Molecular-based characterization and bioengineering of Sorghum bicolor to enhance iron deficiency tolerance in iron-limiting calcareous soils.

Plant biomass can significantly contribute to alternative energy sources. Sorghum bicolor is a promising plant for producing energy, but is susceptible to iron deficiency, which inhibits its cultivation in iron-limiting calcareous soils. The molecular basis for the susceptibility of sorghum to iron deficiency remains unclear. Here, we explored the sorghum genome to identify genes involved in iron uptake and translocation. Iron deficiency-responsive gene expression was comparable to that in other graminaceous plants. A nicotianamine synthase gene, SbNAS1, was induced in response to iron deficiency, and SbNAS1 showed enzyme activity. Sorghum secreted 2'-deoxymugineic acid and other phytosiderophores under iron deficiency, but their levels were relatively low. Intercropping of sorghum with barley or rice rescued iron deficiency symptoms of sorghum. To produce bioengineered sorghum with enhanced tolerance to iron deficiency, we introduced four cassettes into sorghum: 35S promoter-OsIRO2 for activation of iron acquisition-related gene expression, SbIRT1 promoter-Refre1/372 for enhanced ferric-chelate reductase activity, and barley IDS3, and HvNAS1 genomic fragments for enhanced production of phytosiderophores and nicotianamine. The resultant single sorghum line exhibited enhanced secretion of phytosiderophores, increased ferric-chelate reductase activity, and improved iron uptake and leaf greenness compared with non-transformants under iron-limiting conditions. Similar traits were also conferred to rice by introducing the four cassettes. Moreover, these rice lines showed similar or better tolerance in calcareous soils and increased grain iron accumulation compared with previous rice lines carrying two or three comparable cassettes. These results provide a molecular basis for the bioengineering of sorghum tolerant of low iron availability in calcareous soils.

The basic leucine zipper transcription factor OsbZIP83 and the glutaredoxins OsGRX6 and OsGRX9 facilitate rice iron utilization under the control of OsHRZ ubiquitin ligases.

Under low iron availability, plants induce the expression of various genes for iron uptake and translocation. The rice (Oryza sativa) ubiquitin ligases OsHRZ1 and OsHRZ2 cause overall repression of these iron-related genes at the transcript level, but their protein-level regulation is unclear. We conducted a proteome analysis to identify key regulators whose abundance was regulated by OsHRZs at the protein level. In response to iron deficiency or OsHRZ knockdown, many genes showed differential regulation between the transcript and protein levels, including the TGA-type basic leucine zipper transcription factor OsbZIP83. We also identified two glutaredoxins, OsGRX6 and OsGRX9, as OsHRZ-interacting proteins in yeast and plant cells. OsGRX6 also interacted with OsbZIP83. Our in vitro degradation assay suggested that OsbZIP83, OsGRX6 and OsGRX9 proteins are subjected to 26S proteasome- and OsHRZ-dependent degradation. Proteome analysis and our in vitro degradation assay also suggested that OsbZIP83 protein was preferentially degraded under iron-deficient conditions in rice roots. Transgenic rice lines overexpressing OsGRX9 and OsbZIP83 showed improved tolerance to iron deficiency. Expression of iron-related genes was affected in the OsGRX9 and OsGRX6 knockdown lines, suggesting disturbed iron utilization and signaling. OsbZIP83 overexpression lines showed enhanced expression of OsYSL2 and OsNAS3, which are involved in internal iron translocation, in addition to OsGRX9 and genes related to phytoalexin biosynthesis and the salicylic acid pathway. The results suggest that OsbZIP83, OsGRX6 and OsGRX9 facilitate iron utilization downstream of the OsHRZ pathway.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Multicenter retrospective study of nivolumab for recurrent/metastatic oral squamous cell carcinoma.

OBJECTIVES: Immunotherapy with nivolumab for patients with recurrent/metastatic oral squamous cell carcinoma has not been evaluated. Here, we aimed to examine the efficacy, safety, and prognostic factors of nivolumab in these patients. MATERIALS AND METHODS: This multicenter retrospective observational study involved patients who received nivolumab between April 2017 and June 2019. The patient characteristics were evaluated for association with progression-free and overall survival. Progression-free and overall survival rates were calculated; parameters that were significant in the univariate analysis were used as explanatory variables. Independent factors for progression-free and overall survival were identified using multivariate analysis. RESULTS: Totally, 143 patients were included. The overall response and disease control rates were 27.3% and 46.2%, respectively. The median, 1- and 2-year progression-free survival rates were 2.7 months, 25.4%, and 19.2%, respectively; those for overall survival were 11.2 months, 47.3%, and 33.6%, respectively. The independent factors affecting progression-free survival were performance status and immune-related adverse event occurrence, whereas those affecting overall survival were performance status, target disease, and number of previous lines of systemic cancer therapy. Eight patients reported grade ≥3 immune-related adverse events. CONCLUSION: Nivolumab was effective for recurrent/metastatic oral squamous cell carcinoma treatment and was well tolerated by patients.

Palatoplasty for the Patient With Campomelic Dysplasia-Report of a Case and Review of the Literature.

Campomelic dysplasia (CMPD) is a skeletal disorder resulting from SOX9 gene mutations. Palatoplasty is rare due to a high lethality rate in infants from respiratory distress. Our patient had characteristic symptoms of CMPD, including short bowed limbs, macrocephaly, low-set ears, short palpebral fissures, hypertelorism, a flat nasal bridge, a long philtrum, micrognathia, and a cleft palate. We performed a Furlow palatoplasty when the patient was 2 years 9 months of age, after respiratory conditions had stabilized. We reviewed the literature of CMPD cases that underwent palatoplasty and discussed the optimal timing and surgical methods.

Iron deficiency-inducible peptide-coding genes OsIMA1 and OsIMA2 positively regulate a major pathway of iron uptake and translocation in rice.

Under low iron (Fe) availability, plants transcriptionally induce various genes responsible for Fe uptake and translocation to obtain adequate amounts of Fe. Although transcription factors and ubiquitin ligases involved in these Fe deficiency responses have been identified, the mechanisms coordinating these pathways have not been clarified in rice. Recently identified Fe-deficiency-inducible IRON MAN (IMA)/FE UPTAKE-INDUCING PEPTIDE (FEP) positively regulates many Fe-deficiency-inducible genes for Fe uptake in Arabidopsis. Here, we report that the expression of two IMA/FEP genes in rice, OsIMA1 and OsIMA2, is strongly induced under Fe deficiency, positively regulated by the transcription factors IDEF1, OsbHLH058, and OsbHLH059, as well as OsIMA1 and OsIMA2 themselves, and negatively regulated by HRZ ubiquitin ligases. Overexpression of OsIMA1 or OsIMA2 in rice conferred tolerance to Fe deficiency and accumulation of Fe in leaves and seeds. These OsIMA-overexpressing rice exhibited enhanced expression of all of the known Fe-deficiency-inducible genes involved in Fe uptake and translocation, except for OsYSL2, a Fe-nicotianamine transporter gene, in roots but not in leaves. Knockdown of OsIMA1 or OsIMA2 caused minor effects, including repression of some Fe uptake- and translocation-related genes in OsIMA1 knockdown roots. These results indicate that OsIMA1 and OsIMA2 play key roles in enhancing the major pathway of the Fe deficiency response in rice.

Roles of subcellular metal homeostasis in crop improvement.

Improvement of crop production in response to rapidly changing environmental conditions is a serious challenge facing plant breeders and biotechnologists. Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrients for plant growth and reproduction. These minerals are critical to several cellular processes including metabolism, photosynthesis, and cellular respiration. Regulating the uptake and distribution of these minerals could significantly improve plant growth and development, ultimately leading to increased crop production. Plant growth is limited by mineral deficiency, but on the other hand, excess Fe, Mn, Cu, and Zn can be toxic to plants; therefore, their uptake and distribution must be strictly regulated. Moreover, the distribution of these metals among subcellular organelles is extremely important for maintaining optimal cellular metabolism. Understanding the mechanisms controlling subcellular metal distribution and availability would enable development of crop plants that are better adapted to challenging and rapidly changing environmental conditions. Here, we describe advances in understanding of subcellular metal homeostasis, with a particular emphasis on cellular Fe homeostasis in Arabidopsis and rice, and discuss strategies for regulating cellular metabolism to improve plant production.

Application of heavy-ion-beam irradiation to breeding large rotifer.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

Defects in the rice aconitase-encoding OsACO1 gene alter iron homeostasis.

KEY MESSAGE: Rice aconitase gene OsACO1 is involved in the iron deficiency-signaling pathway for the expression of iron deficiency-inducible genes, either thorough enzyme activity or possible specific RNA binding for post-transcriptional regulation. Iron (Fe) is an essential element for virtually all living organisms. When plants are deficient in Fe, Fe acquisition systems are activated to maintain Fe homeostasis, and this regulation is mainly executed at the gene transcription level. Many molecules responsible for Fe uptake, translocation, and storage in plants have been identified and characterized. However, how plants sense Fe status within cells and then induce a transcriptional response is still unclear. In the present study, we found that knockdown of the OsACO1 gene, which encodes an aconitase in rice, leads to the down-regulation of selected Fe deficiency-inducible genes involved in Fe uptake and translocation in roots, and a decrease in Fe concentration in leaves, even when grown under Fe-sufficient conditions. OsACO1 knockdown plants showed a delayed transcriptional response to Fe deficiency compared to wild-type plants. In contrast, overexpression of OsACO1 resulted in the opposite effects. These results suggest that OsACO1 is situated upstream of the Fe deficiency-signaling pathway. Furthermore, we found that the OsACO1 protein potentially has RNA-binding activity. In vitro screening of RNA interactions with OsACO1 revealed that RNA potentially forms a unique stem-loop structure that interacts with OsACO1 via a conserved GGUGG motif within the loop structure. These results suggest that OsACO1 regulate Fe deficiency response either thorough enzyme activity catalyzing isomerization of citrate, or specific RNA binding for post-transcriptional regulation.

OsbHLH058 and OsbHLH059 transcription factors positively regulate iron deficiency responses in rice.

KEY MESSAGE: Subgroup IVc basic helix-loop-helix transcription factors OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in rice in a similar but distinct manner, putatively under partial control by OsHRZs. Under low iron availability, plants transcriptionally induce the expression of genes involved in iron uptake and translocation. OsHRZ1 and OsHRZ2 ubiquitin ligases negatively regulate this iron deficiency response in rice. The basic helix-loop-helix (bHLH) transcription factor OsbHLH060 interacts with OsHRZ1, and positively regulates iron deficiency-inducible genes. However, the functions of three other subgroup IVc bHLH transcription factors in rice, OsbHLH057, OsbHLH058, and OsbHLH059, have not yet been characterized. In the present study, we investigated the functions of OsbHLH058 and OsbHLH059 related to iron deficiency response. OsbHLH058 expression was repressed under iron deficiency, whereas the expression of OsbHLH057 and OsbHLH060 was moderately induced. Yeast two-hybrid analysis indicated that OsbHLH058 interacts with OsHRZ1 and OsHRZ2 more strongly than OsbHLH060, whereas OsbHLH059 showed no interaction. An in vitro ubiquitination assay detected no OsbHLH058 and OsbHLH060 ubiquitination by OsHRZ1 and OsHRZ2. Transgenic rice lines overexpressing OsbHLH058 showed tolerance for iron deficiency and higher iron concentration in seeds. These lines also showed enhanced expression of many iron deficiency-inducible genes involved in iron uptake and translocation under iron-sufficient conditions. Conversely, OsbHLH058 knockdown lines showed susceptibility to iron deficiency and reduced expression of many iron deficiency-inducible genes. OsbHLH059 knockdown lines were also susceptible to iron deficiency, and formed characteristic brownish regions in iron-deficient new leaves. OsbHLH059 knockdown lines also showed reduced expression of many iron deficiency-inducible genes. These results indicate that OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in a similar but distinct manner, and that this function may be partially controlled by OsHRZs.

Understanding the Complexity of Iron Sensing and Signaling Cascades in Plants.

Under iron-deficient conditions, plants induce the expression of a set of genes involved in iron uptake and translocation. This response to iron deficiency is regulated by transcriptional networks mediated by transcription factors (TFs) and protein-level modification of key factors by ubiquitin ligases. Several of the basic helix-loop-helix TFs and the HRZ/BTS ubiquitin ligases are conserved across graminaceous and non-graminaceous plants. Other regulators are specific, such as IDEF1 and IDEF2 in graminaceous plants and FIT/FER and MYB10/72 in non-graminaceous plants. IMA/FEP peptides positively regulate the iron-deficiency responses in a wide range of plants by unknown mechanisms. Direct binding of iron or other metals to some key regulators, including HRZ/BTS and IDEF1, may be responsible for intracellular iron-sensing and -signaling events. In addition, key TFs such as FIT and IDEF1 interact with various proteins involved in signaling pathways of plant hormones, oxidative stress and metal abundance. Thus, FIT and IDEF1 might function as hubs for the integration of environmental signals to modulate the responses to iron deficiency. In addition to local iron signaling, root iron responses are modulated by shoot-derived long-distance signaling potentially mediated by phloem-mobile substances such as iron, iron chelates and IMA/FEP peptides.

Rice HRZ ubiquitin ligases are crucial for response to excess iron.

Iron is essential for virtually all organisms but is toxic when present in excess. To acquire the proper amount of iron, plants induce expression of various genes involved in iron uptake and translocation in response to low iron availability. Two iron-binding ubiquitin ligases, OsHRZ1 and OsHRZ2, negatively regulate such iron deficiency responses in rice (Oryza sativa). Transgenic rice plants with repressed expression of OsHRZ1 and OsHRZ2 (HRZ knockdown lines) are tolerant to low iron availability and accumulate iron in shoots and seeds under both iron-sufficient and -deficient conditions without a growth penalty. Although the expression of OsHRZ1 and OsHRZ2 is transcriptionally upregulated under iron-deficient conditions, the physiological relevance of this induction is not known. In the present study, we analyzed the response of HRZ knockdown lines to excess iron. In the presence of severe excess iron, the HRZ knockdown lines grew worse than non-transformants. The HRZ knockdown lines showed stunted shoot and root growth and more severe leaf bronzing compared to non-transformants. Moreover, these lines accumulated more iron in shoots and exhibited severely elevated expression of various genes involved in iron uptake and translocation as well as jasmonate signaling compared to non-transformants. These results indicate that HRZ ubiquitin ligases are crucial for repressing iron deficiency responses and protecting cells from iron toxicity in the presence of excess iron. These results support the possibility that HRZs are intracellular Fe sensors and provide clues for developing plants tolerant of either iron deficiency or excess with higher iron contents in edible parts.

The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains.

KEY MESSAGE: Rice OsYSL9 is a novel transporter for Fe(II)-nicotianamine and Fe(III)-deoxymugineic acid that is responsible for internal iron transport, especially from endosperm to embryo in developing seeds. Metal chelators are essential for safe and efficient metal translocation in plants. Graminaceous plants utilize specific ferric iron chelators, mugineic acid family phytosiderophores, to take up sparingly soluble iron from the soil. Yellow Stripe 1-Like (YSL) family transporters are responsible for transport of metal-phytosiderophores and structurally similar metal-nicotianamine complexes. Among the rice YSL family members (OsYSL) whose functions have not yet been clarified, OsYSL9 belongs to an uncharacterized subgroup containing highly conserved homologs in graminaceous species. In the present report, we showed that OsYSL9 localizes mainly to the plasma membrane and transports both iron(II)-nicotianamine and iron(III)-deoxymugineic acid into the cell. Expression of OsYSL9 was induced in the roots but repressed in the nonjuvenile leaves in response to iron deficiency. In iron-deficient roots, OsYSL9 was induced in the vascular cylinder but not in epidermal cells. Although OsYSL9-knockdown plants did not show a growth defect under iron-sufficient conditions, these plants were more sensitive to iron deficiency in the nonjuvenile stage compared with non-transgenic plants. At the grain-filling stage, OsYSL9 expression was strongly and transiently induced in the scutellum of the embryo and in endosperm cells surrounding the embryo. The iron concentration was decreased in embryos of OsYSL9-knockdown plants but was increased in residual parts of brown seeds. These results suggested that OsYSL9 is involved in iron translocation within plant parts and particularly iron translocation from endosperm to embryo in developing seeds.

The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice.

Iron is an essential metal element for all living organisms. Graminaceous plants produce and secrete mugineic acid family phytosiderophores from their roots to acquire iron in the soil. Phytosiderophores chelate and solubilize insoluble iron hydroxide in the soil. Subsequently, plants take up iron-phytosiderophore complexes through specific transporters on the root cell membrane. Phytosiderophores are also thought to be important for the internal transport of various transition metals, including iron. In this study, we analyzed TOM2 and TOM3, rice homologs of transporter of mugineic acid family phytosiderophores 1 (TOM1), a crucial efflux transporter directly involved in phytosiderophore secretion into the soil. Transgenic rice analysis using promoter-ß-glucuronidase revealed that TOM2 was expressed in tissues involved in metal translocation, whereas TOM3 was expressed only in restricted parts of the plant. Strong TOM2 expression was observed in developing tissues during seed maturation and germination, whereas TOM3 expression was weak during seed maturation. Transgenic rice in which TOM2 expression was repressed by RNA interference showed growth defects compared with non-transformants and TOM3-repressed rice. Xenopus laevis oocytes expressing TOM2 released (14)C-labeled deoxymugineic acid, the initial phytosiderophore compound in the biosynthetic pathway in rice. In onion epidermal and rice root cells, the TOM2-GFP fusion protein localized to the cell membrane, indicating that the TOM2 protein is a transporter for phytosiderophore efflux to the cell exterior. Our results indicate that TOM2 is involved in the internal transport of deoxymugineic acid, which is required for normal plant growth.

Jasmonate signaling is activated in the very early stages of iron deficiency responses in rice roots.

Under low iron availability, plants induce the expression of various genes involved in iron uptake and translocation at the transcriptional level. This iron deficiency response is affected by various plant hormones, but the roles of jasmonates in this response are not well-known. We investigated the involvement of jasmonates in rice iron deficiency responses. High rates of jasmonate-inducible genes were induced during the very early stages of iron deficiency treatment in rice roots. Many jasmonate-inducible genes were also negatively regulated by the ubiquitin ligases OsHRZ1 and OsHRZ2 and positively regulated by the transcription factor IDEF1. Ten out of 35 genes involved in jasmonate biosynthesis and signaling were rapidly induced at 3 h of iron deficiency treatment, and this induction preceded that of known iron deficiency-inducible genes involved in iron uptake and translocation. Twelve genes involved in jasmonate biosynthesis and signaling were also upregulated in HRZ-knockdown roots. Endogenous concentrations of jasmonic acid and jasmonoyl isoleucine tended to be rapidly increased in roots in response to iron deficiency treatment, whereas these concentrations were higher in HRZ-knockdown roots under iron-sufficient conditions. Analysis of the jasmonate-deficient cpm2 mutant revealed that jasmonates repress the expression of many iron deficiency-inducible genes involved in iron uptake and translocation under iron sufficiency, but this repression is partly canceled under an early stage of iron deficiency. These results indicate that jasmonate signaling is activated during the very early stages of iron deficiency, which is partly regulated by IDEF1 and OsHRZs.

Transcriptomic features associated with energy production in the muscles of Pacific bluefin tuna and Pacific cod.

Bluefin tuna are high-performance swimmers and top predators in the open ocean. Their swimming is grounded by unique features including an exceptional glycolytic potential in white muscle, which is supported by high enzymatic activities. Here we performed high-throughput RNA sequencing (RNA-Seq) in muscles of the Pacific bluefin tuna (Thunnus orientalis) and Pacific cod (Gadus macrocephalus) and conducted a comparative transcriptomic analysis of genes related to energy production. We found that the total expression of glycolytic genes was much higher in the white muscle of tuna than in the other muscles, and that the expression of only six genes for glycolytic enzymes accounted for 83.4% of the total. These expression patterns were in good agreement with the patterns of enzyme activity previously reported. The findings suggest that the mRNA expression of glycolytic genes may contribute directly to the enzymatic activities in the muscles of tuna.

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tuna's RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

Nicotianamine synthase 2 localizes to the vesicles of iron-deficient rice roots, and its mutation in the YXXφ or LL motif causes the disruption of vesicle formation or movement in rice.

Graminaceous plants release mugineic acid family phytosiderophores (MAs) to acquire iron from the soil. Here, we show that deoxymugineic acid (DMA) secretion from rice roots fluctuates throughout the day, and that vesicles accumulate in roots before MAs secretion. We developed transgenic rice plants that express rice nicotianamine (NA) synthase (NAS) 2 (OsNAS2) fused to synthetic green fluorescent protein (sGFP) under the control of its own promoter. In root cells, OsNAS2-sGFP fluorescence was observed in a dot-like pattern, moving dynamically within the cell. This suggests that these vesicles are involved in NA and DMA biosynthesis. A tyrosine motif and a di-leucine motif, which have been reported to be involved in cellular transport, are conserved in all identified NAS proteins in plants. OsNAS2 mutated in the tyrosine motif showed NAS activity and was localized to the vesicles; however, these vesicles stuck together and did not move. On the other hand, OsNAS2 mutated in the di-leucine motif lost NAS activity and did not localize to these vesicles. The amounts of NA and DMA produced and the amount of DMA secreted by OsNAS2-sGFP plants were significantly higher than in non-transformants and domain-mutated lines, suggesting that OsNAS2-sGFP, but not the mutated forms, was functional in vivo. Overall, the localization of NAS to vesicles and the transport of these vesicles are crucial steps in NA synthesis, leading to DMA synthesis and secretion in rice.

Full-genome sequence of a novel myovirus, GF-2, infecting Edwardsiella tarda: comparison with other Edwardsiella myoviral genomes.

Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious infectious diseases in both marine and freshwater fish farms worldwide. Previously, we reported the complete genome sequences of three E. tarda-lytic bacteriophages (two podoviruses and a myovirus), which were isolated from fish tissues and fish-rearing seawater. Further genomic information regarding E. tarda phages is important for understanding phage-host interactions as well as for applications of the phages for the control of disease. Here, we report the complete genome sequence of a novel E. tarda phage (GF-2) of myovirus morphology (family Myoviridae), isolated from tissue homogenates of a cultured Japanese flounder (Paralichthys olivaceus) that succumbed to edwardsiellosis in Japan. The size of the entire genome was 43,129 bp, with a GC content of 51.3 % and containing 82 open reading frames (ORFs). The GF-2 genome possesses lysogeny-related genes that have not been found in the reported Edwardsiella phage genomes. Comparative genomics of Edwardsiella myophages suggest that the C-terminal domains of the tail fiber proteins have relevance to their host specificity. Thus, GF-2 genome information provides a novel resource for our understanding of the molecular mechanisms involved in their host specificity and for detection of E. tarda in aquaculture environments.

A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication.

BACKGROUND: Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. RESULTS: We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. CONCLUSIONS: The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.