Flagellin from Pseudomonas syringae pv. tabaci induced hrp-independent HR in tomato

We have previously shown that flagellin of Pseudomonas syringae pv. tabaci is an elicitor that induces a hypersensitive reaction (HR) in nonhost tomato cells. Flagellin is the major HR elicitor produced by this pathogen, as shown by the inability of a flagellin-defective mutant, ΔfliC, to induce HR. Also, a ΔfliD mutant that secretes large amounts of monomer flagellins induces a strong HR in tomato. In this study, the possible involvement of an Hrp type III secretion system (TTSS) in flagellin-induced HR was investigated using flagella-defective mutants or Hrp TTSS-defective mutants. The hrcC gene encodes HrcC protein, which is required for Hrp pilus formation in the outer membrane. An hrcC mutation, introduced into the wild-type, ΔfliC, and ΔfliD mutants of P. syringae pv. tabaci did not affect swimming motility or flagellin secretion, whereas all ΔhrcC, ΔfliC, and ΔfliD mutants lost the ability to cause disease on host tobacco leaves. However, the ΔhrcC mutant and the ΔfliD/ΔhrcC double mutant were still able to induce HR cell death, expression of one of the defense-related genes hsr203J, and the generation of hydrogen peroxide in nonhost tomato cells. Thus, flagellin is required for both pathogenicity in host tobacco and HR in nonhost tomato. On the other hand, hrp TTSS is necessary for pathogenicity on host tobacco but is not indispensable to induce HR in nonhost tomato. These results clearly show that flagellin-induced HR is hrp-independent in tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB049570     

Evaluation of the allelopathic activity of five Oxalidaceae cover plants and the demonstration of potent weed suppression by Oxalis species

Laboratory and field experiments were conducted to evaluate the usefulness of Oxalis spp. as allelopathic ground-cover plants for weed management. Some Oxalis spp. have previously been reported to possess strong allelopathic activities but few studies have been conducted on their activities in fields. This study aimed to investigate allelopathic activities and the possibility of weed suppression in five species of common Oxalis : shamrock oxalis ( Oxalis articulata Savigny), Bowie's woodsorrel ( Oxalis bowiei Lindl.), trefoil ( Oxalis brasiliensis Lodd. ex Knowl. et West.), lucky clover ( Oxalis deppei Lodd. ex Sweet) and Oxalis hirta L. The effects of the leachates from dry leaves and the exudates from living roots of these plant species were tested in laboratory experiments. The leachates from O. articulata , O. bowiei , O. deppei and O. hirta and the exudates from O. deppei caused > 84% inhibition of the radicle elongation of lettuce seedlings, but no effect was observed on the seed germination of lettuce. In the field experiment, O. deppei significantly reduced the weed population in July. A significant relationship was observed between the weed population and the percentage ground coverage of Oxalis spp. In contrast to the weed population, a significant relationship was observed between the weed above-ground biomass and the allelopathic activity of exudates from Oxalis spp.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

Hormone injections enhance the tolerance of land-locked ayu spermatozoa to cryopreservation

We evaluated the effects of maturation-stimulating hormones on the post-thaw motility of land-locked form ayu ( Plecoglossus altivelis ) spermatozoa. Male ayu were administered three intraperitoneal injections of either salmon pituitary extract (SPE; 0.2 or 0.6 mg g⁻¹ BW day⁻¹) or of 17, 20β-dihydroxy-4-pregnen-3-one (DHP; 2 or 10 μg g⁻¹ BW day⁻¹), the maturation-inducing steroid (MIS) in ayu. Before cryopreservation, the motility of spermatozoa of the SPE- and DHP-treated groups was significantly higher than that of the control group. Similarly, the comparative post-thaw motility (presented as a percentage of the motility obtained before cryopreservation) was significantly higher in the SPE group than in the control; however, there was no significant difference between the DHP group and the control. The effect of SPE and DHP on pre- and post-cryopreservation motility was not dose dependent. Our results suggest that the hormone(s) present in salmon pituitary are effective in enhancing the tolerance of ayu sperm cells to cryopreservation and that the MIS (DHP) is not involved in this process.     

Automatic counting of FISH-labeled microbes by an LED illuminated detecting apparatus

ABSTRACT:   The Bioplorer (BP) (Matsushita Ecology Systems Co. Ltd, Kasugai, Aichi-ken, Japan) is an apparatus that consists of a light-emitting diode and optical and image analysis systems. This instrument has been developed and used for the semi-automatic counting of total microbes, mainly in food, cosmetics and industrial products. In this study, the applicability of BP to the detection and enumeration of eukaryotic and prokaryotic cells, after the fluorescent in situ hybridization (FISH) procedure, was examined. The cells of a yeast ( Candida albicans ) and a bacterium ( Escherichia coli ) were specifically labeled using an oligonucleotide probe with fluorochrome and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The numbers of cells after FISH treatment and DAPI staining agreed well, indicating that the BP optical device and image analysis system had enough sensitivity to detect and quantify eukaryotic and prokaryotic cells. Because of its simplicity and reliability, BP can be a new tool for quantification of specifically labeled target microorganisms, both in industrial products and natural samples.     

Extracellular cyclophilin A possesses chemotaxic activity in cattle

Cyclophilin A (CyPA) was originally discovered in bovine thymocytes as a cytosolic binding protein of the immunosuppressive drug cyclosporine A. Recent studies have revealed that in mice and humans, CyPA is secreted from cells in injured or infected tissues and plays a role in recruiting inflammatory cells in those tissues. Here we found that in cattle abundant level of extracellular CyPA was observed in tissues with inflammation. To aid in investigating the role of extracellular CyPA in cattle, we generated recombinant bovine CyPA (rbCyPA) and tested its biological activity as an inflammatory mediator. When bovine peripheral blood cells were treated with rbCyPA in vitro, we observed that rbCyPA reacts with the membranous surface of granulocytes, monocytes and lymphocytes. Chemotaxis analysis showed that the granulocytes migrate toward rbCyPA and the migration is inhibited by pre-treatment with an anti-bovine CyPA antibody. These results indicate that, as for mice and humans, extracellular CyPA possesses chemotactic activity to recruit inflammatory cells (e.g., granulocytes) in cattle, and could thus be a potential therapeutic target for the treatment of inflammation.     

CEP peptide induces susceptibility of Arabidopsis thaliana to non-adapted pathogens

CEP peptide was synthesized and tested for induction of disease susceptibility using Arabidopsis Col-0. When Colletotrichum tropicale was used as a non-adapted fungal pathogen, the conidia germinated to form hyphal-like structures, which successfully penetrated epidermis, eventually causing disease symptoms. In such case, PEN2-, but not PEN3-dependent resistance was likely suppressed by CEP peptide. Similarly, the CEP peptide-mediated disease susceptibility was also effective to a non-adapted bacterial pathogen. Notably, such induced susceptibility was also evident on Arabidopsis mutants lacking the previously identified receptors, suggesting that the CEP peptide modulates Arabidopsis immunity through an unidentified receptor(s).

     

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.     

The Cassiopeia A supernova was of type IIb

Cassiopeia A is the youngest supernova remnant known in the Milky Way and a unique laboratory for supernova physics. We present an optical spectrum of the Cassiopeia A supernova near maximum brightness, obtained from observations of a scattered light echo more than three centuries after the direct light of the explosion swept past Earth. The spectrum shows that Cassiopeia A was a type IIb supernova and originated from the collapse of the helium core of a red supergiant that had lost most of its hydrogen envelope before exploding. Our finding concludes a long-standing debate on the Cassiopeia A progenitor and provides new insight into supernova physics by linking the properties of the explosion to the wealth of knowledge about its remnant.