Mingwen Zhao

Current progress in the study on biosynthesis and regulation of ganoderic acids

Ganoderic acids (GAs) isolated from Ganoderma lucidum, which have shown remarkable pharmacological activities and a variety of therapeutic effects on a number of human diseases, have provided an important resource for the development of new medicines. The yield of GAs in field cultivation is still limited, which is mainly due to a scarcity of information regarding its biosynthesis pathway and its regulation. Here, we review the recent publication that has been made in the biosynthesis and regulation of GAs. From these studies, researchers have identified part of the biosynthesis pathway of GAs named mevalonate pathway. They have cloned and characterized the genes involved in the biosynthesis pathway. Additionally, they found that expression of the genes involved in GA biosynthesis is closely related to the impact of environmental factors through transcriptional profiling analysis. Moreover, this review focuses on suggesting new directions for studying GAs and attempts to gain some insights for better understanding of the biosynthesis and regulation of GAs.

Molecular Cloning, Characterization, and Differential Expression of a Farnesyl-Diphosphate Synthase Gene from the Basidiomycetous Fungus Ganoderma lucidum

A farnesyl-diphosphate synthase gene, designated GlFPS, was isolated from a triterpene-producing basidiomycetous fungus, Ganoderma lucidum. The GlFPS cDNA was found to contain an open reading frame of 1,083 bp, encoding a protein of 360 amino acids with a calculated molecular mass of 41.27 kDa. The deduced amino acid sequence of the GlFPS cDNA exhibited a high homology with other fungal FPS genes, and contained four conserved domains. Phylogenetic analysis showed that GlFPS belonged to the basidiomycete FPS group. Competitive PCR revealed that GlFPS was constitutively expressed in the mycelium growth stage, whereas the transcripts of GlFPS accumulated to high levels rapidly during the process of mushroom primordia. Treatment of mycelia with exogenous methyl jasmonate also caused a large accumulation of GlFPS mRNA. Subsequently, promoter analysis indicated that the 5′ upstream region of GlFPS possessed various potential regulatory elements associated with physiological and environmental factors. Functional complementation of GlFPS in an ERG20-disrupted yeast strain indicated that the cloned cDNA encoded a farnesyl-diphosphate synthase.

Functions of the nicotinamide adenine dinucleotide phosphate oxidase family in Ganoderma lucidum: an essential role in ganoderic acid biosynthesis regulation, hyphal branching, fruiting body development, and oxidative-stress resistance

Ganoderma lucidum has drawn worldwide interest with regard to its secondary metabolism and pharmaceutical activity. However, the development of such research has been limited because of a lack of basic biological knowledge. Nicotinamide adenine dinucleotide phosphate oxidases (Nox) have recently been highlighted because of 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, we identified two Nox isoforms (NoxA and NoxB) and a regulator, NoxR. RNA interference was used, and silencing of the Nox isoforms and NoxR expression indicated a central role for these genes in hyphal branching, fruiting body development, reactive oxygen species (ROS) generation, ROS resistance and ganoderic acid biosynthesis regulation. Further mechanistic investigation revealed that Nox-generated ROS elevated cytosolic Ca(2+) levels by activating a plasma membrane Ca(2+) influx pathway, thereby inducing the Ca(2+) signal pathway to regulate ganoderic acid biosynthesis and hyphal branching. Importantly, our results highlight the Nox functions in signal crosstalk between ROS and Ca(2+), and these findings provide an excellent opportunity to identify the potential pathway linking ROS networks to calcium signalling in fungi and suggest that plants, animals and fungi share a conserved signal-crosstalk mechanism.

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

Ganoderma lucidum 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 (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 URA3 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 URA3 and laccase in 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 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.

Cloning and Characterization of a Gene Encoding HMG-CoA Reductase from Ganoderma lucidum and Its Functional Identification in Yeast

A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) was isolated from a triterpene-producing fungus, Ganoderma lucidum (Reishi or Lingzhi). This report provides the complete nucleotide sequence of the full-length cDNA encoding HMGR and its genomic DNA sequence. The cDNA of the HMGR (GenBank Accession no., EU263989) was found to contain an open reading frame (ORF) of 3,681 bp encoding a 1,226-amino-acid polypeptide, whereas the HMGR genomic DNA sequence (GenBank Accession no., EU263990) consisted of 4,262 bp and contained seven exons and six introns. The deduced amino acid sequence of G. lucidum HMGR showed significant homology to the known HMGRs from Ustilago maydis and Cryptococcus neoformans, and contained four conserved domains. Gene expression analysis showed that the expression level was relatively low in mycelia incubated for 10, 12, and 14 d, and reached the highest level in the primordia. Functional complementation of Gl-HMGR in a HMGR-deficient mutant yeast strain indicated that the cloned cDNA encoded a HMG-CoA reductase.

Molecular cloning, characterization, and differential expression of a lanosterol synthase gene from Ganoderma lucidum.

A homology-based PCR method was used to clone a cDNA encoding lanosterol synthase (LS) from Ganoderma lucidum (G. lucidum), which produces triterpenes. The cDNA of the LS (GenBank accession no. GQ169528) was found to contain an open reading frame (ORF) of 2,181 bp encoding a 726 amino acid polypeptide, whereas the LS genomic DNA sequence (GenBank accession no. GQ169529) consists of 2,924 bp. Functional complementation of G. lucidum LS (Gl-LS) in an erg7 yeast strain lacking LS activity demonstrated that the cloned cDNA encoded a functional LS. Analysis of the Gl-LS transcript profiles revealed a positive correlation between the pattern of LS gene expression and triterpenes content changes in G. lucidum during development. Up-regulation of expression of the Gl-LS gene by methyl jasmonate (MeJA) in the mycelia was also demonstrated by real-time RT-PCR. Up-regulation of the Gl-LS promoter activity by MeJA was also investigated.

Functional analysis of the role of glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal branching and the regulation of ganoderic acid biosynthesis in Ganoderma lucidum

Ganoderma lucidum, a hallmark of traditional Chinese medicine, has been widely used as a pharmacologically active compound. Although numerous research studies have focused on the pharmacological mechanism, fewer studies have explored the basic biological features of this species, restricting the further development and application of this important mushroom. Because of the ability of this mushroom to reduce and detoxify the compounds produced by various metabolic pathways, glutathione peroxidase (GPx) is one of the most important antioxidant enzymes with respect to ROS. Although studies in both animals and plants have suggested many important physiological functions of GPx, there are few systematic research studies concerning the role of this enzyme in fungi, particularly in large basidiomycetes. In the present study, we cloned the GPx gene and created GPx-silenced strains by the down-regulation of GPx gene expression using RNA interference. The results indicated an essential role for GPx in controlling the intracellular H2O2 content, hyphal branching, antioxidant stress tolerance, cytosolic Ca(2+) content and ganoderic acid biosynthesis. Further mechanistic investigation revealed that GPx is regulated by intracellular H2O2 levels and suggested that crosstalk occurs between GPx and intracellular H2O2. Moreover, evidence was obtained indicating that GPx regulation of hyphal branching via ROS might occur independently of the cytosolic Ca(2+) content. Further mechanistic investigation also revealed that the effects of GPx on ganoderic acid synthesis via ROS are regulated by the cytosolic Ca(2+) content. Taken together, these findings indicate that ROS have a complex influence on growth, development and secondary metabolism in fungi and that GPx serves an important function. The present study provides an excellent framework to identify GPx functions and highlights a role for this enzyme in ROS regulation.

The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity

Verticillium wilt in cotton caused by Verticillium dahliae is one of the most serious plant diseases worldwide. Because no known fungicides or cotton cultivars provide sufficient protection against this pathogen, V. dahliae causes major crop yield losses. Here, an isolated cotton endophytic bacterium, designated Bacillus amyloliquefaciens 41B-1, exhibited greater than 50% biocontrol efficacy against V. dahliae in cotton plants under greenhouse conditions. Through high-performance liquid chromatography and mass analysis of the filtrate, we found that the antifungal compounds present in the strain 41B-1 culture filtrate were a series of isoforms of iturins. The purified iturins suppressed V. dahliae microsclerotial germination in the absence or presence of cotton. Treatment with the iturins induced reactive oxygen species bursts, Hog1 mitogen-activated protein kinase (MAPK) activation and defects in cell wall integrity. The oxidative stress response and high-osmolarity glycerol pathway contribute to iturins resistance in V. dahliae. In contrast, the Slt2 MAPK pathway may be involved in iturins sensitivity in this fungus. In addition to antagonism, iturins could induce plant defence responses as activators and mediate pathogen-associated molecular pattern-triggered immunity. These findings suggest that iturins may affect fungal signalling pathways and mediate plant defence responses against V. dahliae.

Profiling and Quantifying Differential Gene Transcription Provide Insights into Ganoderic Acid Biosynthesis in Ganoderma lucidum in Response to Methyl Jasmonate

Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum.

The regulation of methyl jasmonate on hyphal branching and GA biosynthesis in Ganoderma lucidum partly via ROS generated by NADPH oxidase.

Ganoderma lucidum is one of the best known medicinal basidiomycetes because it produces many pharmacologically active compounds, and methyl jasmonate (MeJA) was previously reported to induce the biosynthesis of ganoderic acids (GA) in G. lucidum. In this study, we found that MeJA not only increased the amount of GA but also increased the distance between hyphal branches by approximately 1.2-fold. Further analysis showed that MeJA could increase the intracellular ROS (reactive oxygen species) content by approximately 2.2-2.7-fold. Furthermore, the hyphal branching and GA biosynthesis regulated by MeJA treatment could be abolished by ROS scavengers to a level similar to or lower than that of the control group. These results indicated that the regulation of hyphal branching and GA biosynthesis by MeJA might occur via a ROS signaling pathway. Further analysis revealed that NADPH oxidase (NOX) plays an important role in MeJA-regulated ROS generation. Importantly, our results highlight that NOX functions in signaling cross-talk between ROS and MeJA. In addition, these findings provide an excellent opportunity to identify potential pathways linking ROS networks to MeJA signaling in fungi and suggest that plants and fungi share a conserved signaling-crosstalk mechanism.