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1.
Heredity (Edinb) ; 127(2): 167-175, 2021 08.
Article in English | MEDLINE | ID: mdl-34175895

ABSTRACT

Deformities in cultured fish species may be genetic, and identifying causative genes is essential to expand production and maintain farmed animal welfare. We previously reported a genetic deformity in juvenile red sea bream, designated a transparent phenotype. To identify its causative gene, we conducted genome-wide linkage analysis and identified two single nucleotide polymorphisms (SNP) located on LG23 directly linked to the transparent phenotype. The scaffold on which the two SNPs were located contained two candidate genes, duox and duoxa, which are related to thyroid hormone synthesis. Four missense mutations were found in duox and one in duoxa, with that in duoxa showing perfect association with the transparent phenotype. The mutation of duoxa was suggested to affect the transmembrane structure and thyroid-related traits, including an enlarged thyroid gland and immature erythrocytes, and lower thyroxine (T4) concentrations were observed in the transparent phenotype. The transparent phenotype was rescued by T4 immersion. Loss-of-function of duoxa by CRISPR-Cas9 induced the transparent phenotype in zebrafish. Evidence suggests that the transparent phenotype of juvenile red sea bream is caused by the missense mutation of duoxa and that this mutation disrupts thyroid hormone synthesis. The newly identified missense mutation will contribute to effective selective breeding of red sea bream to purge the causative gene of the undesirable phenotype and improve seed production of red sea bream as well as provide basic information of the mechanisms of thyroid hormones and its related diseases in fish and humans.


Subject(s)
Sea Bream , Animals , Genetic Linkage , Humans , Phenotype , Sea Bream/genetics , Thyroid Hormones , Zebrafish
2.
Nat Microbiol ; 4(10): 1654-1660, 2019 10.
Article in English | MEDLINE | ID: mdl-31235957

ABSTRACT

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that depend on living host plants to complete their life cycle1,2. This feature, which leads to their unculturability in the absence of plants, strongly hinders basic research and agricultural application of AM fungi. However, at least one AM fungus can grow and develop fertile spores independently of a host plant in co-culture with the bacterium Paenibacillus validus3. The bacteria-derived substances are thought to act as stimulants or nutrients for fungal sporulation, but these molecules have not been identified. Here, we show that (S)-12-methyltetradecanoic acid4,5, a methyl branched-chain fatty acid isolated from bacterial cultures, stimulates the branching of hyphae germinated from mother spores and the formation of secondary spores in axenic culture of the AM fungus Rhizophagus irregularis. Extensive testing of fatty acids revealed that palmitoleic acid induces more secondary spores than the bacterial fatty acid in R. irregularis. These induced spores have the ability to infect host plant roots and to generate daughter spores. Our work shows that, in addition to a major source of organic carbon6-9, fatty acids act as stimulants to induce infection-competent secondary spores in the asymbiotic stage and could provide the key to developing the axenic production of AM inoculum.


Subject(s)
Fatty Acids/pharmacology , Glomeromycota/drug effects , Mycorrhizae/drug effects , Culture Media, Conditioned , Fatty Acids/metabolism , Fatty Acids, Monounsaturated/pharmacology , Gene Expression Regulation, Fungal , Glomeromycota/genetics , Glomeromycota/growth & development , Glomeromycota/physiology , Hyphae/drug effects , Hyphae/genetics , Hyphae/growth & development , Hyphae/physiology , Mycorrhizae/genetics , Mycorrhizae/growth & development , Mycorrhizae/physiology , Paenibacillus/metabolism , Plant Roots/microbiology , Spores, Fungal/drug effects , Spores, Fungal/genetics , Spores, Fungal/growth & development , Spores, Fungal/physiology
4.
PLoS Genet ; 15(1): e1007865, 2019 01.
Article in English | MEDLINE | ID: mdl-30605473

ABSTRACT

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis.


Subject(s)
Lotus/genetics , Medicago truncatula/genetics , Mycorrhizae/growth & development , Symbiosis/genetics , Gene Expression Regulation, Plant/genetics , Lotus/growth & development , Lotus/microbiology , Medicago truncatula/growth & development , Medicago truncatula/microbiology , Mutation , Mycorrhizae/genetics , Plant Proteins/genetics , Plant Root Nodulation/genetics , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/microbiology , Rhizobium/genetics , Rhizobium/growth & development , Root Nodules, Plant/genetics , Root Nodules, Plant/growth & development , Root Nodules, Plant/microbiology
5.
Plant Divers ; 40(1): 41-44, 2018 Feb.
Article in English | MEDLINE | ID: mdl-30159540

ABSTRACT

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii, occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii, and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean = 3.2) and 0.13 to 0.82 (mean = 0.45), respectively. All loci were in Hardy-Weinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.

6.
Microbes Environ ; 33(3): 340-344, 2018 Sep 29.
Article in English | MEDLINE | ID: mdl-30146542

ABSTRACT

PCR clamping by locked nucleic acid (LNA) oligonucleotides is an effective technique for selectively amplifying the community SSU rRNA genes of plant-associated bacteria. However, the original primer set often shows low amplification efficiency. In order to improve this efficiency, new primers were designed at positions to compete with LNA oligonucleotides. Three new sets displayed higher amplification efficiencies than the original; however, efficiency varied among the primer sets. Two new sets appeared to be available in consideration of bacterial profiles by next-generation sequencing. One new set, KU63f and KU1494r, may be applicable to the selective gene amplification of plant-associated bacteria.


Subject(s)
Bacteria/genetics , Bacterial Typing Techniques/methods , Microbiota/genetics , Oligonucleotides/genetics , Plants/microbiology , Polymerase Chain Reaction , Ribosome Subunits, Small, Bacterial/genetics , Bacteria/classification , Bacteria/isolation & purification , DNA Primers/genetics , DNA, Bacterial/genetics , High-Throughput Nucleotide Sequencing , Mitochondria/genetics , Plants/genetics , Plastids/genetics , Sequence Analysis, DNA
7.
Nat Commun ; 9(1): 499, 2018 02 05.
Article in English | MEDLINE | ID: mdl-29403008

ABSTRACT

Legumes and rhizobia establish symbiosis in root nodules. To balance the gains and costs associated with the symbiosis, plants have developed two strategies for adapting to nitrogen availability in the soil: plants can regulate nodule number and/or stop the development or function of nodules. Although the former is accounted for by autoregulation of nodulation, a form of systemic long-range signaling, the latter strategy remains largely enigmatic. Here, we show that the Lotus japonicus NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1) gene encoding a NIN-LIKE PROTEIN transcription factor acts as a key regulator in the nitrate-induced pleiotropic control of root nodule symbiosis. NRSYM1 accumulates in the nucleus in response to nitrate and directly regulates the production of CLE-RS2, a root-derived mobile peptide that acts as a negative regulator of nodule number. Our data provide the genetic basis for how plants respond to the nitrogen environment and control symbiosis to achieve proper plant growth.


Subject(s)
Lotus/metabolism , Nitrates/metabolism , Plant Proteins/metabolism , Acetylene/chemistry , Cell Nucleus/metabolism , Cell Size , Fabaceae , Gene Expression Regulation, Plant , Mutation , Plant Proteins/genetics , Plant Root Nodulation , Plants, Genetically Modified , Rhizobium/physiology , Root Nodules, Plant/metabolism , Symbiosis
8.
Appl Plant Sci ; 5(5)2017 May.
Article in English | MEDLINE | ID: mdl-28529833

ABSTRACT

PREMISE OF THE STUDY: Microsatellite markers were developed and characterized for the critically endangered birch Betula chichibuensis (Betulaceae) to investigate the genetic structure of this species for conservation purposes. METHODS AND RESULTS: Sixteen microsatellite markers with di-, tri-, and tetranucleotide repeat motifs were developed and optimized using MiSeq paired-end sequencing. Of these, 14 were polymorphic, with two to five alleles per locus, in 47 individuals from two newly discovered populations of B. chichibuensis in Japan. Observed and unbiased expected heterozygosities per locus ranged from 0.000 to 0.617 and from 0.000 to 0.629, respectively. These markers were tested for cross-species amplification in B. maximowicziana, B. platyphylla var. japonica, and B. schmidtii. CONCLUSIONS: This set of microsatellite markers, the first developed for B. chichibuensis, will help elucidate spatial patterns of gene flow and levels of inbreeding in this species to aid its conservation.

9.
J Plant Res ; 129(5): 909-919, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27294965

ABSTRACT

Cell-to-cell communication, principally mediated by short- or long-range mobile signals, is involved in many plant developmental processes. In root nodule symbiosis, a mutual relationship between leguminous plants and nitrogen-fixing rhizobia, the mechanism for the autoregulation of nodulation (AON) plays a key role in preventing the production of an excess number of nodules. AON is based on long-distance cell-to-cell communication between roots and shoots. In Lotus japonicus, two CLAVATA3/ESR-related (CLE) peptides, encoded by CLE-ROOT SIGNAL 1 (CLE-RS1) and -RS2, act as putative root-derived signals that transmit signals inhibiting further nodule development through interaction with a shoot-acting receptor-like kinase HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1). Here, an in silico search and subsequent expression analyses enabled us to identify two new L. japonicus CLE genes that are potentially involved in nodulation, designated as CLE-RS3 and LjCLE40. Time-course expression patterns showed that CLE-RS1/2/3 and LjCLE40 expression is induced during nodulation with different activation patterns. Furthermore, constitutive expression of CLE-RS3 significantly suppressed nodule formation in a HAR1-dependent manner. TOO MUCH LOVE, a root-acting regulator of AON, is also required for the CLE-RS3 action. These results suggest that CLE-RS3 is a new component of AON in L. japonicus that may act as a potential root-derived signal through interaction with HAR1. Because CLE-RS2, CLE-RS3 and LjCLE40 are located in tandem in the genome and their expression is induced not only by rhizobial infection but also by nitrate, these genes may have duplicated from a common gene.


Subject(s)
Gene Expression Regulation, Plant , Genes, Plant , Lotus/genetics , Plant Proteins/genetics , Plant Root Nodulation/genetics , Amino Acid Sequence , Cytokinins/pharmacology , Gene Expression Regulation, Plant/drug effects , Lotus/drug effects , Nitrates/pharmacology , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Root Nodulation/drug effects , Plants, Genetically Modified , Real-Time Polymerase Chain Reaction , Root Nodules, Plant/drug effects , Root Nodules, Plant/metabolism , Transformation, Genetic
10.
New Phytol ; 211(4): 1202-8, 2016 09.
Article in English | MEDLINE | ID: mdl-27136716

ABSTRACT

Arbuscular mycorrhizal fungi translocate polyphosphate through hyphae over a long distance to deliver to the host. More than three decades ago, suppression of host transpiration was found to decelerate phosphate delivery of the fungal symbiont, leading us to hypothesize that transpiration provides a primary driving force for polyphosphate translocation, probably via creating hyphal water flow in which fungal aquaporin(s) may be involved. The impact of transpiration suppression on polyphosphate translocation through hyphae of Rhizophagus clarus was evaluated. An aquaporin gene expressed in intraradical mycelia was characterized and knocked down by virus-induced gene silencing to investigate the involvement of the gene in polyphosphate translocation. Rhizophagus clarus aquaporin 3 (RcAQP3) that was most highly expressed in intraradical mycelia encodes an aquaglyceroporin responsible for water transport across the plasma membrane. Knockdown of RcAQP3 as well as the suppression of host transpiration decelerated polyphosphate translocation in proportion to the levels of knockdown and suppression, respectively. These results provide the first insight into the mechanism underlying long-distance polyphosphate translocation in mycorrhizal associations at the molecular level, in which host transpiration and the fungal aquaporin play key roles. A hypothetical model of the translocation is proposed for further elucidation of the mechanism.


Subject(s)
Aquaporins/metabolism , Gene Silencing , Lotus/microbiology , Mycorrhizae/physiology , Nicotiana/microbiology , Plant Viruses/metabolism , Polyphosphates/metabolism , Symbiosis , Aquaporins/genetics , Biological Transport , Gene Knockdown Techniques , Genes, Fungal , Glomeromycota/genetics , Glomeromycota/physiology , Models, Biological , Mycelium/metabolism , Phylogeny , Plant Transpiration/physiology
11.
Mol Plant Microbe Interact ; 29(4): 277-86, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26757243

ABSTRACT

Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plants and fungi. To provide novel insights into the molecular mechanisms of AM symbiosis, we screened and investigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants. R. irregularis genes involved in the infection were explored by RNA-sequencing (RNA-seq) analysis. One of the identified genes was then characterized by a reverse genetic approach using host-induced gene silencing (HIGS), which causes RNA interference in the fungus via the host plant. The RNA-seq analysis revealed that 19 genes are up-regulated by both treatment with strigolactone (SL) (a plant symbiotic signal) and symbiosis. Eleven of the 19 genes were predicted to encode secreted proteins and, of these, SL-induced putative secreted protein 1 (SIS1) showed the largest induction under both conditions. In hairy roots of Medicago truncatula, SIS1 expression is knocked down by HIGS, resulting in significant suppression of colonization and formation of stunted arbuscules. These results suggest that SIS1 is a putative secreted protein that is induced in a wide spatiotemporal range including both the presymbiotic and symbiotic stages and that SIS1 positively regulates colonization of host plants by R. irregularis.


Subject(s)
Fungal Proteins/genetics , Gene Expression Regulation, Plant , Glomeromycota/genetics , Medicago truncatula/microbiology , Mycorrhizae/genetics , Symbiosis , Transcriptome , Fungal Proteins/metabolism , Gene Knockdown Techniques , Glomeromycota/physiology , Lactones/metabolism , Mycorrhizae/physiology , Plant Roots/microbiology , Sequence Analysis, RNA
12.
Plant Signal Behav ; 10(6): e1028706, 2015.
Article in English | MEDLINE | ID: mdl-26024424

ABSTRACT

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.


Subject(s)
Gibberellins/metabolism , Host-Pathogen Interactions , Mycorrhizae/growth & development , Plant Diseases/microbiology , Colony Count, Microbial , Gene Expression Regulation, Plant , Hyphae/physiology , Lotus/genetics , Lotus/microbiology , Models, Biological , Plant Diseases/genetics , Signal Transduction
13.
Plant Cell Physiol ; 56(8): 1490-511, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26009592

ABSTRACT

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Co-regulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis.


Subject(s)
Gene Expression Regulation, Plant , Glomeromycota/physiology , Lotus/genetics , Mycorrhizae/physiology , Transcriptome , Base Sequence , Fungal Proteins/genetics , High-Throughput Nucleotide Sequencing , Lotus/microbiology , Molecular Sequence Data , Plant Proteins/genetics , Plant Roots/genetics , Plant Roots/microbiology , RNA, Fungal/genetics , RNA, Plant/genetics , Root Nodules, Plant/genetics , Root Nodules, Plant/microbiology , Seedlings/genetics , Seedlings/microbiology , Sequence Analysis, RNA , Symbiosis , Transcription Factors/genetics
14.
Plant Physiol ; 167(2): 545-57, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25527715

ABSTRACT

Arbuscular mycorrhiza is a mutualistic plant-fungus interaction that confers great advantages for plant growth. Arbuscular mycorrhizal (AM) fungi enter the host root and form symbiotic structures that facilitate nutrient supplies between the symbionts. The gibberellins (GAs) are phytohormones known to inhibit AM fungal infection. However, our transcriptome analysis and phytohormone quantification revealed GA accumulation in the roots of Lotus japonicus infected with AM fungi, suggesting that de novo GA synthesis plays a role in arbuscular mycorrhiza development. We found pleiotropic effects of GAs on the AM fungal infection. In particular, the morphology of AM fungal colonization was drastically altered by the status of GA signaling in the host root. Exogenous GA treatment inhibited AM hyphal entry into the host root and suppressed the expression of Reduced Arbuscular Mycorrhization1 (RAM1) and RAM2 homologs that function in hyphal entry and arbuscule formation. On the other hand, inhibition of GA biosynthesis or suppression of GA signaling also affected arbuscular mycorrhiza development in the host root. Low-GA conditions suppressed arbuscular mycorrhiza-induced subtilisin-like serine protease1 (SbtM1) expression that is required for AM fungal colonization and reduced hyphal branching in the host root. The reduced hyphal branching and SbtM1 expression caused by the inhibition of GA biosynthesis were recovered by GA treatment, supporting the theory that insufficient GA signaling causes the inhibitory effects on arbuscular mycorrhiza development. Most studies have focused on the negative role of GA signaling, whereas our study demonstrates that GA signaling also positively interacts with symbiotic responses and promotes AM colonization of the host root.


Subject(s)
Gene Expression Regulation, Plant , Gibberellins/metabolism , Glomeromycota/physiology , Lotus/genetics , Lotus/microbiology , Mycorrhizae/physiology , Symbiosis , Biosynthetic Pathways/genetics , Colony Count, Microbial , Gene Expression Regulation, Plant/drug effects , Genes, Plant , Gibberellins/biosynthesis , Gibberellins/pharmacology , Glomeromycota/drug effects , Glomeromycota/growth & development , Hyphae/drug effects , Lotus/drug effects , Models, Biological , Mycorrhizae/drug effects , Mycorrhizae/growth & development , Organ Specificity/genetics , Plant Growth Regulators/metabolism , Plant Roots/genetics , Plant Roots/microbiology , Signal Transduction/drug effects , Signal Transduction/genetics , Symbiosis/drug effects , Symbiosis/genetics , Up-Regulation/genetics
15.
New Phytol ; 204(3): 638-649, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25039900

ABSTRACT

Arbuscular mycorrhizal (AM) fungi accumulate a massive amount of phosphate as polyphosphate to deliver to the host, but the underlying physiological and molecular mechanisms have yet to be elucidated. In the present study, the dynamics of cationic components during polyphosphate accumulation were investigated in conjunction with transcriptome analysis. Rhizophagus sp. HR1 was grown with Lotus japonicus under phosphorus-deficient conditions, and extraradical mycelia were harvested after phosphate application at prescribed intervals. Levels of polyphosphate, inorganic cations and amino acids were measured, and RNA-Seq was performed on the Illumina platform. Phosphate application triggered not only polyphosphate accumulation but also near-synchronous and near-equivalent uptake of Na(+) , K(+) , Ca(2+) and Mg(2+) , whereas no distinct changes in the levels of amino acids were observed. During polyphosphate accumulation, the genes responsible for mineral uptake, phosphate and nitrogen metabolism and the maintenance of cellular homeostasis were up-regulated. The results suggest that inorganic cations play a major role in neutralizing the negative charge of polyphosphate, and these processes are achieved by the orchestrated regulation of gene expression. Our findings provide, for the first time, a global picture of the cellular response to increased phosphate availability, which is the initial process of nutrient delivery in the associations.


Subject(s)
Fungi/metabolism , Gene Expression Regulation, Fungal/physiology , Lotus/microbiology , Mycorrhizae/metabolism , Polyphosphates/metabolism , Transcriptome , Amino Acids/metabolism , Biological Transport , Cations/metabolism , Nitrogen/metabolism , RNA, Fungal/genetics , RNA, Fungal/metabolism
16.
Nucleic Acids Res ; 42(5): 3152-63, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24322300

ABSTRACT

The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a non-stop mRNA, and for ribosome-binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. Additionally, we determined the solution structure of the GGQ domain of YaeJ from E. coli using nuclear magnetic resonance spectroscopy. YaeJ and a human homolog, ICT1, had similar levels of PTH activity, despite various differences in sequence and structure. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129 and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. The equivalent residues were also found in the C-terminal extension of ICT1, allowing an appropriate sequence alignment between YaeJ and ICT1 proteins from various species. Single amino acid substitutions for each of these residues significantly decreased ribosome-binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.


Subject(s)
Carboxylic Ester Hydrolases/chemistry , Escherichia coli Proteins/chemistry , Ribosomes/metabolism , Amino Acid Sequence , Amino Acid Substitution , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Codon , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Proteins/chemistry , RNA, Transfer, Amino Acyl/metabolism , Ribosomal Proteins , Sequence Alignment
17.
Proc Natl Acad Sci U S A ; 110(50): 20117-22, 2013 Dec 10.
Article in English | MEDLINE | ID: mdl-24277808

ABSTRACT

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.


Subject(s)
Evolution, Molecular , Genome, Fungal/genetics , Glomeromycota/genetics , Mycorrhizae/genetics , Plants/microbiology , Symbiosis/genetics , Base Sequence , Molecular Sequence Data , Sequence Analysis, DNA
18.
Nucleic Acids Res ; 39(5): 1739-48, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21051357

ABSTRACT

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl-tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , RNA, Transfer, Amino Acyl/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Amino Acid Motifs , Carboxylic Ester Hydrolases/chemistry , Codon , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Hydrolysis , Protein Binding , Ribosomal Proteins/chemistry
19.
J Mol Biol ; 404(2): 260-73, 2010 Nov 26.
Article in English | MEDLINE | ID: mdl-20869366

ABSTRACT

The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69-162 of the 206-residue full-length protein forms a structure with a ß1-ß2-α1-ß3-α2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an α-helix (α1), instead of a ß-turn, is inserted between strand ß2 and strand ß3. A characteristic groove formed between α1 and the three-stranded antiparallel ß-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function.


Subject(s)
Mitochondrial Proteins/chemistry , Amino Acid Sequence , Animals , Base Sequence , Catalytic Domain , Cell Survival , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , In Vitro Techniques , Mice , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/genetics , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Termination Factors/chemistry , Peptide Termination Factors/genetics , Protein Structure, Tertiary , Proteins/antagonists & inhibitors , Proteins/chemistry , Proteins/genetics , RNA, Small Interfering/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Ribosomal Proteins , Sequence Homology, Amino Acid , Species Specificity
20.
Pain ; 117(3): 271-279, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16150545

ABSTRACT

Gangliosides are a family of sialic acid-containing glycosphingolipids that are highly enriched in the mammalian nervous system. In particular, b- and c-series gangliosides, all of which contain alpha-2,8 sialic acids, have been considered to play important roles in adhesion, toxin-binding, neurite extension, cell growth and apoptosis. However, the neurobiological functions of these series of gangliosides remain largely unknown. To clarify the function of b- and c-series gangliosides in pain sensation in vivo, we generated mice in whom the gene for the alpha-2,8-sialyltransferase (GD3 synthase), which is responsible for the generation of all b-series gangliosides as well as c-series gangliosides, was disrupted. Compared to the wild-type mice, the mutant mice exhibited increased sensory responses to thermal and mechanical stimuli as measured by a hot plate test and von Frey test. In contrast, the mutant mice showed decreased responses during the late phase of the formalin test. Paw edema and Fos expression in the spinal cord after formalin injection were significantly decreased in the mutant mice compared to the wild-type mice. No significant differences in the conduction velocity of the sciatic nerve, and no apparent morphologic differences in the spinal cord and the sciatic nerve were detected between the wild-type and the mutant mice. These results suggested that b- and c-series gangliosides are critical in the development and/or maintenance of the sensory nervous system responsible for the transmission of acute pain sensation and pain modulation. Moreover, they play an important role in the development of hyperalgesia induced by inflammation.


Subject(s)
Hyperalgesia/genetics , Hyperalgesia/physiopathology , Pain/psychology , Sialyltransferases/deficiency , Analysis of Variance , Animals , Behavior, Animal , Cell Count/methods , Formaldehyde , Gangliosides/analysis , Hot Temperature , Immunohistochemistry/methods , Mice , Mice, Knockout , Neural Conduction/physiology , Oncogene Proteins v-fos/metabolism , Pain/chemically induced , Pain/metabolism , Pain Measurement/methods , Pain Threshold/physiology , Physical Stimulation/adverse effects , Reaction Time/genetics , Sciatic Nerve/physiopathology , Sialyltransferases/physiology , Skin/innervation , Skin/metabolism , Spinal Cord/metabolism , Spinal Cord/pathology , Time Factors
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