Recognition of pathogen-derived sphingolipids in Arabidopsis.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors, which induce defense responses. Here, we show that the ceramide Phytophthora infestans-ceramide D (Pi-Cer D) from the plant pathogenic oomycete P. infestans triggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2). 9-Methyl-branched sphingoid base is specific to microbes and induces plant immune responses by physically interacting with RDA2. Loss of RDA2 or NCER2 function compromised Arabidopsis resistance against an oomycete pathogen. Thus, we elucidated the recognition mechanisms of pathogen-derived lipid molecules in plants.

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

INTRODUCTION: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. MATERIALS AND METHODS: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. RESULTS: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. DISCUSSION: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases.

Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor.

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.

Glutamine protects articular chondrocytes from heat stress and NO-induced apoptosis with HSP70 expression.

OBJECTIVE: To investigate the effect of l-glutamine (Gln) on stress responses of chondrocytes exposed to heat stress or nitric oxide (NO). METHODS: Cultures of articular chondrocytes were established from rabbit joints, and treated for 12h with various concentrations of Gln (0-20 mM). In some experiments, cells were also treated with quercetin (Que), a heat shock protein 70 (HSP70) inhibitor. Heat stress (43 degrees C) was applied to the cells for 0-120 min. Apoptosis was induced by 0.5mM sodium nitroprusside (SNP) dihydrate that produces NO. After stress loading, HSP70 expression was detected by Western blot analysis. Cell viability was assessed by lactate dehydrogenase (LDH) release and tetrazolium salt-based assays, while apoptosis was evaluated by Hoechst 33342 staining, TUNEL methods and active caspase-3 determination. RESULTS: Gln demonstrated dose-dependent enhancing effect on stress-mediated induction of HSP70, while in the absence of any stress HSP70 was not induced by Gln alone. After heating or SNP loading, chondrocytes showed severe reduction in viability, while the cytotoxic outcome was almost completely abrogated by conditioning with Gln. The protective effect of Gln was significantly blocked by Que that effectively suppressed stress-induced HSP70 expression in chondrocytes. The Gln also rendered chondrocytes unsusceptible to NO-induced apoptosis that was frequently seen in SNP-treated culture. CONCLUSION: This study demonstrated that the treatment of chondrocytes with Gln protected the cells from heat stress and NO-induced apoptosis. These chondroprotective effects of Gln may be mediated by HSP70.

Mechanical stress induces expression of cytokines in human periodontal ligament cells.

OBJECTIVE: Periodontal tissue has a unique structure in that the human periodontal ligament (hPDL) lies between the hard tissues of cementum and alveolar bone. Although the role of cytokines in hPDL function is not clearly understood, we investigated the effect of mechanical stress as hydrostatic pressure (HP) on cytokine expression in hPDL cells. MATERIALS AND METHODS: The hPDL cells were obtained from a healthy maxillary third molar. After the third to fourth passage, the cells were exposed to HP ranging from 1 to 6 MPa as previously described. Total RNA was extracted and the expression of cytokine mRNA was determined by RT-PCR. RESULTS: The exposure to 6 MPa of HP caused no morphological changes of hPDL cells, and did not affect the cellular viability. No expression of IL-1beta, IL-6, IL-8, TNF-alpha, receptor activator of NF-lambdaB (RANK), receptor activator of NF-lambdaB ligand (RANKL), or osteoprotegerin mRNA was observed in the control cells under atmospheric pressure, whereas, in hPDL cells treated with HP, a pressure-dependent enhancement of IL-6, IL-8, RANKL, and OPG mRNA expression was observed between 10 and 60 min after the exposure to HP. CONCLUSION: These results suggest that hPDL cells may play a role in the production of cytokines in response to mechanical stress in vivo.

Genetic control of weediness traits and the maintenance of sympatric crop-weed polymorphism in pearl millet (Pennisetum glaucum).

Pearl millet (Pennisetum glaucum), a diploid outcrossing crop widely grown in semiarid tropics, provides a unique extant material for the study of crop-weed interactive evolution. Co-occurrence of a weedy, shattering type of pearl millet with the cultivated one is the rule in the traditional agro-ecosystem in the Sahel zone of Africa. Selfed progeny of weed-type plants invariably segregated into distinct weed and crop types in an approximately 3:1 ratio. Genetic analysis using a cleaved amplified polymorphic sequence (CAPS) marker strongly suggested that a series of differences between the crop and the weed types are determined by a single putative supergene that has two allelic types, C and W. The crop-type plants are CC homozygotes, and the weed-type plants are CW heterozygotes. WW homozygotes are sterile and rare in the field. Thus, the CW weed plants recurrently arise from crosses between the crop and the weed, as well as from crosses among the weed-type plants. The weed type appears to have a sufficiently high fitness to maintain the W allele in the pearl millet population, resulting in the perpetuation of this unique crop-weed polymorphism.

Serial analysis of gene expression (SAGE) of host-plant resistance to the cassava mosaic disease (CMD).

Cassava mosaic disease (CMD) is a viral disease of the important tropical staple crop cassava (Manihot esculenta) and preferred management involves use of host-plant resistance. The best available resistance is controlled by a single dominant gene. Serial analysis of gene expression (SAGE) was used to analyze the gene expression pattern in a bulk of 40 each of CMD resistant and susceptible genotypes drawn from a gene mapping progeny. Messenger RNA used for the SAGE analysis came from plants that were exposed to heavy disease pressure over a period of 2 years in the field. A total of 12,786 tags were studied, divided into 5733 and 7053 tags from the resistant and susceptible genotypes, respectively. Tag annotation was by PCR amplification using the tag sequence as sense primer and 4000 cassava ESTs generated from the bulk of CMD resistant genotypes. Annotation of more than 30 differentially expressed tags revealed several genes expressed during systemic acquired resistance (SAR) in plants and other genes involved in cell-to-cell and cytoplasm-to-nucleus virus trafficking. Differential expression of the most abundantly expressed tag, corresponding to a beta-tubulin gene, was confirmed by Northern Analysis. RFLP analysis of the tags in the parents and bulks of the CMD mapping progeny revealed only one tag, a WRKY transcription factor, associated with the region bearing the dominant CMD gene.

Serial Analysis of Gene Expression (SAGE) of Magnaporthe grisea: genes involved in appressorium formation.

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.

Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen, but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana.

Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens. We isolated cDNAs for these MAPKs (NbWIPKand NbSIPK) from Nicotiana benthamiana. The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen (Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N. benthamiana. Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P. cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.

Overexpression of Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza sativa L) cells.

Treatment of suspension-cultured cells of rice (Oryza sativa L.) with cell wall extract of rice blast fungus (Magnaporthe grisea) elicits a rapid generation of H2O2, alkalinization of culture medium, and eventual cell death. To elucidate genes involved in these processes, we exploited SAGE (Serial Analysis of Gene Expression) technique for the molecular analysis of cell death in suspension-cultured cells treated with the elicitor. Among the downregulated genes in the elicitor-treated cells, a BI-1 gene coding for Bax inhibitor was identified. Transgenic rice cells overexpressing Arabidopsis BI-1 gene showed sustainable cell survival when challenged with M. grisea elicitor. Thus, the plant Bax inhibitor plays a functional role in regulating cell death in the rice cell culture system.

Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana.

SUMMARY Mitogen-activated protein kinases (MAPKs) play pivotal roles in the signal transduction pathway of plant defence responses against pathogens. A search for MAPK-interacting proteins revealed an interaction between a Nicotiana benthamiana MAPK, SIPK (NbSIPK) and cytosolic Hsp90 (NbHsp90c-1) in yeast two-hybrid assay. To study the function of Hsp90 in disease resistance, we silenced NbHsp90c-1 in N. benthamiana by virus-induced gene silencing (VIGS) with Potato virus X (PVX). NbHsp90c-1 silenced plants exhibited: (1) a stunted phenotype, (2) no hypersensitive response (HR) development after infiltration with the Phytophthora infestans protein INF1 and a non-host pathogen Pseudomonas cichorii that normally triggers HR in N. benthamiana, (3) compromised non-host resistance to P. cichorii, and (4) consistently reduced transcription levels of PR (pathogenesis related) protein genes. Similar phenotypes were observed also for plants in which a cytosolic Hsp70 (NbHsp70c-1), a gene for another class of molecular chaperon, was silenced. Hsp90 was isolated as a MAPK-interacting protein in yeast two-hybrid assay, therefore we tested the effect of NbHsp90c-1 silencing as well as NbHsp70c-1 silencing on the HR development caused by infiltration of a hyperactive potato MAPKK (StMEK1(DD)). No difference in the timing or extent of HR was found among NbHsp90c-1 silenced, NbHsp70c-1 silenced and control plants. This result indicates that observed impairment of INF1- and P. cichorii-mediated HR development in NbHsp90c-1 silenced and NbHsp70c-1 silenced plants was not caused by the abrogation in MAPK function downstream of active MAPKK that leads to HR. These findings suggest essential roles of Hsp90 and Hsp70 in plant defence signal transduction pathway upstream or independent of the MAPK cascade.

A male and hermaphrodite specific RAPD marker for papaya ( Carica papayaL.).

The random amplified polymorphic DNA (RAPD) technique was used to determine the sex of a dioecious species, Carica papayaL., with three sex types, male, female and hermaphrodite. A 450 bp marker fragment, named PSDM(Papaya Sex Determination Marker), exists in all male and hermaphrodite plants but not in the female plants so far analyzed. The DNA sequence of PSDM exhibited no significant similarity to previously reported sequences. A sequence-characterized amplified region (SCAR) marker, SCARps, was developed from PSDM to determine the sex of papaya. Southern hybridization, using PSDM as a probe, showed that PSDM exists in the male and hermaphrodite genomes, but not in the female genome. This result strongly suggests that PSDM is located on the chromosome region that is specific to the male and the hermaphrodite. SCARps is a suitable marker for the precise and rapid diagnosis of sex in papaya.

Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus ( Magnaporthe grisea) in transgenic rice.

Transgenic rice ( Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.

Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus X vector.

A recombinant plasmid, pTXS.TH, was constructed to express the gene-encoding wasabi (Wasabia japonica) defensin with the potato virus X (PVX) vector. pTXS.TH allows the expression of defensin in the host Nicotiana benthamiana, and the defensin protein WT1 can be purified from virus-infected leaves by heat treatment and affinity chromatography. WT1 exhibits strong antifungal activity toward the phytopathogenic fungi Magnaporthe grisea (50% inhibitory concentration [IC50] = 5 microg/ml) and Botrytis cinerea (IC50 = 20 microg/ml) but is weakly active against the phytopathogenic bacterium Pseudomonas cichorii. This virus-mediated expression system is a rapid and efficient method to produce and characterize antimicrobial proteins in plants. It is particularly useful for the study of proteins that are difficult to produce with other expression systems.

Radial magnetic resonance imaging and pathological findings of acetabular labrum in dysplastic hips.

Radial MRI findings and pathological changes were comparatively examined in the acetabular labrum of 11 hips of 11 patients, who underwent total hip arthroplasty for osteoarthritis due to acetabular dysplasia. Diffuse high signal pattern on the radial MR images corresponded to histological degeneration of the labrum. High signal pattern which was equivalent to the synovial fluid, showed an intralabral tear. In the obscure areas of MR images, severe impairment of the labrum such as rupture, detachment, and displacement were found.

Rapid isolation of promoter sequences by TAIL-PCR: the 5'-flanking regions of Pal and Pgi genes from yams (Dioscorea).

Using a modified TAIL-PCR technique, the 5'-flanking regions of the phenylalanine ammonia lyase (Pal) genes of a yam species, Dioscorea bulbifera, and the phosphoglucose isomerase (Pgi) gene of D. tokoro were successfully isolated. Two novel modifications of the TAIL-PCR procedure introduced here, namely (1) the use of a battery of random 10-mers (RAPD primers) as short arbitrary primers, and (2) the use of a total of five nested, gene-specific primers, allow the rapid isolation of the 5'-flanking region of any gene from organisms with large genomes. Isolated 5'-flanking regions were fused to the gus gene, and tested for transient expression in tobacco BY2 cells. All the isolated 5'-flanking regions were shown to drive reporter gene expression. Three Pal promoters responded to salicylic acid, presumably as a result of the binding of a MYB transcriptional activator to the multiple MREs (Myb Recognition Elements) present in these regions.

Requirement of regulated activation of Ras for response of MDCK cells to hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.

cDNA cloning and molecular analysis of papaya small GTP-binding protein, pgp1.

In the course of papaya EST collection, one clone (pRA4-3) encoding partial sequence of papaya small GTP-binding protein gene, pgp1, was obtained. Based on the sequence information of pRA4-3, the entire coding region of pgp1 was cloned using the 3'RACE PCR technique. ORF of pgp1 is 636bp long and deduced molecular weight of the protein is 23,311. Phylogenetic analysis showed that PGP1 belongs to YPT/RAB group of the small GTP-binding protein and is a homologue of RAB2. Southern analysis showed that there are several pgp1-related genes in papaya genome. Northern analysis showed that pgp1 was expressed equally in stems of seedlings that were grown under light and dark conditions. This result shows that PGP1 is not involved in the phytochrome-mediated signal transduction as an auxin signal transducer in stems of papaya seedlings.

A method for estimating nucleotide diversity from AFLP data.

A method for estimating the nucleotide diversity from AFLP data is developed by using the relationship between the number of nucleotide changes and the proportion of shared bands. The estimation equation is based on the assumption that GC-content is 0.5. Computer simulations, however, show that this method gives a reasonably accurate estimate even when GC-content deviates from 0.5, as long as the number of nucleotide changes per site (nucleotide diversity) is small. As an example, the nucleotide diversity of the wild yam, Dioscorea tokoro, was estimated. The estimated nucleotide diversity is 0.0055, which is larger than estimations from nucleotide sequence data for Adh and Pgi.