Quality control of photosystem II: direct imaging of the changes in the thylakoid structure and distribution of FtsH proteases in spinach chloroplasts under light stress

　Under light stress, the reaction center-binding protein D1 of PSII is photo-oxidatively damaged and removed from PSII complexes by proteases located in the chloroplast. A protease considered to be responsible for degradation of the damaged D1 protein is the metalloprotease FtsH. We showed previously that the active hexameric FtsH protease is abundant at the grana margin and the grana end membranes, and this homo-complex removes the photodamaged D1 protein in the grana. Here, we showed a change in the distribution of FtsH in spinach thylakoids during excessive illumination by transmission electron microscopy (TEM) and immunogold labeling of FtsH. The change in distribution of the protease was accompanied by structural changes to the thylakoids, which we detected using spinach leaves by TEM after chemical fixation of the samples. Quantitative analyses showed several characteristic changes in the structure of the thylakoids, including shrinkage of the grana, outward bending of the marginal portions of the thylakoids and an increase in the height of the grana stacks under excessive illumination. The increase in the height of the grana stacks may include swelling of the thylakoids and an increase in the partition gaps between the thylakoids. These data strongly suggest that excessive illumination induces partial unstacking of the thylakoids, which enables FtsH to access easily the photodamaged D1 protein. Finally three-dimensional tomography of the grana was recorded to observe the effect of light stress on the overall structure of the thylakoids

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of E-rns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane. Author summaryIt is difficult to characterize the function of NS1 in the post-genome replication stages in the virus life cycle of flaviviruses. Here, by means of protein-protein interaction network analyses and mutagenesis scanning, we identified a unique mutation in NS1 by which the protein loses its secretory capacity while retaining its genome replication activity. Physicochemical assays using the mutant NS1 revealed that oligomerization of NS1 is responsible for the lipid association and secretion of NS1. In addition, we established a complementation assay that can evaluate the particle formation of Flaviviridae viruses. By using recombinant flaviviruses possessing the identified mutation in NS1, we clarified that NS1 is involved in particle formation. Our findings reveal that the flavivirus NS1 has at least two roles in the virus life cycles-namely, a role in infectious particle formation and a role in viral genome replication

A new pollination system: brood-site pollination by flower bugs in Macaranga (Euphorbiaceae)

†Background and Aims Macaranga (Euphorbiaceae) is a large genus of dioecious trees with approx. 260 species. To date, only one pollination study of the genus has reported brood-site pollination by thrips in M. hullettii. In this study, the pollination system of Macaranga tanarius is reported. †Methods The study was conducted on Okinawa and Amami Islands, Japan. Flower visitors on M. tanarius were collected and their pollen load and behaviour on the flowers examined, as well as inflorescence structure and reward for the pollinators. †Key Results The most abundant flower visitors found on the male and female inflorescences were Orius atratus (Anthocoridae, Hemiptera), followed by Decomioides schneirlai (Miridae, Hemiptera). Pollen load on O. atratus from flowering pistillate inflorescences was detected as well as from staminate flowers. Orius atratus and D. schneirlai are likely to use the enclosed chambers formed by floral bracts as breeding sites before and during flower anthesis, and feed on nectar on the adaxial surface of flower bracts. The extrafloral nectary has a ball-shaped structure and the contained nectar is not exposed; the hemipterans pierce the ball to suck out the nectar. †Conclusions The results indicate that the plant is pollinated by flower bugs breeding on the inflorescences. This study may be the first report of pollination systems in which flower bugs are the main pollinators. Similarity o

Possible models for RyDEN’s mechanism of action in the suppression of DENV infection.

<p>During DENV replication, PABPC1 and LARP1 are recruited to viral RNA, form a closed-loop structure of viral RNA with a cap-binding complex that includes eIF4G and eIF4E, and serve as positive regulators for the translation of viral proteins. RyDEN, whose expression is upregulated by IFN, specifically recognizes the DENV translation complex via interaction with viral RNA and PABPC1/LARP1. This interaction may interfere with the protein translation machinery of DENV RNA. Additionally, functions of PABPC1 and LARP1 in the regulation of mRNA turnover may be enhanced by interaction with RyDEN, resulting in the degradation of viral RNA in cytoplasmic foci such as P-bodies.</p

Decreased translation efficiency of DENV reporter constructs by RyDEN.

<p>(A) Puromycin labeling to monitor global protein synthesis. HepG2 cells expressing V5-RyDEN (left four lanes) or V5-DHFR (right four lanes) were cultured in the presence (20 μg/ml [CHX 20] and 10 μg/ml [CHX 10]) or absence (CHX 0) of cycloheximide. After 1 h incubation, 10 μg/ml of puromycin was added to the culture. Cells were harvested 40 min after puromycin pulse, and the cell lysate was subjected to immunoblotting (IB) using anti-puromycin antibody (top panel) and anti-actin antibody (bottom panel). (B) <i>In vitro</i> transcribed RNA of DENrepPAC2A-Rluc WT and its RdRp mutant, DENrepPAC2A-Rluc GVD, were transfected to V5-RyDEN (gray bars) or V5-DHFR (white bars)-expressing HepG2 cells, and cells were lysed and subjected to luciferase assay at 4 and 8 h after transfection. Luciferase activity in the cell lysate was normalized to total protein concentration. Statistical significance was determined by two-way ANOVA.</p

Interaction of RyDEN with PABPC1 and LARP1.

<p>(A) Affinity purification analysis for RyDEN. TAP tag-fused RyDEN (or BAP control protein) was expressed in HepG2 cells by lentiviral vector transduction, and protein complexes containing the TAP-RyDEN (or TAP-BAP) were isolated from cell lysates using IgG Sepharose beads. Purified proteins were visualized by silver staining on an SDS-PAGE gel and analyzed by MALDI TOF-TOF MS. MW, molecular weight marker. Note that protein band of TAP-BAP was overlapped with that of IgG heavy chain, both which were detected as 50 kDa bands in the gel. (B, C, D, and E) HepG2 cells were transfected with siRNA duplex against PABPC1 (siPABPC1, B and D) and LARP1 (siLARP1, C and E) or nonspecific siRNA duplex (siCtrl). Two days after transfection, cells were infected with DENV-2 at an MOI of 1. Two more days after infection, cells and culture supernatants were harvested for total RNA isolation (B and C) and plaque assay to quantify the virus titer (D and E), respectively. Levels of PABPC1 and LARP1 mRNA quantified by qRT-PCR were normalized with GAPDH mRNA levels. Statistical significance was determined by Student’s <i>t</i> test.</p

Critical contribution of RyDEN in IFN-mediated anti-DENV response.

<p>(A and B) Induction of RyDEN expression by IFN treatments. HeLa (A) and shRNA-expressing HepG2 (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.</p