A novel gene, Le-Dd10, is involved in fruiting body formation of Lentinula edodes

Abstract

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd1 ~ 21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with an 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

Keywords: Dictyostelium discoideum; Fruiting body; Shiitake mushroom; Transcription factor; Transformation.

Fig. 1

Knockout mutants of cDNA clones, SSJ301, SSJ337, and SSK864 exhibited aberrant fruiting bodies. A AX2 Control; B SSJ301; C SSJ337; D SSK864 SSJ301, a fruiting body with a tiny sorus; SSJ337, a fruiting body with an abnormal stalk; SSK864, a tiny fruiting body Scale bars are 50 μm

Fig. 2

Southern blot analysis of transformants with Le-Dd10, Le-Dd11, Le-Dd14, or Le-Dd18. Southern blotting was performed using Le-Dd10, Le-Dd11, Le-Dd14, or Le-Dd18 cDNA and hph (hygromycin resistance gene) as a probe. The symbol # indicates a clone of each transformant. The genomic DNA of L. edodes mycelia was digested with BamH I or Dra I. M, λDNA digested with Hind III; C, Mori 252; B, BamH I; D, Dra I

Fig. 3

Northern blot analysis of pLG-RasF1/Le-Dd5, Le-Dd6, Le-Dd7, Le-Dd9, Le-Dd10, Le-Dd11, Le-Dd12, Le-Dd13, Le-Dd14, and Le-Dd18. Northern blotting was performed using insert cDNA as a probe. The symbol # indicates a clone of each transformant. Total RNA was isolated from transformant mycelia cultured at 25 °C for 2 weeks C, Mori 252

Fig. 4

Northern blot analysis of transformants with RNAi vector for Le-Dd10. Northern blotting was performed using Le-Dd10 cDNA as a probe. The symbol # indicates a clone of each transformant. Total RNA was isolated from each mycelium transformed with the RNAi vector cultured at 25 °C for 2 weeks C, Mori 252

Fig. 5

Mapping of Le-Dd10 on the linkage of Lentinula edodes. Mapping of Le-Dd10 was carried out by SSCP analysis. An arrow shows the position of Le-Dd10

Fig. 6

SDS-PAGE of GST:Le-Dd10 fusion proteins. The transformants with the GST:Le-Dd10 fusion expression vector were incubated with 0.5 mM IPTG at 25 °C or 37 °C for 5, 10, or 16 h. They were applied on a 10% gel and the gel was stained with Coomassie brilliant blue (A). The PVDF membrane for Western blotting analysis was incubated with anti-GST antibody diluted 1:1000 as a primary antibody and then incubated with peroxidase-conjugated goat anti-rabbit IgG diluted 1:500 as a secondary antibody (B). M, molecular weight marker; B, Escherichia coli BL21- Gold (DE3) without expression vector; G with GST expression vector; 1–6 with GST:Le-Dd10 fusion expression; 1–3 at 25 °C, 4–6 at 37 °C; 1, 4 for 5 h; 2, 5 for 10 h; 3, 6 for 16 h. Arrows show the position of a fusion protein band

Fig. 7

Western blot analysis of Hokken 600. Each sample was prepared as described in Materials and Methods. Arrows show the positions of 56-kDa, 31-kDa, 27-kDa, 24-kDa, and 14-kDa bands. M, SDS-PAGE marker; 1, mycelium; 2, primordium; 3, fruiting body stipe; 4, fruiting body pileus