SummaryTo show the involvement of microfilaments and microtubules in non-host resistance of barley, partially dissected coleoptiles which had been inoculated with a non-pathogen, Erysiphe pisi, were treated with several actin and tubulin inhibitors. If the coleoptiles were not treated with any of the inhibitors, the non-pathogen always failed to penetrate the coleoptile cells. However, when coleoptiles were treated with actin or tubulin polymerization or depolymerization inhibitors, the non-pathogen was able to penetrate successfully and to form haustoria in coleoptlie cells of a non-host plant, barley. Actin polymerization inhibitors, cytochalasins, were more effective in causing an increase in penetration efficiency of E. pisithan tubulin inhibitors. The effects of cytochalasins depended on the kind of cytochalasin; the strength of the actin depolymerizing activity correlated significantly with the efficiency of increasing the penetration of the non-pathogen. When both actin and tubulin inhibitors were added simultaneously, the polarization of defense-related responses, such as massive cytoplasmic aggregation, deposition of papillae and accumulation of autofluorescent compounds, at fungal penetration sites was suppressed. Actin inhibitors did not affect arrangement and stability of microtubules and vice versa, and a double treatment of coleoptile cells with both microfilament and microtubule inhibitors showed an additive effect in increasing the penetration efficiency of E. pisL Furthermore, cytochalasin A treatment allowed other non-pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non-host barley cells. These results strongly suggest that microfilaments and microtubules might play important roles in the expression of non-host resistance of barley.

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The filamentous fungus Fusarium oxysporum is a soil-borne facultative parasite that causes economically important losses in a wide variety of crops. F. oxysporum exhibits filamentous growth on agar media and undergoes asexual development producing three kinds of spores: microconidia, macroconidia, and chlamydospores. Ellipsoidal microconidia and falcate macroconidia are formed from phialides by basipetal division; globose chlamydospores with thick walls are formed acrogenously from hyphae or by the modification of hyphal cells. Here we describe rensa, a conidiation mutant of F. oxysporum, obtained by restrictionenzyme-mediated integration mutagenesis. Molecular analysis of rensa identified the affected gene, REN1, which encodes a protein with similarity to MedA of Aspergillus nidulans and Acr1 of Magnaporthe grisea. MedA and Acr1 are presumed transcription regulators involved in conidiogenesis in these fungi. The rensa mutant and REN1-targeted strains lack normal conidiophores and phialides and form rod-shaped, conidium-like cells directly from hyphae by acropetal division. These mutants, however, exhibit normal vegetative growth and chlamydospore formation. Nuclear localization of Ren1 was verified using strains expressing the Ren1-green fluorescent protein fusions. These data strongly suggest that REN1 encodes a transcription regulator required for the correct differentiation of conidiogenesis cells for development of microconidia and macroconidia in F. oxysporum.

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Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as "biogenous iron oxide (BIOX)". The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.

In an aquatic environment, the genus Leptothrix produces an extracellular Fe- or Mn-encrusted tubular sheath composed of a complex hybrid of bacterial exopolymers and aqueous-phase inorganic elements. This ultrastructural study investigated initial assemblage of bacterial saccharic fibrils and subsequent deposition of aqueous-phase inorganic elements to form the immature sheath skeleton of cultured Leptothrix sp. strain OUMS1. After one day of culture, a globular and/or thread-like secretion was observed on the surface of the bacterial cell envelope, and secreted bodies were transported across the intervening space away from the cell to form an immature sheath skeleton comprising assembled and intermingled fibrils. Energy dispersive X-ray microanalysis and specific Bi-staining detected a distinguishable level of P, trace Si, and a notable amount of carbohydrates in the skeleton, but not Fe. By the second day, the skeleton was prominently thickened with an inner layer of almost parallel aligned fibrils, along with low level of Fe deposition, whereas an outer intermingled fibrous layer exhibited heavy deposition of Fe along with significant deposition of P and Si. These results indicate that basic sheath-construction proceeds in two steps under culture conditions: an initial assemblage of bacterial saccharic fibrils originated from the cell envelope and the subsequent deposition of aqueous-phase Fe, P, and Si

The so-called Fe/Mn-oxidizing bacteria have long been recognized for their potential to form extracellular iron hydroxide or manganese oxide structures in aquatic environments. Bacterial species belonging to the genus Gallionella, one type of such bacteria, oxidize iron and produce uniquely twisted extracellular stalks consisting of iron oxide-encrusted inorganic/organic fibers. This paper describes the ultrastructure of Gallionella cells and stalks and the visualized structural and spatial localization of constitutive elements within the stalks. Electron microscopy with energy-dispersive X-ray microanalysis showed the export site of the stalk fibers from the cell and the uniform distribution of iron, silicon, and phosphorous in the stalks. Electron energy-loss spectroscopy revealed that the stalk fibers had a central carbon core of bacterial exopolymers and that aquatic iron interacted with oxygen at the surface of the carbon core, resulting in deposition of iron oxides at the surface. This new knowledge of the structural and spatial associations of iron with oxygen and carbon provides deeper insights into the unique inorganic/organic hybrid structure of the stalks.Some microorganisms convert metal ions to their oxidized forms by enzymatic removal of electrons, causing physicochemical reactions associated with the resulting change of the local pH (19). The so-called Fe/Mn-oxidizing bacteria have long been recognized for their potential to form extracellular iron hydroxide or manganese oxide structures in aquatic environments (5,8,12,15,16,17). Bacteria belonging to the genus Gallionella are ubiquitous inhabitants of ocherous deposits that form in bodies of freshwater (5). They form a uniquely twisted extracellular iron oxide-encrusted bundle of fibers (commonly called a twisted stalk) (5). It is presently thought that the extracellular polysaccharides from the cell, which are the major organic components of the stalk, are closely linked with its mineralization by Fe, Si, and P (2, 4, 5, 7) and other minor elements (5). However, the structural origin and the presence of the stalk polysaccharides and the spatial association of elements within their structure remain unsolved in spite of a number of ultrastructural studies (2,7,11,14,18).Iron-oxidizing bacteria such as Gallionella, Leptothrix, Mariprofundus, and Rhodobacter (3,5,8,12,15,16,17), with the ability to form extracellular iron oxides, have evoked great interest in biological and geochemical fields of research. The potential for future industrial use of these biologically derived iron oxides clearly indicates the need for detailed systematic study of the interactions of the biological organics with the aquatic metals and minerals in the stalks. The aim of this study is to examine the ultrastructure of Gallionella cells and stalks and to define the structural and spatial localization of constitutive elements within the stalks. Our analyses included the use of scanning electron microscopy (SEM)/transmission electron microscopy (TEM) with energy-dispersive ...

New arrangements of microtubules and actin filaments in coleoptile cells of barley that had been inoculated with either a nonpathogen, Erysiphe pisi, or a pathogen, E. graminis, were observed by cytochemistry and confocal laser scanning microscopy. In uninoculated coleoptile cells, microtubules were oriented almost obliquely or transversely to the long axis of the cells and actin filaments almost obliquely or longitudinally. A thick actin bundle was located beneath approximately 70% of appressoria of E. pisi when the appressoria matured 3–4 h before they attempted penetration. This phenomenon occurred below approximately 30% of appressoria of E. graminis. Microtubules were gathered beneath the appressoria when and after the inoculated fungi induced cytoplasmic aggregation. This phenomenon also occurred more frequently below appressoria of E. pisi than those of E. graminis. Confocal laser scanning microscopy confirmed the localization of microtubules and actin filaments in a cortical region of the coleoptile cell beneath the appressorium. The time-course study revealed that the new arrangement of actin filaments was initiated 3–4 h prior to the fungal penetration attempt, whereas that of microtubules began at the time of initiation of cytoplasmic aggregation. The incidence of cells with newly arranged cytoskeletons was distinctly higher when E. pisi rather than E. graminis was used as inoculum. The possibilities that actin filaments might be involved in sensing the presence of the fungi and that both microtubules and actin filaments might be involved in localized resistance mechanisms are discussed. Key words: microtubule, F-actin, Erysiphe pisi, E. graminis, resistance mechanism.