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 findings of epiduroscopy corresponded to the symptoms. Epiduroscopy may reduce low back and leg pain in elderly patients with degenerative lumbar spinal stenosis, particularly those with radiculopathy.

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.Extraction of genomic DNA is an essential step for molecular analyses of fungi. The standard method to prepare fungal DNA consists of lyophilization of mycelia, disruption of cell walls by grinding, extraction of DNA in buffers containing sodium dodecyl sulfate, removal of proteins with a mixture of phenol and chloroform, and precipitation of DNA with 2-propanol ( Kawabe et al. 2004). This method is suitable to obtain a large amount of pure DNA, but is time consuming, labor intensive, and pollutes water with phenol and chloroform. Therefore, a rapid and simple method for DNA preparation on a small scale has been needed for polymerase chain reaction (PCR)-based identification and determination of genotypes such as mating types or drug resistance, and for screening transformants to obtain isolates with a targeted gene modification. Generally, a fungus isolated from the field or a mixture of transformants is grown in liquid culture before DNA extraction. If DNA can be successfully extracted directly from thalli on agar media, the total time for the identification or screening will be markedly decreased. Several methods for rapid DNA extraction have been proposed (they were not fully suitable for this purpose.In this report, a simple and reliable method was developed for the mini preparation of fungal DNA from thalli on agar media, based on the method of Liu et al. (2000). This method has several advantages:1. Preculture in liquid media is not necessary. 2. The duration of culture is more flexible and can be chosen before the preparation. 3. Potentially harmful organic solvents, such as phenol and chloroform, which are troublesome to discard, are excluded as exhaustively as possible. 4. DNA is less damaged by mechanical forces because freezing and homogenizing the thalli are not needed. 5. The minimized number of preparation steps and the lyophilized powder-free procedure reduce the risk of crosscontamination. 6. Total time of the procedure is less than 1 h.Magnaporthe grisea was cultured on Misato-Hara agar medium (0.2% yeast extract, 1% soluble starch, 1.5% agar) at 26°C. Fusarium oxysporum was cultured on potatosucrose agar medium [20% (w/v) potato extract, 0.5% sucrose, 1.5% agar] at 26°C. A small piece of mycelia (7-15 mm on a side) with agar medium (100-300 mg total m...

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.

Histological criteria for subclassification of intraductal papillary mucinous tumor (IPMT) and mucinous cystic tumor (MCT) of the pancreas remain ambiguous in the absence of apparent invasion or metastasis. To elucidate this issue, we evaluated clonality and K- ras mutations in 11 cystic tumors of the pancreas from female patients, including 7 IPMTs and 4 MCTs. The analyses were performed on DNA from laser microdissected epithelia showing different degrees of atypia as well as normal-appearing epithelia (NAE) in the individual tumors. The grades of atypia were classified into three groups on conventional hematoxylin-eosin staining. Clonality was assessed using the methylation-induced polymorphic inactivation of the X-linked phosphoglycerate kinase gene. The incidence of monoclonality increased with the grades of atypia: 27% for NAE, 43% for grade 1, and 100% for grades 2 and 3. In three of four MCTs, foci of NAE were polyclonal, while monoclonality was seen in each one of grades 1 and 2. The frequency of K- ras mutation depended on the grades of atypia: 0% for NAE, 29% for grade 1, 50% for grade 2, and 75% for grade 3. Polyclonal epithelia were devoid of K- ras mutation in 92% of sites, while monoclonality was associated with both wild and mutational types in an approximately equal ratio. Both IPMT and MCT seem to arise from polyclonal epithelia and to be replaced by monoclonal neoplastic cells as they undergo dysplastic changes and K- ras mutation. These data suggest that the monoclonal expansion precedes K- ras mutation.