[Show abstract][Hide abstract] ABSTRACT: In our previous study we found that the expression of stlA showed peaks both in the early and last stages of development and that a product of SteelyA, 4-methyl-5-pentylbenzene-1,3-diol (MPBD), controlled Dictyostelium spore maturation during the latter. In this study we focused on the role of SteelyA in early stage development.
PLoS ONE 09/2014; 9(9):e106634. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28 °C than at 22 °C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.
Journal of plant physiology 03/2014; 171(6):382–388. · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fatty acids are fundamental cellular components, and provide essential building blocks for membrane biosynthesis. Although the use of gene knockout mutants is a robust method for examining the function of specific cellular metabolic networks, fatty acid synthase knockout mutants are extremely difficult to isolate. In the Dictyostelium discoideum genome, we found two putative fatty acid synthase genes, and we created a knockout mutant for one of them to examine the physiological consequences. In this study, we found that a continuous fatty acid supply was necessary for normal development, and the fatty acid synthase knockout mutant showed severe developmental delay. This developmental defect was corrected in chimeras composed of wild type cells and knockout mutant cells (3:7, respectively). The knockout mutant also showed aberrant expression of fatty acid biosynthesis genes. These results showed that D. discoideum needs correct fatty acid synthesis for normal development.
Journal of oleo science 02/2014; · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of SteelyA enzyme, controls Dictyostelium spore maturation. Since the expression of stlA split the in early and terminal stages, we cannot exclude the possibility that MPBD regulates spore differentiation from the early stage by creating a bias between the cells. 1-(3,5-Dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-on (DIF-1), a product of SteelyB, was identified as the major stalk cell inducer by in vitro assay, but in vivo assay revealed that DIF-1 induces only prestalkB (pstB) and prestalkO (pstO) cells and, that the major prestalkA (pstA) cells differentiated without DIF-1. In order to determine mechanism of polyketide regulated pattern formation, we examined the spatial expression patterns of prestalk and prespore markers in stlA and stlB knockout mutants. We found that MPBD regulates spore maturation at the culmination stage. We also found that the stlA and stlB double-knockout mutant lost pstA marker gene expression.
Bioscience Biotechnology and Biochemistry 10/2013; · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The signalling molecule 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one (DIF-1) is required for differentiation and pattern formation in Dictyostelium discoideum development. DIF-1 is synthesized by three enzymes, a hybrid polyketide synthase, a flavin-dependent halogenase, and a des-methyl-DIF-1 methyltransferase. The genome data on the related species D. purpureum are now public. Using this genome information, des-methyl-DIF-1 methyltransferase of D. purpureum was identified, and was named Dp dmtA. Overexpression of Dp dmtA complemented the defects in basal disc formation and lower cup formation in a dmtA knock-out mutant of D. discoideum. This indicates that Dp dmtA has the same function as D. discoideum dmtA and compensates for loss of the dmtA gene in the D. discoideum dmtA mutant. The materials released in the medium by D. purpureum contained stalk-inducing activity with the same retention time as that of DIF-1 in HPLC fractionation. This indicates that the stalk-inducing signal of DIF-1 and des-methyl-DIF-1 methyltransferase are conserved in D. purpureum.
Bioscience Biotechnology and Biochemistry 09/2012; 76(9):1672-6. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protein encoded by the activated disease resistance 1-like1 (ADR1-L1) gene (locus name, At4g33300) belongs to the activated disease resistance 1 (ADR1) family of coiled-coil nucleotide-binding
site leucine-rich repeat-type disease resistance proteins. This family contains four proteins and they have specific features
in their amino acid sequences. It has been reported that ADR1 protein belongs to the ADR1 family, which is related to not
only defense response but also drought tolerance. We found that transgenic plants overexpressing the ADR1-L1 gene showed a dwarf phenotype and morphological change in leaves. The expression levels of defense-related genes and the
resistance to Pseudomonas syringae pv. tomato DC3000 were increased in transgenic plants. However, enhancement of drought tolerance and activation of abiotic response
genes were not observed. When the growth temperature was changed from 22°C to 28°C, the expression of defense-related genes
and the enhancement of resistance to a bacterial pathogen were suppressed and the dwarf phenotype and morphological change
of leaves recovered.
–Disease resistance–Dwarf phenotype–
R gene–SA signaling
Journal of Plant Biology 06/2011; 54(3):172-179. · 1.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genome of Dictyostelium contains two novel hybrid-type polyketide synthases (PKSs) known as 'Steely'; the Steely enzyme is formed by the fusion of type I and type III PKSs. One of these enzymes, SteelyB, is known to be responsible for the production of the stalk cell-inducing factor DIF-1 in vivo. On the other hand, the product(s) and expression pattern of SteelyA are not clearly understood, because there are two different reports associated with the in vitro products of SteelyA and its expression pattern. To solve this problem, we first examined the expression pattern using two different primer sets and found that it was quite similar to that shown in the dictyExpress database. stlA expression peaked at approximately 3 h and declined, but showed a small peak around the end of development. Next, we examined the in vivo product of SteelyA using a stlA null mutant and found that the mutant lacked 4-methyl-5-pentylbenzene-1,3-diol (MPBD). This null mutant showed aberrant, glassy sori, and most of the cells in the sori remained amoeba-like without a cell wall. This defect was restored by adding 200 nM of MPBD to the agar. These results indicate that SteelyA produces MPBD in vivo and induces spore maturation.
[Show abstract][Hide abstract] ABSTRACT: The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum.
We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict.
The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.
[Show abstract][Hide abstract] ABSTRACT: NAC proteins comprise one of the largest families of transcription factors in the plant genome. They are known to be involved in various aspects of plant development, but the functions of most of them have not yet been determined. ANAC036, a member of the Arabidopsis NAC transcription factor family, contains unique sequences that are conserved among various NAC proteins found in other plant species. Expression analysis of the ANAC036 gene indicated that this gene was strongly expressed in leaves. Transgenic plants overexpressing the ANAC036 gene showed a semidwarf phenotype. The lengths of leaf blades, petioles and stems of these plants were smaller than those in wild-type plants. Microscopy revealed that cell sizes in leaves and stems of these plants were smaller than those in wild-type plants. These findings suggested that ANAC036 and its orthologues are involved in the growth of leaf cells.
Journal of plant physiology 12/2009; 167(7):571-7. · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Comparative analysis of nucleotide sequences of the genomic region located around 100 map unit of chromosome 1 using two accessions,
Columbia (Col) and Landsberg erecta (Ler), of Arabidopsis thaliana was performed. High divergence was detected between them, and the length of the Ler sequence was half of corresponding sequence of Col. This divergence occurred by tandem duplication, deletion of large regions,
and insertion of unrelated sequences. These events led to the high polymorphism of plant disease resistant genes, which are
located in the analyzed region. It is highly probable that two-round duplication occurred, and the insertion sequences are
transposable elements. The data suggest that the analyzed region had been evolving until quite recently.
Journal of Plant Biology 12/2009; 52(6):616-624. · 1.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The collective motion of cells in a biological tissue originates from their individual responses to chemical and mechanical signals. The Dictyostelium slug moves as a collective of up to 100,000 cells with prestalk cells in the anterior 10-30% and prespore cells, intermingled with anterior-like cells (AL cells), in the posterior. We used traction force microscopy to measure the forces exerted by migrating slugs. Wild-type slugs exert frictional forces on their substratum in the direction of motion in their anterior, balanced by motive forces dispersed down their length. StlB- mutants lack the signal molecule DIF-1 and hence a subpopulation of AL cells. They produce little if any motive force in their rear and immediately break up. This argues that AL cells, but not prespore cells, are the motive cells in the posterior zone. Slugs also exert large outward radial forces, which we have analyzed during "looping" movement. Each time the anterior touches down after a loop, the outward forces rapidly develop, approximately normal to the almost stationary contact lines. We postulate that these forces result from the immediate binding of the sheath to the substratum and the subsequent application of outward "pressure," which might be developed in several different ways.
Cell Motility and the Cytoskeleton 09/2009; 66(12):1073-86. · 4.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The polyketide DIF-1 induces Dictyostelium amoebae to form stalk cells in culture. To better define its role in normal development, we examined the phenotype of a mutant blocking the first step of DIF-1 synthesis, which lacks both DIF-1 and its biosynthetic intermediate, dM-DIF-1 (des-methyl-DIF-1). Slugs of this polyketide synthase mutant (stlB(-)) are long and thin and rapidly break up, leaving an immotile prespore mass. They have approximately 30% fewer prestalk cells than their wild-type parent and lack a subset of anterior-like cells, which later form the outer basal disc. This structure is missing from the fruiting body, which perhaps in consequence initiates culmination along the substratum. The lower cup is rudimentary at best and the spore mass, lacking support, slips down the stalk. The dmtA(-) methyltransferase mutant, blocked in the last step of DIF-1 synthesis, resembles the stlB(-) mutant but has delayed tip formation and fewer prestalk-O cells. This difference may be due to accumulation of dM-DIF-1 in the dmtA(-) mutant, since dM-DIF-1 inhibits prestalk-O differentiation. Thus, DIF-1 is required for slug migration and specifies the anterior-like cells forming the basal disc and much of the lower cup; significantly the DIF-1 biosynthetic pathway may supply a second signal - dM-DIF-1.
[Show abstract][Hide abstract] ABSTRACT: Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.
Nature Chemical Biology 10/2006; 2(9):494-502. · 13.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes - the pstA cells - as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.
Biochimica et Biophysica Acta 06/2006; 1760(5):754-61. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The AtRE1retrotransposon of Arabidopsis thaliana is transcribed in both directions and the amount of sense RNA is much less than that of antisense RNA. The characterization of one cDNA derived from an antisense transcript showed that the antisense RNA is complementary to one half of the open reading frame and 5′-LTR. In order to study the function of this antisense RNA, we characterized the promoter activity for the antisense transcript. Primer extension analysis indicated that the antisense RNA is produced from an internal promoter and indicated the possibility of a plural initiation site of transcription. Promoter analysis using β-glucuronidase (GUS) gene as a reporter gene showed that the promoter sequence is localized within an approximately 200 bp region upstream from initiation sites of antisense RNA. However, no typical promoter sequences were detected in this region. Histochemical staining of GUS activity indicated that antisense RNAs are transcribed in pollens and actively proliferative regions of calluses. This result suggests that the expression of AtRE1 is repressed by antisense RNA but this mechanism functions in only limited organs.
[Show abstract][Hide abstract] ABSTRACT: Membrane fluidity is critical for proper membrane function and is regulated in part by the proportion of unsaturated fatty acids present in membrane lipids. The proportion of these lipids in turn varies with temperature and may contribute to temperature adaptation in poikilothermic organisms. The fundamental question posed in this study was whether the unsaturation of fatty acids contributes to the ability to adapt to temperature stress in Dictyostelium. First, fatty acid composition was analysed and it was observed that the relative proportions of dienoic acids changed with temperature. To investigate the role of dienoic fatty acids in temperature adaptation, null mutants were created in the two known Delta5 fatty acid desaturases (FadA and FadB) that are responsible for the production of dienoic fatty acids. The fadB null mutant showed no significant alteration in fatty acid composition or in phenotype. However, the disruption of fadA resulted in a large drop in dienoic fatty acid content from 51.2 to 4.1 % and a possibly compensatory increase in monoenoic fatty acids (40.9-92.4 %). No difference was detected in temperature adaptation with that of wild-type cells during the growth phase. However, surprisingly, mutant cells developed more efficiently than the wild-type at elevated temperatures. These results show that the fatty acid composition of Dictyostelium changes with temperature and suggest that the regulation of dienoic fatty acid synthesis is involved in the development of Dictyostelium at elevated temperatures, but not during the growth phase.
[Show abstract][Hide abstract] ABSTRACT: Dictyostelium is a favored model for studying problems in cell and developmental biology. To comprehend the genetic potential and networks that direct growth and multicellular development, we are performing a large-scale analysis of Dictyostelium cDNAs. Here, we newly determine 7720 nucleotide sequences of cDNAs from the multicellular, slug stage (S) and 10 439 from the unicellular, vegetative stage (V). The combined 26 954 redundant ESTs were computer assembled using the PHRAP program to yield 5381 independent sequences. These 5381 predicted genes represent about half of the estimated coding potential of the organism. One-third of them were classified into 12 functional categories. Although the overall classification patterns of the V and S libraries were very similar, stage-specific genes exist in every category. The majority of V-specific genes function in some aspect of protein translation, while such genes are in a minority in the S-specific and common populations. Instead, genes for signal transduction and multicellular organization are enriched in the population of S-specific genes. Genes encoding the enzymes of basic metabolism are mainly found in the common gene population. These results therefore suggest major differences between growing and developing Dictyostelium cells in the nature of the genes transcribed.
Nucleic Acids Research 02/2004; 32(5):1647-53. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.
[Show abstract][Hide abstract] ABSTRACT: Beta-oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or D-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB, encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Such a persistent accumulation was not detected in wild-type cells or axenically grown mutant cells. Bacterial phosphatidylethanolamine that contains abundant cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while dipalmitoyl phosphatidylethanolamine did not. These results suggest that MFE1 protects the cells from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes cellular lipid composition for proper development. Hence, we propose that this enzyme plays an irreplaceable role in the survival strategy of Dictyostelium cells to form spores for their efficient dispersal in nature.
[Show abstract][Hide abstract] ABSTRACT: When Dictyostelium cells starve, they express genes necessary for aggregation. Using insertional mutagenesis, we have isolated a mutant that does not aggregate upon starvation and that forms small plaques on bacterial lawns, thus indicating slow growth. Sequencing of the mutated locus showed a strong similarity to the catalytic domain of cdc2-related kinase genes. Phylogenetic analysis further indicated that the amino acid sequence was more close to cyclin-dependent kinase 8 than to the sequence of other cyclin-dependent kinases. Thus, we designated this gene as Ddcdk8. The Ddcdk8-null cells do not aggregate and grow somewhat more slowly than parental cells when being shaken in axenic medium or laid on bacterial plates. To confirm whether these defective phenotypes were caused by disruption of this gene, the Ddcdk8-null cells were complemented with DdCdk8 protein expressed from an endogenous promoter, but not an actin promoter, and when the complemented cells were then allowed to grow on a bacterial lawn, they began to aggregate as the food supply was depleted and finally became fruiting bodies. The results suggest that properly regulated DdCdk8 activity is essential for aggregation. Because, when starved, Ddcdk8-null cells do not express the acaA transcripts required for aggregation, we deduce that Ddcdk8 is epistatic for acaA expression, indicating that the DdCdk8 products may regulate expression of acaA and/or other genes.