Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence.

Lentinula edodes is one of the most important edible mushrooms, but no method for analyzing its molecular genetics has yet been established. RNA interference (RNAi) is a mechanism that inhibits expression of specific genes at the post-transcriptional stage and has been used to analyze the genetics of several fungal species. RNAi was used to examine the expression of the laccase (EC 1.10.3.2) gene lcc1 of L. edodes, which encodes a lignin-degrading enzyme. Vector pChG'-ivrL1 that expressed a 40 bp homologous inverted repeat sequence from lcc1 was constructed. This was transformed into L. edodes using the restriction enzyme mediated integration method (REMI). Lcc1 protein was not detected in two of 57 transformants (ivrL1#26, ivrL1#32) where the lcc1 transcription levels were suppressed. Thus, a 40 bp inverted repeat sequence expression vector suppressed expression of the target gene in L. edodes. Lcc1 downregulated transformants (ivrL1#32) did not form a thick aerial mycelium mat on agar medium. Electron microscopy showed hyphae of ivrL1#32 had many short branches with low mycelial density, a thin cell wall, and few fibrous layers as compared to the host strain. These morphological phenotypes would be caused by the absence of Lcc1, and that provides some clue to resolve the biological function of Lcc1 in L. edodes. Our results show that RNAi can be used for gene silencing in L. edodes.