Construction of a Binary Vector for Knockout and Expression Analysis of Rice Blast Fungus Genes

Saitoh, K; Nishimura, Mika; Kubo, Yasuyuki; Hayashi, Nagao; Minami, Eiichi

doi:10.1271/bbb.70834

Cited by 16 publications

(14 citation statements)

References 8 publications

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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.…”

Section: Discussionmentioning

confidence: 99%

“…Vectors and Agrobacterium-mediated transformation Vectors used to knockout ChBD genes and for Agrobacterium-mediated transformation of M. oryzae were constructed using the GFP-tagging gene knockout (GGKO) method of Saitoh et al (2008). Table 1 lists the primers used to construct the knockout vectors.…”

Section: Rt-pcr Assaymentioning

confidence: 99%

“…We selected ChBD-1 and ChBD-15 from the three lectintype ChBD genes expressed in the inoculated rice sheath and generated gene-targeted knockout mutants of each using the GGKO method of Saitoh et al (2008). Successful gene-targeted disruption was confirmed by PCR (Fig.…”

Section: Knockout Of Chbd Genesmentioning

confidence: 99%

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Localization of probe-accessible chitin and characterization of genes encoding chitin-binding domains during rice–Magnaporthe oryzae interactions

2011

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We examined the localization of probeaccessible 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.

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Section: Discussionmentioning

confidence: 99%

Section: Rt-pcr Assaymentioning

confidence: 99%

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Localization of probe-accessible chitin and characterization of genes encoding chitin-binding domains during rice–Magnaporthe oryzae interactions

2011

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“…Agrobacterium ‐mediated transformation of M. oryzae was performed according to Saitoh et al. (). At least three transformants were selected for each vector construct, based on fluorescence intensity, growth, and conidiation on the media plate, and virulence.…”

Section: Methodsmentioning

confidence: 99%

Live‐cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe oryzae

2015

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The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live‐cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane‐rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High‐resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first‐invaded cell. Furthermore, a newly developed, long‐term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first‐invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection.

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“…To elucidate the function of the isolated genes, we generated null mutants of each gene and characterized their phenotypes (Kamakura et al 2002;Saitoh et al 2003;Kanamori et al 2005). In addition, to enhance the analysis of the genes, we developed a GFP-tagging gene knockout (GGKO) method that enables expression and functional analysis of a gene simultaneously (Saitoh et al 2008) and devised a simple method to obtain fungal DNA for molecular biological analyses (Saitoh et al 2006). …”

Section: Exploration Of the Genes Related To Appressorium Formationmentioning

confidence: 99%

Molecular biological studies on appressorium formation in the rice blast fungus

Saitoh

2010

J Gen Plant Pathol

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Construction of a Binary Vector for Knockout and Expression Analysis of Rice Blast Fungus Genes

Cited by 16 publications

References 8 publications

Localization of probe-accessible chitin and characterization of genes encoding chitin-binding domains during rice–Magnaporthe oryzae interactions

Localization of probe-accessible chitin and characterization of genes encoding chitin-binding domains during rice–Magnaporthe oryzae interactions

Live‐cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe oryzae

Molecular biological studies on appressorium formation in the rice blast fungus

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