We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD-sC-). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD-sC-adeA-) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD-sC-DeltaargB adeA- or niaD-sC-DeltaargB adeB-) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD-sC-DeltaargB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.

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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 ⌬Aopex11-1 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 ⌬Aopex11-1 strain. Furthermore, distribution of AoHex1 was found to be peripheral in the enlarged peroxisome or junctional in dumbbell-shaped peroxisomes. Electron microscopy of the ⌬Aopex11-1 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 ⌬Aopex11-1 strain. In conclusion, the present study describes the role of Pex11 in Woronin body differentiation for the first time.

Most known basic-region helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors often involved in the control of growth and differentiation. Therefore, inappropriate expression of genes encoding bHLH proteins is frequently associated with developmental dysfunction. In our previously reported study, a novel bHLH protein-encoding gene (AO090011000215) of Aspergillus oryzae was identified. The genedisrupted strain was found to produce dense conidia, but sparse sclerotia, relative to the parent strain. Here, to further analyze its function, we generated an overexpressing strain using the A. oryzae amyB gene promoter. Genetic overexpression led to a large number of initial hyphal aggregations and then the formation of mature sclerotia; it was therefore designated sclR (sclerotium regulator). At the same time, the sclR-overexpressing strain also displayed both delayed and decreased conidiation. Scanning electron microscopy indicated that the aerial hyphae of the sclR-overexpressing strain were extremely branched and intertwined with each other. In the generation of the SclR-enhanced green fluorescent protein (EGFP) expression strain, the SclR-EGFP protein fusion was conditionally detected in the nuclei. In addition, the loss of sclR function led to rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. Taken together, these observations indicate that SclR plays an important role in hyphal morphology, asexual conidiospore formation, and the promotion of sclerotial production, even retaining normal cell function, at least in submerged liquid culture.

Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs), 41,690 into 58 gene ontology (GO) terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium.