Lentinula edodes (shiitake mushroom) is a very popular edible, cultivated mushroom in Japan. There are post-storage problems with shiitake mushrooms, such as gill browning and cell wall lysis of the fruiting body, which can result in loss of fresh food quality and consequent loss of value. Lentinan is a cell wall component of beta-1, 3-linked-D-glucan with beta-1, 6 branches, which was isolated as an anti-tumor active-substance from L. edodes. Lentinan content decreases following harvest as a result of increased glucanase activity. We isolated one exo-glucanase encoding genes, exg21) and two endo-glucanase encoding gene tlg12) and glu1 from L. edodes. Transcription level of the exg2, tlg1 and glu1 gene increased after harvesting. Enzymes encoded by the genes have lentinan degrading activity, therefore, these genes are involved in lentinan degradation after harvesting. We also identified several cell wall degradation- related enzyme-encoding genes3), such as mixed-linked glucanase (mlg1), chitinases (chi1, chi29), chitin deacetylase (chd1), and chitosanase (cho1). It is revealed that transcriptional levels of these genes increased after harvesting, by real-time PCR. Glucanase and chitinase activity increased following harvest as results of increased transcription of these cell wall degradation-related enzyme-encoding genes. Increase of these cell wall degradation- related enzyme activities would cause cell wall lysis and lentinan degradation during post-harvest preservation. We identified laccase and tyrosinase encoding genes (lcc4 and tyr, respectively) by PCR-subtraction. The lcc4 was a novel laccase-encoding gene in L. edodes. Transcription levels of lcc4 and tyr increased after harvesting, and these genes would be involved in browning of the fruiting body. 1) Sakamoto et al. (2005) Current Genetics, 48: 195-203 2) Sakamoto et al. (2006) Plant Physiology 141: 793-801 3) Sakamoto et al. (2009) Current Genetics 55: 409-423

Laccase (Lcc; EC 1.10.3.2) belongs to a group of polyphenol oxidases, which catalyzed the oxidation of single-electron from phenolic substrates or aromatic amines. Many organisms possess several lcc encoding genes, and their biological functions diverge into many branches. There are many studies on biochemical function of Lccs, however, there are few studies about biological functions of one Lcc in detail. We researched on biological function of Lcc1, which is most abundantly secreted enzyme from vegetative mycelia into liquid culture in L. edodes. In our previous study, lcc1 gene was down regulated by RNAi method in L. edodes, and then ivrL1#32 was selected as a completely lcc1 dowaregulated transformant. We revealed that fruiting body development in ivrL1#32 was significantly suppressed compared to wild type strain. In this study, we observed the hyphal morphology of ivrL1#32. IvrL1#32 did not form thick aerial mycelium mat when grown on MYPG agar plate. From the observation using scanning electron microscope (SEM), hyphae of ivrL1#32 had many abnormal short branches and their mycelial density was lower than that of wild type strain. From transmission electron microscope observation (TEM), ivrL1#32 lacked obviously distinguishable outer and inner layer in their cell wall. In addition, the fibrous layer of ivrL1#32, which connected hyphae, obviously decreased. These morphological phenotypes would be caused by the absence of Lcc1 in L. edodes. Our results provide a clue to resolve of the biological function of Lcc1 in L. edodes.