Construction of a binary vector for knockout and expression analysis of rice blast fungus genes.

Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Bioscience Biotechnology and Biochemistry (Impact Factor: 1.27). 06/2008; 72(5):1380-3. DOI: 10.1271/bbb.70834
Source: PubMed

ABSTRACT We developed an efficient method to analyze gene function and expression of the rice blast fungus. We constructed a GATEWAY binary vector, which generates a gene-targeted disruptant carrying a green fluorescent protein gene under the native promoter of the target gene. Using this method, the knockout efficiency and expression patterns of two hypothetical genes were determined.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Site-specific recombination mediates the rearrangement of nucleic acids by the virtue of an recombinase acting on specific recognition sequences. Recombining activities belong either to the tyrosine- or serine-type group, based on the presence of specific residues in the catalytic centre, which can be further subdivided into families due to additional criteria. The most prominent systems are the λ phage integrase acting on att sites; the Cre recombinase from bacteriophage P1 with its loxP attachment sites; the FLP/FTR system of fungal origin, where it is required for 2-μm plasmid replication/amplification in yeast; and the prokaryotic β-recombinase that recombines six sites specifically in cis. Each of these has been exploited in fungal hosts of biotechnological, medical or general relevance, mainly for cloning projects, approaches of gene targeting, genome modification or screening purposes. With their precise and defined mode of action are site-specific recombination systems eminently suited for genetic tasks in fungi, like they are executed in functional studies at high throughput or modern approaches of synthetic biology.
    Applied Microbiology and Biotechnology 01/2014; DOI:10.1007/s00253-013-5480-y · 3.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The environmental conditions under which rhizodia are grown, strongly influence their symbiotic and growth behaviour. Chemostat is an appropriate culture system for studying organisms response to nutrient-limited environments. The type of nutrient limitation can severely affect the efficiency of bacterial growth yields. It was previously reported that Mn2+ affected rhizobial growth. The objective of this study was to obtain more information about the effect of Mn2+ on growth characteristics of rhizobia. Rhizobium etli was grown in chemostat culture under either carbon (mannitol) or nitrogen (ammonium) limiting conditions using a synthetic culture medium. Both types of culture were carried out without added MnSO4 or with a medium containing 1.0 μM of this salt. Carbon- or nitrogen-limited cultures without added MnSO4 in the medium were shown to be growing under a double nutrient limitation.The absence of Mn2+ had a great effect on the growth characteristics of R. etli. Yields on carbon substrate and on oxygen were much higher when cells were grown in a culture medium containing MnSO4. R. etli seemingly possesses two NADH dehydrogenases, which, in analogy with similar enzymes of Escherichia coli, have been tentatively named NDH-1 and NDH-2. Cells grown without added MnSO4 lacked NDH-1 activity, whereas in cells grown in the presence of 1.0 μM MnSO4 this enzyme was active.
    Microbiological Research 12/1997; 152(4):367-371. DOI:10.1016/S0944-5013(97)80053-5 · 1.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein, CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins, LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. GFP-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.
    Molecular Plant-Microbe Interactions 06/2014; DOI:10.1094/MPMI-03-14-0068-R · 4.46 Impact Factor