is promoted through interaction between the Fc region of immunoglobulin G1 (IgG1) and Fcγ receptor IIIa (FcγRIIIa), depending on N -glycosylation of these glycoproteins. In particular, core fucosylation of IgG1-Fc N -glycans negatively affects this interaction and thereby compromises ADCC activity. To address the mechanisms of this effect, we performed replica-exchange molecular dynamics simulations based on crystallographic analysis of a soluble form of FcγRIIIa (sFcγRIIIa) in complex with IgG1-Fc. Our simulation highlights increased conformational fluctuation of the N -glycan at Asn162 of sFcγRIIIa upon fucosylation of IgG1-Fc, consistent with crystallographic data giving no interpretable electron density for this N -glycan, except for the innermost part. The fucose residue disrupts optimum intermolecular carbohydrate-carbohydrate interactions, rendering this sFcγRIIIa glycan distal from the Fc glycan. Moreover, our simulation demonstrates that core fucosylation of IgG1-Fc affects conformational dynamics and rearrangements of surrounding amino acid residues, typified by Tyr296 of IgG1-Fc, which was more extensively involved in the interaction with sFcγRIIIa without Fc core fucosylation. Our findings offer a structural foundation for designing and developing therapeutic antibodies with improved ADCC activity.

The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium -mediated transformation system using M. polymorpha sporelings. The targeting efficiency was evaluated by knocking out the NOP1 gene, which impaired air-chamber formation. Homologous recombination was observed in about 2% of the thalli that passed the positive/negative selection. With the advantage of utilizing the haploid gametophytic generation, this strategy should facilitate further molecular genetic analysis of M. polymorpha , in which many of the mechanisms found in land plants are conserved, yet in a less complex form.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil , and analysing the dwarf gene, CONTRACTED , located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families. Japanese morning glory ( Ipomoea nil ) has diverse flowering traits. Here, the authors describe the reference genome sequence of I. nil , annotations of genes and transposons, and compare evolution of the I. nil genome to other Convolvulaceae and Solanales genomes.

Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability. By swapping the constant regions of IgG1 and IgG4, we determined that the constant heavy chain (CH3) domain is critical for aggregate formation, but a core-hinge-stabilizing S228P mutation in IgG4 is insufficient for preventing aggregation. To identify the aggregation-prone amino acid, we substituted the CH3 domain of IgG4 with that of IgG1, changing IgG4 Arg409 to a Lys, thereby preventing the aggregation of the IgG4 variant as effectively as in IgG1. A stabilizing effect was also recorded with other variable-region variants. Analysis of thermal stability using differential scanning calorimetry revealed that the R409K substitution increased the Tm value of CH3, suggesting that the R409K mutation contributed to the structural strengthening of the CH3-CH3 interaction. The R409K mutation did not influence the binding to antigens/human Fcγ receptors; whereas, the concurrent S228P and R409K mutations in IgG4 suppressed Fab-arm exchange drastically and as effectively as in IgG1, in both in vitro and in vivo in mice models. Our findings suggest that the IgG4 R409K variant represents a potential therapeutic IgG for use in low-effector-activity format that exhibits increased stability.

Main conclusion UDP-glucoserflavonoid 3-O-glucosyltransferase is essential for maintaining proper production quantity, acylation, and glucosylation of anthocyanin, and defects cause pale and dull flower pigmentation in morning glories. The Japanese (Ipomoea nil) and the common (I. purpurea) morning glory display bright blue and dark purple flowers, respectively. These flowers contain acylated and glucosylated anthocyanin pigments, and a number of flower color mutants have been isolated in I. nil. Of these, the duskish mutants of I. nil produce pale-and dull-colored flowers. We found that the Duskish gene encodes UDP-glucoseiflavonoid 3-O-glucosyltransferase (3GT). The duskish-1 mutation is a frameshift mutation caused by a 4-bp insertion, and duskish-2 is an insertion of a DNA transposon, Tpn10, at 1.3 kb upstream of the 3GT start codon. In the duskish-2 mutant, excision of Tpn10 is responsible for restoration of the expression of the 3GT gene. The recombinant 3GT protein displays expected 3GT enzymatic activities to catalyze 3-O-glucosylation of anthocyanidins in vitro. Anthocyanin analysis of a duskish-2 mutant and its germinal revertant showing pale and normal pigmented flowers, respectively, revealed that the mutation caused around 80 % reduction of anthocyanin accumulation. We further characterized two I. purpurea mutants showing pale brownish-red flowers, and found that they carry the same frameshift mutation in the 3GT gene. Most of the flower anthocyanins in the mutants were previously found to be anthocyanidin 3-O-glucosides lacking several caffeic acid and glucose moieties that are attached to the anthocyanins in the wild-type plants. These results indicated that 3GT is essential not only for production, but also for proper acylation and glucosylation, of anthocyanin in the morning glories.

While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17. These MET1a/MET1a met1b/met1b homozygotes exhibited abnormal seed phenotypes, which is associated with either viviparous germination or early embryonic lethality. They also displayed decreased levels of DNA methylation at repetitive CentO sequences and at the FIE1 gene locus in the embryos. In addition, independently isolated knock-in-targeted plants, in which the promoterless GUS reporter gene was fused with the endogenous MET1b promoter, showed the reproducible, dosage-dependent, and spatiotemporal expression patterns of GUS. The genotyping analysis of selfed progeny of heterozygous met1a met1b null mutants indicated that weakly active MET1a seems to serve as a genetic backup mechanism in rice met1b gametophytes, although the stochastic and uncoordinated activation of epigenetic backup mechanisms occurred less efficiently in the met1b homozygotes of rice than in the met1 homozygotes of Arabidopsis. Moreover, passive depletion of CG methylation during the postmeiotic DNA replication in the haploid nuclei of the met1a met1b gametophytes in rice results in early embryonic lethality. This situation somewhat resembles that of the met1 gametophytes in Arabidopsis.

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.

The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting (GT), has broad implications for basic and applied research. Rice (Oryza sativa), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, and the single-copy gene Waxy on chromosome 6 has been modified with a frequency of 1% per surviving callus by GT using a strong positive-negative selection. Because the strategy is independent of gene-specific selection or screening, it is in principle applicable to any gene. However, a gene in the multigene family or a gene carrying repetitive sequences may preclude efficient homologous recombination-promoted GT due to the occurrence of ectopic recombination. Here, we describe an improved GT procedure whereby we obtained nine independent transformed calli having the alcohol dehydrogenase2 (Adh2) gene modified with a frequency of approximately 2% per surviving callus and subsequently isolated eight fertile transgenic plants without the concomitant occurrence of undesirable ectopic events, even though the rice genome carries four Adh genes, including a newly characterized Adh3 gene, and a copy of highly repetitive retroelements is present adjacent to the Adh2 gene. The results indicate that GT using a strong positive-negative selection can be widely applicable to functional genomics in rice and presumably in other higher plants.

Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans. In this study, we aimed to establish a surrogate animal model that can be used for anti-EBOV and -MARV drug screening under BSL-2 conditions by focusing on the replication-competent recombinant vesicular stomatitis virus (rVSV) pseudotyped with the envelope glycoprotein (GP) of EBOV (rVSV/EBOV) and MARV (rVSV/MARV), which has been investigated as vaccine candidates and thus widely used in BSL-2 laboratories. We first inoculated mice, rats, and hamsters intraperitoneally with rVSV/EBOV and found that only hamsters showed disease signs and succumbed within 4 days post-infection. Infection with rVSV/MARV also caused lethal infection in hamsters. Both rVSV/EBOV and rVSV/MARV were detected at high titers in multiple organs including the liver, spleen, kidney, and lungs of infected hamsters, indicating acute and systemic infection resulting in fatal outcomes. Therapeutic effects of passive immunization with an anti-EBOV neutralizing antibody were specifically observed in rVSV/EBOV-infected hamsters. Thus, this animal model is expected to be a useful tool to facilitate in vivo screening of anti-filovirus drugs targeting the GP molecule.