Shuichi Yamashita

Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth.

The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor‐like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA‐repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth.

Structure of phytotoxin syringomycin produced by a sugar cane isolate of Pseudomonas syringae pv. syringae

Abstract The structure of the phytotoxin syringomycin produced by a sugar cane isolate of Pseudomonas syringae pv. syringae was determined as 1 with NMR and mass spectrometry.

Class I and Class II Chitin Synthases Are Involved in Septum Formation in the Filamentous Fungus Aspergillus nidulans

ABSTRACT The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a ΔchsA ΔchsC double mutant (ΔAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the ΔAC mutant, electron-transparent regions were thickened. Septa of the ΔAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.

Structure and stereochemistry of three phytotoxins, syringomycin, syringotoxin and syringostatin, produced by pseudomonas syringae pv. syringae

The structures of two phytotoxins, syringomycin and syringotoxin, produced by Pseudomonas syringae pv. syringae, were determined. Several amino acid residues of syringomycin were different from those in the syringostatins. Syringotoxin B proved to be [Gly3]syringostatin A. The three kinds of phytotoxins showed close structural similarity, and the stereochemistry of their components was deduced and compared.

Structures of syringostatins A and B, novel phytotoxins produced by pseudomonas syringae pv. syringae isolated from lilac blights

Abstract The structures of syringostatins A and B produced by Pseudomonas syringae pv. syringae SY12 were determined as 1 and 2 , respectively, from NMR and Mass spectrometry.

Disruption of the Aopex11-1 Gene Involved in Peroxisome Proliferation Leads to Impaired Woronin Body Formation in Aspergillus oryzae

ABSTRACT The Woronin body, a unique organelle found in the Pezizomycotina, plugs the septal pore upon hyphal damage to prevent excessive cytoplasmic bleeding. Although it was previously shown that the Woronin body buds out from the peroxisome, the relationship between peroxisomal proliferation/division and Woronin body differentiation has not been extensively investigated. In this report, we examined whether Pex11 required for peroxisomal proliferation participates in Woronin body formation in Aspergillus oryzae. A. oryzae contained two orthologous PEX11 genes that were designated Aopex11-1 and Aopex11-2. Deletion of Aopex11 genes revealed that only the ΔAopex11-1 strain showed reduced growth and enlarged peroxisomes in the presence of oleic acid as a sole carbon source, indicating a defect in peroxisomal function and proliferation. Disruption of Aopex11-1 gene impaired the Woronin body function, leading to excessive loss of the cytosol upon hyphal injury. Dual localization analysis of the peroxisome and Woronin body protein AoHex1 demonstrated that Woronin bodies fail to fully differentiate from peroxisomes in the ΔAopex11-1 strain. Furthermore, distribution of AoHex1 was found to be peripheral in the enlarged peroxisome or junctional in dumbbell-shaped peroxisomes. Electron microscopy of the ΔAopex11-1 strain revealed the presence of Woronin bodies that remained associated with organelles resembling peroxisomes, which was supported from the sucrose gradient centrifugation confirming that the Woronin body protein AoHex1 overlapped with the density-shifted peroxisome in the ΔAopex11-1 strain. In conclusion, the present study describes the role of Pex11 in Woronin body differentiation for the first time.

Class III Chitin Synthase ChsB of Aspergillus nidulans Localizes at the Sites of Polarized Cell Wall Synthesis and Is Required for Conidial Development

ABSTRACT Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.

The nucleotide sequence and genome organization of Magnaporthe oryzae virus 1.

SummaryMagnaporthe oryzae virus 1 (MoV1) found in Magnaporthe oryzae, the pathogenic fungus responsible for rice blast, is a small icosahedral virus with a nonsegmented double-stranded RNA genome. The viral genome has two open reading frames (ORF 1 and 2). The deduced amino acid sequences of both ORF 1 and ORF 2 show a significant similarity to those of capsid protein and RdRp, respectively, of members of the family Totiviridae. Both a comparison of genome organization and phylogenic analysis have indicated that MoV1 is closely related to some of the totiviruses that infect filamentous fungi. These results suggest that MoV1 belongs to the family Totiviridae.

Isolation and structural elucidation of syringostatins, phytotoxins produced by Pseudomonas syringae pv. syringae lilac isolate

A bacterial strain of Pseudomonas syringae pv. syringae isolated from lilac was found to produce a homologous mixture of phytotoxins different from syringomycin and syringotoxin. The toxins were termed Syringostatins and the structures of the main components, Syringostatins A and B, were determined by 2D-NMR spectroscopy and mass spectrometry. Minor component structures were elucidated from mass/mass spectra.

The nucleotide sequence and genome organization of Japanese iris necrotic ring virus, a new species in the genus Carmovirus.

Summary. The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5′-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5′-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3′-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF. JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5′ end of the viral RNA is uncapped. The 3′ end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae.