| Ganoderma lucidum is one of the most important medicinal mushrooms in China and Southeast Asia. Modern researches showed that the chemical composition of G. lucidum complex have a wide range of pharmacological activities. Triterpenes in G. lucidum, also called ganoderic acid, which is one kind of the secondary metabolites, are considered as the main active ingredient. They have important medicinal values, such as:anti-tumor inhibits histamine release, anti-HIV and absorption effects and so on. Ganoderic acids (GAs) have the most important pharmacological activity, as a results, their contents have been taken as an important measurement of the quality of G. lucidum. Currently, the completed genome sequence for Ganoderma lucidum has generated widespread interest in investigating this organism, which is becoming a potential model system for the study of complicated growth patterns and the regulation of secondary metabolic pathways in medicinal mushrooms. Nonetheless, the scarcity of basic biology studies has hindered the further development of its commercial value.RNA interference (RNAi) is a convenient reverse genetic method that has been widely used in several types of organisms for studying functional genomics.. In this study, we established the transformation procedure based on the electroporation of mycelial fragments, which is a convenient method for introducing transgenes into G. lucidum and results in hygromycin B resistance and GFP expression in the transformants. The endogenous orotidine5’-monophosphate decarboxylase gene (ura3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of ura3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of ura3silencing compared with other vectors (up to81.9%). 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 ura3and laccase1in G. lucidum. 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 should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species for our further studies.Reactive oxygen species (ROS) have long been considered to be deleterious by-products of aerobic metabolism. However, recent studies on NADPH oxidases (Noxs), a large family of enzymes dedicated to ROS production, have highlighted the many important biological roles of ROS. NADPH oxidases have recently been highlighted due to the many important biological roles in plants and animals; however, the exact functions of Nox are still not fully understood in fungi. In this study, based on the completed genome sequence of G. lucidum, we identified two Nox isoforms (NoxA and NoxB) and a regulator, NoxR. RNA interference (RNAi) was used to examine the function of the Nox family, and silencing of the Nox isoforms and NoxR expression indicated a central role for this gene family in ROS generation to regulate ganoderic acid biosynthesis and hyphal growth. Additionally, the Nox genes are required for fruiting body development and ROS resistance.Oxidants are known to trigger the generation of Ca2+signals, in part through the activation of calcium channels in mammal cells and plants to activate a series of biological and physiological processes. However, this mechanism in fungi remains to be determined. To explore whether Nox-generated ROS could activate Ca2+signaling in G. lucidum and whether this activation would regulate GA biosynthesis. We detected the intracellular Ca2+levels of the Nox silenced strains using the Ca2+-fluorescent probe Fluo-3AM. The NoxAB silenced strains, NoxR silenced strains and DPI treatment caused a significant reduction in the cytosolic Ca2+levels. Further mechanistic investigation revealed that Nox-generated ROS elevated cytosolic Ca2+levels by activating a plasma membrane Ca2+influx pathway, thereby inducing the Ca2+signal pathway to regulate GA biosynthesis and hyphal branching. On transcription level, most of the Ca2+signaling genes exhibited an early response to ROS at30min, with some being up-regulated at3h and most genes down-regulated at12h. These results suggest a Ca2+-signal pathway in G. lucidum, and that the signaling genes respond to H2O2. Importantly, our results highlight the Nox functions in the Basidiomycetes G. lucidum, and we provide evidence that Noxs, as in plants and animals, might activate Ca2+signaling during cell physiological processes in this fungus. These findings provide an excellent opportunity to identify the potential pathway linking ROS networks to calcium signaling in fungi and suggest that plants, animals, and fungi share a conserved signal-crosstalk mechanism. |
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