Sterility is common in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice (Oryza sativa). Although multiple loci for plant hybrid sterility have been identified, it remains unknown how alleles of the loci interact at the molecular level. Here we show that a locus for indicajaponica hybrid male sterility, Sa, comprises two adjacent genes, SaM and SaF, encoding a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively. Most indica cultivars contain a haplotype SaM ؉ SaF ؉ , whereas all japonica cultivars have SaM ؊ SaF ؊ that diverged by nucleotide variations in wild rice. Male semi-sterility in this heterozygous complex locus is caused by abortion of pollen carrying SaM ؊ . This allele-specific gamete elimination results from a selective interaction of SaF ؉ with SaM ؊ , a truncated protein, but not with SaM ؉ because of the presence of an inhibitory domain, although SaM ؉ is required for this male sterility. Lack of any one of the three alleles in recombinant plants does not produce male sterility. We propose a two-gene/threecomponent interaction model for this hybrid male sterility system. The findings have implications for overcoming male sterility in inter-subspecific hybrid rice breeding.allelic interaction ͉ gamete selection ͉ hybrid sterility ͉ reproductive barrier ͉ two-gene/three-component model

Autotetraploid rice has greater genetic variation and higher vigor than diploid rice, but low pollen fertility is one of the major reasons for low yield of autotetraploid rice. Very little is known about the molecular mechanisms of low pollen fertility of autotetraploid rice. In this study, cytological observations and microarray analysis were used to assess the genetic variation during pollen development in autotetraploid and diploid rice. Many abnormal chromosome behaviors, such as mutivalents, lagged chromosomes, asynchronous cell division, and so on, were found during meiosis in autotetraploid. Microsporogenesis and microgametogenesis in autotetraploid rice was similar to diploid rice, but many different kinds of abnormalities, including microspores degeneration, multi-aperture, and abnormal cell walls, were found in autotetraploid rice. Compared with diploid rice, a total of 1,251 genes were differentially expressed in autotetraploid rice in pollen transcriptome, among them 1,011 and 240 genes were up-regulated and down-regulated, respectively. 124 and 6 genes were co-up-regulated and co-down-regulated during three pollen development stages, respectively. These results suggest that polyploidy induced up-regulation for most of the genes during pollen development. Quantitative RT-PCR was done to validate 12 differentially expressed genes selected from functional categories based on the gene ontology analysis. These stably expressed genes not only related to the pollen development genes, but also involved in cell metabolism, cell physiology, binding, catalytic activity, molecular transducer activity, and transcription regulator activity. The present study suggests that differential expression of some key genes may lead to complex gene regulation and abnormal pollen development in autotetraploid rice.

Xenotransplantation from pigs could provide a potential solution to the severe shortage of allogeneic donor organs. Because xenogeneic tissues are subject to vigorous immune rejection, tolerance induction is likely to be essential to the success of clinical xenotransplantation. Here we explore the possibility of inducing human T-cell tolerance to porcine xenografts through mixed chimerism. We previously showed that NOD/SCID-Tg mice expressing porcine cytokine transgenes permit the induction of durable porcine hematopoietic chimerism. In this study we achieved human T-cell development in these mice by engrafting human fetal thymus/liver tissues. In porcine hematopoietic chimeras, human thymus grafts were populated with porcine class II high cells in addition to human cells, and human T cells were tolerant of the porcine hematopoietic donor as measured by mixed lymphocyte reaction assay and skin grafting. This study proves the principle that porcine chimerism induces tolerance of xenoreactive human T cells. IntroductionThe severe shortage of allogeneic organ donors currently limits the number of transplantations performed. 1,2 This supply-demand disparity could be corrected by the ability to use organs from other species (xenografts). In view of the ethical issues and impracticalities associated with the use of nonhuman primates, interest has focused on other species, in particular the pig, as the most suitable organ donor species for humans. In addition to organ size and physiologic similarities to humans, the ability to rapidly breed and inbreed pigs makes them particularly amenable to genetic modifications that could improve their ability to function as organ donors to humans. [3][4][5][6][7] However, organ transplantations across discordant species barriers are subject to vigorous immunologic rejection. 1,8 One might, therefore, expect the amount of nonspecific immunosuppression that would be required to overcome xenograft rejection to be so great that recipients would succumb to infections. Thus, it would be highly desirable to eliminate the immune response to xenografts through the induction of tolerance.Mixed chimerism has been proven to be a powerful and reliable approach for tolerance induction across allogeneic and closely related xenogeneic barriers. 9-14 Our recent studies showed that this approach also induces tolerance in a pig-to-mouse, highly disparate xenogeneic combination. 15 However, the ability of mixed chimerism to induce human T-cell tolerance to porcine xenografts has yet to be explored because of the lack of a suitable model system. It has been unclear whether or not genetic incompatibilities in immune cytokines, 16,17 accessory molecules, [17][18][19] and other unknown molecules between the 2 species might impede the induction of human T-cell tolerance to porcine xenografts through mixed chimerism. To address these questions, we have developed a murine model that permits the induction of both sustained porcine hematopoietic chimerism and active human thymopoiesis. Our results demonstrate...

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

The purification and characterization of a novel extracellular beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila J18 were studied. The strain produced the maximum level of extracellular beta-glucanase (135.6 U mL(-1)) when grown in a medium containing corncob (5%, w/v) at 50 degrees C for 4 days. The crude enzyme solution was purified by 122.5-fold with an apparent homogeneity and a recovery yield of 8.9%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 38.6 kDa. The molecular masses were 34.6 kDa and 31692.9 Da when detected by gel filtration and mass spectrometry, respectively, suggesting that it is a monomeric protein. The enzyme was a glycoprotein with a carbohydrate content of 19.0% (w/w). Its N-terminal sequence of 10 amino acid residues was determined as H2N-A(?)GYVSNIVVN. The purified enzyme was optimally active at pH 7.0 and 70 degrees C. It was stable within pH range 4.0-10.0 and up to 65 degrees C, respectively. Substrate specificity studies revealed that the enzyme is a true beta-1,3-1,4-D-glucanase. The K m values determined for barley beta-D-glucan and lichenan were 2.46 and 1.82 mg mL(-1), respectively. The enzyme hydrolyzed barley beta-D-glucan and lichenan to yield bisaccharide, trisaccharide, and tetrasaccharide as the main products. Circular dichroism studies indicated that the protein contains 28% alpha-helix, 24% beta-sheet, and 48% random coil. Circular dichroism spectroscopy is also used to investigate the thermostability of the purified enzyme. This is the first report on the purification and characterization of a beta-1,3-1,4-glucanase from Paecilomyces sp. These properties make the enzyme highly suitable for industrial applications.

MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice.

The AAA protein Msp1 extracts mislocalized tail-anchored membrane proteins and targets them for degradation, thus maintaining proper cell organization. How Msp1 selects its substrates and firmly engages them during the energetically unfavorable extraction process remains a mystery. To address this question, we solved cryo-EM structures of Msp1-substrate complexes at near-atomic resolution. Akin to other AAA proteins, Msp1 forms hexameric spirals that translocate substrates through a central pore. A singular hydrophobic substrate recruitment site is exposed at the spiral’s seam, which we propose positions the substrate for entry into the pore. There, a tight web of aromatic amino acids grips the substrate in a sequence-promiscuous, hydrophobic milieu. Elements at the intersubunit interfaces coordinate ATP hydrolysis with the subunits’ positions in the spiral. We present a comprehensive model of Msp1’s mechanism, which follows general architectural principles established for other AAA proteins yet specializes Msp1 for its unique role in membrane protein extraction.

Autotetraploid rice is a useful germplasm that has four chromosome sets and strong biological advantages; however, low fertility limits its commercial utilization. Little information is available about the DNA variation and differential gene expressions associated with low fertility in autotetraploid rice. In the present study, 81 SNPs and 182 InDels were identified in T449 (an autotetraploid rice line with low fertility) compared to E249 (diploid counterpart) by whole-genome re-sequencing. We detected only three non-synonymous SNPs and six large-effect InDels, which were associated with three and six genes, respectively. A total of 75 meiosis-related differentially expressed genes were detected during the meiosis stage by transcriptome analysis, including OsMTOPVIB, which is essential for meiotic DSB formation, and OsMOF, which takes part in homologous chromosome pairing and synapsis. Approximately 20.69% lagging chromosome at metaphase I and 4.65% abnormal tetrad were observed in T449. Moreover, transcriptome analysis revealed down-regulation of a sucrose transporter (OsSUT5) and two monosaccharide transporters (OsMST1 and OsMST8) in T449 at the single microspore stage, and their expression levels were verified by qRT-PCR. Cytological observation of saccharide distribution showed abnormal accumulation of saccharides in T449 and the contents of fructose and glucose were markedly higher in T449 than E249 at the single microspore stage. Our results suggested that polyploidy not only induces abrupt expression changes in the meiosis-related genes that lead to abnormal chromosome behavior, but also causes changes in the saccharide distribution and expression patterns of saccharide-related genes, which jointly causes sterility in the autotetraploid rice.Electronic supplementary materialThe online version of this article (10.1007/s00438-018-1471-0) contains supplementary material, which is available to authorized users.