Ken-ichiro Saitoh

A simple method for a mini-preparation of fungal DNA

A simple method was established to prepare DNA from fungal mycelia cultured on an agar plate. The fungi tested successfully with this method contained Zygomycetes, Ascomycetes, Basidiomycetes, and Oomycetes. This method did not require any time-consuming steps to crush or digest mycelia or fractionation in a phenol–chloroform mixture. The DNA was easily extracted by immersing and dispersing the mycelial plugs in a specific buffer (200 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid, 200 mM NaCl, 1% n-lauroylsarcosine, pH 8.0), then concentrated by ethanol precipitation. The total time to complete the whole procedure was less than 1 h. The quality and quantity were sufficient for polymerase chain reaction amplification and Southern blot analysis.

A Novel Gene, CBP1, Encoding a Putative Extracellular Chitin-Binding Protein, May Play an Important Role in the Hydrophobic Surface Sensing of Magnaporthe grisea During Appressorium Differentiation

The conidial germ tube of the rice blast fungus, Magnaporthe grisea, differentiates a specialized cell, an appressorium, required for penetration into the host plant. Formation of the appressorium is also observed on artificial solid substrata such as polycarbonate. A novel emerging germ tube-specific gene, CBP1 (chitin-binding protein), was found in a cDNA subtractive differential library. CBP1 coded for a putative extracellular protein (signal peptide) with two similar chitin-binding domains at both ends of a central domain with homology to fungal chitin deacetylases and with a C-terminus domain rich in Ser/Thr related extracellular matrix protein such as agglutinin. The consensus sequence of the chitin-binding domain found in CBP1 has never been reported in fungi and is similar to the chitin-binding motif in plant lectins and plant chitinases classes I and IV. CBPI was disrupted in order to identify its function. Null mutants of CBP1 failed to differentiate appressoria normally on artificial surface but succeeded in normally differentiating appressoria on the plant leaf surface. Since the null mutant Cbp1- showed abnormal appressorium differentiation only on artificial surfaces and was sensitive to the chemical inducers, CBP1 seemed to play an important role in the recognition of physical factors on solid surfaces.

The Role of Catalase-Peroxidase Secreted by Magnaporthe oryzae During Early Infection of Rice Cells

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.

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

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.

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

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.

Targeted Gene Disruption of the Neuronal Calcium Sensor 1 Homologue in Rice Blast Fungus, Magnaporthe grisea

We isolated a neuronal calcium sensor 1/frequenin-like gene, Mg-NCS-1, from Magnaporthe grisea and evaluated the phenotypes of null-mutants of the gene. The putative Mg-NCS-1 protein showed high similarity to the other NCS-1 proteins. The null-mutants had normal growth and pathogenicity similar to the parental strain, but their growth was suppressed in high concentrations of Ca2+ or acidic conditions.

Possible roles and functions of LPL1 gene encoding lysophospholipase during early infection by Magnaporthe grisea

The rice blast fungus Magnaporthe grisea differentiates appressoria, which are required to attack its rice plant host. Clone A26, tentatively named LPL1, was previously found to be homologous to the known lysophospholipase genes from our subtractive cDNA library. The LPL1 protein had a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence, and the protein expressed in Escherichia coli had lysophospholipase activity. To clarify the functions and possible roles of LPL1, the gene was disrupted by targeted gene replacement. The ΔLPL1 mutants formed fewer appressoria on the hydrophobic surface of GelBond film, and the appressoria had reduced turgor pressure and penetration into cells of the leaf sheath. The ΔLPL1 mutants and wild-type differentiated normal appressoria on other artificial substrata such as polycarbonate plate and on rice leaf sheath. Cytological analysis of the appressoria indicated that ΔLPL1 mutants had a delay in the disappearance of lipid droplets. These findings imply that LPL1, phospholipid metabolism, or both are involved in glycerol biosynthesis and accumulation to generate turgor pressure in the appressorium. LPL1 was, however, dispensable for full pathogenicity, suggesting that other complementary pathways or similar genes related to phospholipid metabolism probably function in M. grisea.

Chitin-deacetylase activity induces appressorium differentiation in the rice blast fungus Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium to invade rice cells. In this report, we show that CBP1, which encodes a chitin-deacetylase, is involved in the induction phase of appressorium differentiation. We demonstrate that the enzymatic activity of Cbp1 is critical for appressorium formation. M. oryzae has six CDA homologues in addition to Cbp1, but none of these are indispensable for appressorium formation. We observed chitosan localization at the fungal cell wall using OGA488. This observation suggests that Cbp1-catalysed conversion of chitin into chitosan occurs at the cell wall of germ tubes during appressorium differentiation by M. oryzae. Taken together, our results provide evidence that the chitin deacetylase activity of Cbp1 is necessary for appressorium formation.

Identification and characterization of a novel gene encoding the NBS1 protein in Pyricularia oryzae.

The ascomycete Pyricularia oryzae (teleomorph: Magnaporthe oryzae) causes one of the most serious diseases known as rice blast. The Nijmegen breakage syndrome protein (NBS1) is essential for DNA repair; thus, we studied the P. oryzae NBS1 homolog (PoNBS1). A PoNBS1 null mutant exhibited high sensitivity to DNA damage-inducing agents. The mutant also exhibited the retarded hyphal growth, and induced abnormal conidial germination and shape, but showed normal appressorium formation. The phenotypes of the null mutant were complemented by introducing the cDNA of PoNBS1 driven by a TrpC promoter of Aspergillus nidulans. In addition, the null mutant similarly complemented with the PoNBS1 cDNA lacking the FHA domain that had a normal phenotype except for hyphal growth. These results suggest that PoNBS1 is involved in DNA repair and normal development in P. oryzae. Moreover, the FHA domain of PoNBS1 participates in normal hyphal growth. PoNBS1, a Pyricularia oryzae homolog of the Nijmegen breakage syndrome protein (NBS1) gene, is involved in DNA repair and normal development in P. oryzae.

Molecular biological studies on appressorium formation in the rice blast fungus

The rice blast fungus, Magnaporthe oryzae, initiates its infection by forming an appressorium, a specialized structure to penetrate the host plant, at the tip of the germ tube. Appressorium formation is thought to be a prerequisite for initiating the disease. Environmental factors such as the hydrophobicity of the contact surface (Lee and Dean 1994), surface hardness (Xiao et al. 1994), and plant cutin monomers (Gilbert et al. 1996) have been proposed as cues to trigger appressorium differentiation by M. oryzae. For the differentiation of the germ tube into an appressorium, the fungus is thought to perceive these environmental cues, then change its pattern of gene expression and its physiology. In this study, genes specifically expressed during appressorium formation were isolated, and the functions of the genes were analyzed by a reverse genetic approach.