The Development and Application of a Multiple Gene Co-Silencing System Using Endogenous <em>URA3</em> as a Reporter Gene in <em>Ganoderma lucidum</em>

<div><p><em>Ganoderma lucidum</em> is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5′-monophosphate decarboxylase gene (<em>URA3</em>) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into <em>G. lucidum</em> through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of <em>URA3</em>. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of <em>URA3</em> silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing <em>URA3</em> and <em>laccase</em> in <em>G. lucidum</em>. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.</p> </div

Measurement of co-silenced gene expression as determined by real-time PCR and laccase enzyme assays.

<p>(A) Real-time PCR analysis was performed in dual promoter co-silenced transformants. The relative mRNA levels of <i>URA3</i> and <i>lcc1</i> were calculated as the ratio of <i>URA3</i> and <i>lcc1</i> mRNA to endogenous <i>18S</i> rRNA. The upper running title of each panel is the ratio of target genes and <i>18S</i> rRNA mRNA compared with <i>wt</i>. The lower running title of each panel is the silenced transformant, <i>wt</i> and <i>ck2</i> strains. The dotted column is <i>ura3</i>/<i>18s</i>, and the black rectangle column is <i>lcc1</i>/<i>18s</i>. (B) Laccase activity was monitored in the filtrate cultures using an ABTS oxidization test. Wt represents the wild-type strain, <i>ck2</i> strains contain the pAN7-ura3-dual vector, and strains 1, 12, 15, and 16 represent ura3-lcc co-silenced transformants. Values are means ± SE (n = 3). Asterisks represent significant differences among the strains according to a one-way ANOVA test (P<0.01).</p

Oligonucleotide primers used.

*<p>R = A, G; Y = T, C; N = A, T, C or G; H = A, T or C; V = A, C or G</p

Phenotypic assays of the RNAi transformants on MMM plates.

<p><i>URA3</i>-silenced transformants (dual-2), wild-type (<i>wt</i>) and control transformants (CK1, transformed with the pAN7-1 plasmid) were cultured on MMM medium with or without uracil (100 mg/L) for 7 days at 28°C. The upper row (M+uracil) is MMM medium with uracil, and the lower row (MMM) is MMM medium.</p

Number of transformants with silenced and no-silenced.

<p>Number of transformants with silenced and no-silenced.</p

Phenotypic assays of the RNAi transformants on 5-FOA plates.

<p><i>URA3</i>-silenced transformants, wild-type (<i>wt</i>) and control transformants (CK1, transformed with plasmid pAN7-1) were cultured on CYM medium containing 600 µg/mL 5-FOA for ten days. Hp-2 was one of the pAN7-ura3-hp transformants, s-13 was one of the pAN7-ura3-s transformants, as-3 was one of the pAN7-ura3-as transformants, and dual-2 was one of the pAN7-ura3-dual transformants.</p

Measurement of <i>URA3</i> gene expression as determined by real-time PCR.

<p>Real-time PCR analysis was performed on pAN7-ura3-hp (A), pAN7-ura3-s (B), pAN7-ura3-as (C) and pAN7-ura3-dual (D) transformants. The relative mRNA levels of <i>URA3</i> were calculated as the ratio of <i>G. lucidum URA3</i> mRNA to endogenous <i>18S</i> rRNA. The upper running title of each panel is the ratio of <i>URA3</i> mRNA and <i>18S</i> rRNA mRNA compared with <i>wt</i>. The lower running title of each panel is the silenced transformant, <i>wt</i> and <i>ck1</i> strains. Wild type was considered to have a value of 1.0 for all experiments trials because the mRNA expression in all other isolates was relative to wt. Values are the means ± SE (n = 3).</p

Phenotypic assays of co-silenced transformants.

<p> (A) <i>URA3-lcc1</i> co-silenced transformants, wild-type and <i>ck2</i> transformants were cultured on CYM medium containing 600 µg/mL 5-FOA for ten days. Wt represents the wild-type strain, <i>ck2</i> strains contain the pAN7-ura3-dual vector, and lcc-1, 10, 12, 15, 16, and 17 represent ura3-lcc co-silenced transformants. (B) An ABTS-Petri dish was used to determine whether <i>laccase</i> was silenced in the co-silencing transformants. Lcc-1, lcc-10 lcc-12, lcc-15, lcc-16, and lcc-17 had significantly smaller colored circles compared with the <i>wt</i> and control strains.</p

Construction of the <i>URA3</i> RNAi expression cassette plasmids used to silence <i>URA3</i>.

<p>In all plasmids except for the dual promoter plasmid, <i>URA3</i> transcription is driven by the glyceraldehyde-3-phosphate dehydrogenase promoter. pAN7-ura3-hp contains the antisense fragment of <i>URA3</i> from 790-74 bp and the sense fragment from 306-790 bp (A). pAN7-ura3-s contains 484 bp of <i>URA3</i> in the sense orientation (B). Complementary portions of the antisense and sense regions are paired in the 306-790 nucleotide region of the <i>URA3</i> gene, forming a hairpin loop with a 484 bp complementary region (C). pAN7-ura3-as contains <i>URA3</i> in the antisense orientation (D). Maps of the pAN7-1 vector and the inserted RNAi cassettes (E). The dual promoter plasmid contains 484 bp of <i>URA3</i> between the gpd promoter and 35S promoter (F). The ura3-lcc co-silencing vector used the dual promoter method (G). Bar = 1000 bp</p

Expression of GFP in <i>G. lucidum</i>.

<p>A GFP-expressing <i>G. lucidum</i> transformant was cultivated on CYM agar medium at 28°C and examined microscopically. Fluorescence of wild-type <i>G. lucidum</i> (A and C) and the GFP transformant (B and D) was examined. Mycelia were removed from actively growing colonies, suspended in sterile water, and viewed microscopically. Samples shown in panels A and B were examined using bright-field microscopy, and samples in panels C and D were viewed using blue-field microscopy. Bars = 50 µm.</p