BackgroundMycoheterotrophic plants are one of the most difficult plant groups to conserve because they are entirely dependent on symbiotic fungi. Establishment of viable culture systems would greatly aid their conservation. We describe a simple culture system for the mycoheterotrophic orchid, Gastrodia pubilabiata, that does not require laboratory facilities. The orchid is symbiotic with leaf-litter-decomposing fungi.Results Gastrodia pubilabiata seeds were incubated in plastic boxes or glass bottles filled with leaf litter collected from the natural habitat of the species. Seed germination was observed after 35 days and seedling development followed. Fungal isolates from seedlings were identified as Mycenaceae (Basidiomycota), a leaf-litter-decomposing mycorrhizal fungus of Gastrodia species.ConclusionOur method can be used to conserve endangered mycoheterotrophic plants associated with leaf litter-decomposing fungi efficiently, and can also serve as a model system for physiological and molecular studies of such plants.Electronic supplementary materialThe online version of this article (10.1186/s40529-017-0214-6) contains supplementary material, which is available to authorized users.

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.

In vitro symbiotic culture of Gastrodia pubilabiata seeds were conducted with the fungal isolates from G. pubilabiata roots. We obtained five fungal isolates, which belonged to Mycena, Marasmiaceae and Omphalotaceae. Firstly, optimal temperature and culture medium for subculture of these fungal isolates were examined. All five isolates grew the fastest on malt extract agar medium. Mycelial growth rate was highest at 25 °C between 10 °C and 40 °C. Secondly, we evaluated suitable culture vessels and organic materials for symbiotic culture. Seeds germinated well in petri dishes with Quercus leaf disc on water agar medium, and the seed germination process was well observed without dense mycelium. The most developed seedlings were found in glass bottles filled with Japanese cedar leaves, but densely grew mycelium prevent accurate seedling counts. Leaves of Quercus, Japanese cedar and bamboo were used as organic materials for symbiotic culture. All three leaves induced seed germination with Mycena and Marasmiaceae fungi, but material types affected subsequent seedling growth. Our method will contribute to understanding the mycorrhizal association of Gastrodia species and also other mycoheterotrophic plants.