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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: 1.21). 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.

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    • "RT-PCR analysis of the 14 genes indicated that ChBD-1, ChBD-6, ChBD-8, ChBD-13, and ChBD-15 are expressed in the rice sheath. We produced gene-disrupted mutants of the two lectin-type ChBD genes using the GGKO method (Saitoh et al. 2008), which allows observation of stage-and site-specific expression of the target gene using GFP fluorescence. Unlike RT-PCR analysis, GFP fluorescence observations revealed that only ChBD-1 is expressed in rice cells. "
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    ABSTRACT: We examined the localization of probe-accessible chitin in Magnaporthe oryzae, which causes rice blast disease, during the early infection process and the functions of two genes encoding a chitin-binding domain (ChBD). Invasive hyphae in the first-invaded rice cell showed little staining with fluorescently labeled wheat germ agglutinin, a probe to detect chitin. However, in the second-invaded cell, hyphae showed strong fluorescence, and treatment with chitinase diminished the signal. Fourteen ChBD genes encoding family 18 carbohydrate-binding module (CBM18) were isolated from a Japanese strain of M. oryzae, Ina86-137. Reverse transcription-polymerase chain reaction analysis demonstrated that ChBD-1, ChBD-6, ChBD-8, ChBD-13, and ChBD-15 are expressed in the rice sheath. Gene-targeted disruptants of ChBD-1 and ChBD-15 had no significant differences in invasive growth, pathogenicity, or tolerance to chitinase compared to the wild type. These results suggest that M. oryzae has a mechanism to evade being a substrate for the host chitinase in the first-invaded cell, but neither ChBD-1 nor ChBD-15 contributes to this mechanism. KeywordsCBM18–Chitin-binding domain–Chitin elicitor–Fungal cell wall– Magnaporthe oryzae –Rice
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    • "After verifying the amplified sequences, both fragments were cloned into the entry vector pETH to construct pETH-CPXB-KO. pCAMBIA-CPXB-KO was generated by the LR reaction of pETH-CPXB-KO with pCAMBIA- Bar-RfA and introduced into M. oryzae isolate Ina86-137 via Agrobacterium tumefaciens EH105, followed by selection as previously described (Saitoh et al. 2008). For complementation of ΔCPXB, the genomic DNA fragment containing the protein coding region (2,006 bp) and 5′-flank- ing region upstream of putative initiation codon (219 bp) was amplified by PCR. "
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    ABSTRACT: The biological role of a secretory catalase of the rice blast fungus Magnaporthe oryzae was studied. The internal amino acid sequences of the partially purified catalase in the culture filtrate enabled us to identify its encoding gene as a catalase-peroxidase gene, CPXB, among four putative genes for catalase or catalase-peroxidase in M. oryzae. Knockout of the gene drastically reduced the level of catalase activity in the culture filtrate and supernatant of conidial suspension (SCS), and increased the sensitivity to exogenously added H₂O₂ compared with control strains, suggesting that CPXB is the major gene encoding the secretory catalase and confers resistance to H₂O₂ in hyphae. In the mutant, the rate of appressoria that induced accumulation of H₂O₂ in epidermal cells of the leaf sheath increased and infection at early stages was delayed; however, the formation of lesions in the leaf blade was not affected compared with the control strain. These phenotypes were complimented by reintroducing the putative coding regions of CPXB driven by a constitutive promoter. These results suggest that CPXB plays a role in fungal defense against H₂O₂ accumulated in epidermal cells of rice at the early stage of infection but not in pathogenicity of M. oryzae.
    Molecular Plant-Microbe Interactions 11/2010; 24(2):163-71. DOI:10.1094/MPMI-07-10-0175 · 4.46 Impact Factor
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