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

Abstract

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.

Lentinula edodes Genome Survey and Postharvest Transcriptome Analysis

“…2). RNA interference tools are available in Lentinula edodes (40,73), and TILLING, a method for screening mutants that have a mutation in a target gene (74), will be applicable for breeding. Gene expression for wood decay, biofilm formation in sawdust, medium cultivation, and fruiting body primordia in Lentinula edodes has been reported.…”

RNAi silencing: A tool for functional genomics research on fungi

“…In N. crassa, it has been demonstrated that hpRNA gene silencing is more efficient and stable compared to quelling induced by promoterless transgenes (Goldoni et al, 2004;Romano and Macino, 1992). Within the developed vectors for this application, the pSilent-1 vector enables high performance in RNA silencing (Nakayashiki et al, 2005;Shafran et al, 2008). pSilent-1 has a hygromycin resistance cassette and a transcriptional unit for expressing RNA hairpin with a spacer consisting of an intron of cutinase from Magnaporthe oryzae (Nakayashiki et al, 2005).…”

“…RNAi enables researchers the ability to attenuate the expression of specific genes at different levels while keeping the organism viable even when applied to genes vital for cell survival (Nakayashiki et al, 2005). In addition, RNAi enables the silencing of a complete gene family, simultaneously (Salame et al, 2010).…”

“…It is noteworthy that in the molecular subtype (VGII) the pathogenic fungus Cryptococcus gattii RNAi is absent while other species of Cryptococcus RNAi is retained (D'Souza et al, 2011). These results may indicate that RNAi machinery lost during evolution is not an exceptional event and may be dispensable for some organisms where they are not essential for growth and development in the life cycle of these fungal species (Dang et al, 2011;Nakayashiki et al, 2006). For the rest of the fungal kingdom, the comparative phylogenetic analysis shows that the number of genes encoding Dicer, Argonaute and RdRp proteins varies significantly between species, suggesting that the RNA silencing pathways are diversified in the evolution of fungi (Nakayashiki et al, 2006).…”

“…These results may indicate that RNAi machinery lost during evolution is not an exceptional event and may be dispensable for some organisms where they are not essential for growth and development in the life cycle of these fungal species (Dang et al, 2011;Nakayashiki et al, 2006). For the rest of the fungal kingdom, the comparative phylogenetic analysis shows that the number of genes encoding Dicer, Argonaute and RdRp proteins varies significantly between species, suggesting that the RNA silencing pathways are diversified in the evolution of fungi (Nakayashiki et al, 2006). Originally, RNAi was thought to be a host defense mechanism against invading viruses and transposons (Ding and Voinnet, 2007;Wang et al, 2010).…”