Migration of prospindle before the first asymmetric division in germinating spore of Marchantia polymorpha

The development of the plant body starts with spore germination in bryophytes. In many cases, the first division of the spore occurs after germination and cell elongation of the spore. In Marchantia polymorpha, asymmetric division occurs upon spore germination to generate two daughter cells: the larger one retains the ability to divide and develops into the thallus via sporeling or protonema, while the smaller one maintains tip growth and differentiates into the first rhizoid, providing a scaffold for initial development. Although spore germination of M. polymorpha was described in the 19th century, the intracellular processes of the first asymmetric division of the spore have not been well characterized. In this study, we used live-cell imaging analyses to elucidate microtubule dynamics during the first asymmetric division concomitantly with germination. In particular, we demonstrated that the preprophase band was not formed in the spore and that the bipolar prospindle, which is a microtubule structure surrounding the nucleus during prophase, migrated from the center to the periphery in the spore, suggesting that it was the earliest visible sign of cell polarity. We also showed that the occurrence of asymmetric division depended on actin filaments. Our findings regarding the first division of the spore in M. polymorpha will lead to a better model for cell-autonomous asymmetric division in plants.     

Design and Application of a New Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum magneticum

A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.     

Diversification of three <Emphasis Type="Italic">APETALA1/FRUITFULL</Emphasis>-like genes in wheat

The genomes of grass family species have three paralogs of APETALA1/FRUITFULL (AP1/FUL)-like genes (FUL1, FUL2 and FUL3) that are derived from the FUL lineage. In this study, we focus on the different roles of the wheat AP1/FUL-like genes, WFUL1 (identical to VRN1), WFUL2 and WFUL3, during the transition from vegetative to reproductive growth. Sequence analysis indicated that there was a high level of variability in the amino acid sequence of the C-domain among three WFUL genes. Expression analyses using the spring wheat cultivar Chinese Spring indicated that WFUL1/VRN1 was expressed in leaves as well as spike primordia of non-vernalized plants at the vegetative stage just before phase transition, while WFUL2 and WFUL3 were not expressed in leaves. This result indicates that WFUL1/VRN1 performs a distinct role in leaves before phase transition. In young spikes, WFUL1/VRN1 and WFUL3 were expressed in all developing floral organs, whereas WFUL2 expression was restricted in the floral organs to the lemma and palea. Furthermore, yeast two-hybrid and three-hybrid analyses revealed that WFUL2, but not WFUL1/VRN1 or WFUL3, interacted with class B and class E proteins. These results suggest that WFUL2 of wheat has class A functions in specifying the identities of floral meristems and outer floral organs (lemma and palea) through collaboration with class B and class E genes.     

Properties of a fetal multipotent neural stem cell (NEP cell)

Multipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFR alpha), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.     

Construction of a high-resolution linkage map of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene bph2

The brown planthopper (BPH), Nilaparvata lugens Stål, is a significant insect pest of rice (Oryza sa-tiva L.). bph2 is one of the 12 major BPH resistance genes so far identified in several indica cultivars and two wild relatives. We have constructed a high-resolution linkage map as a foundation for map-based cloning of the bph2 locus. An advanced mapping population derived from a cross of ’Tsukushibare’ (a susceptible japonica cultivar) with ’Norin-PL4’ (an authentic bph2-introgression line) was used. Segregation analysis by the mass seedling test showed that bph2 behaved as a single dominant gene. Through bulked segregant analysis and linkage analysis, bph2 was located within a 3.2-cM region containing eight AFLP markers. One marker (KAM4) showed complete co-segregation with bph2, and bph2 was mapped within a 1.0-cM region delimited by KAM3 and KAM5, two flanking markers. KAM4 was converted into a PCR-based sequence-tagged-site (STS) marker and its co-segregation with bph2 was validated.     

Production of alkyl glucoside from cellooligosaccharides using yeast strains displaying Aspergillus aculeatus β-glucosidase 1

For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of β-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl β-d-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond β-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates.     

Negative Regulation of Melanoma Differentiation-associated Gene 5 (MDA5)-dependent Antiviral Innate Immune Responses by Arf-like Protein 5B

RIG-I-like receptors (RLRs), including retinoic acid-inducible gene-I (RIG-I) and MDA5, constitute a family of cytoplasmic RNA helicases that senses viral RNA and mounts antiviral innate immunity by producing type I interferons and inflammatory cytokines. Despite their essential roles in antiviral host defense, RLR signaling is negatively regulated to protect the host from excessive inflammation and autoimmunity. Here, we identified ADP-ribosylation factor-like protein 5B (Arl5B), an Arl family small GTPase, as a regulator of RLR signaling through MDA5 but not RIG-I. Overexpression of Arl5B repressed interferon β promoter activation by MDA5 but not RIG-I, and its knockdown enhanced MDA5-mediated responses. Furthermore, Arl5B-deficient mouse embryonic fibroblast cells exhibited increased type I interferon expression in response to MDA5 agonists such as poly(I:C) and encephalomyocarditis virus. Arl5B-mediated negative regulation of MDA5 signaling does not require its GTP binding ability but requires Arl5B binding to the C-terminal domain of MDA5, which prevents interaction between MDA5 and poly(I:C). Our results, therefore, suggest that Arl5B is a negative regulator for MDA5.