Ai-Liang Jiang

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.

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.

Heat Stress Modulates Mycelium Growth, Heat Shock Protein Expression, Ganoderic Acid Biosynthesis, and Hyphal Branching of Ganoderma lucidum via Cytosolic Ca2+

ABSTRACT Heat stress (HS) influences the growth and development of organisms. Thus, a comprehensive understanding of how organisms sense HS and respond to it is required. Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system due to the complete sequencing of its genome, transgenic systems, and reliable reverse genetic tools. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced the accumulation of ganoderic acid biosynthesis and heat shock proteins (HSPs) in G. lucidum. Our data showed that HS induced a significant increase in cytosolic Ca2+ concentration. Further evidence showed that Ca2+ might be a factor in the HS-mediated regulation of hyphal branching, ganoderic acid (GA) biosynthesis, and the accumulation of HSPs. Our results further showed that the calcium-permeable channel gene (cch)-silenced and phosphoinositide-specific phospholipase gene (plc)-silenced strains reduced the HS-induced increase in HSP expression compared with that observed for the wild type (WT). This study demonstrates that cytosolic Ca2+ participates in heat shock signal transduction and regulates downstream events in filamentous fungi. IMPORTANCE Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system for evaluating how environmental factors regulate the development and secondary metabolism of basidiomycetes. Heat stress (HS) is an important environmental challenge. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced HSP expression and ganoderic acid biosynthesis in G. lucidum. Further evidence showed that Ca2+ might be a factor in the HS-mediated regulation of hyphal branching, GA biosynthesis, and the accumulation of HSPs. This study demonstrates that cytosolic Ca2+ participates in heat shock signal transduction and regulates downstream events in filamentous fungi. Our research offers a new way to understand the mechanism underlying the physiological and metabolic responses to other environmental factors in G. lucidum. This research may also provide the basis for heat shock signal transduction studies of other fungi.

Hydrogen‐rich water regulates effects of ROS balance on morphology, growth and secondary metabolism via glutathione peroxidase in Ganoderma lucidum

Ganoderma lucidum is one of the most important medicinal fungi, but the lack of basic study on the fungus has hindered the further development of its value. To investigate the roles of the redox system in G. lucidum, acetic acid (HAc) was applied as a reactive oxygen species (ROS) stress inducer, and hydrogen-rich water (HRW) was used to relieve the ROS stress in this study. Our results demonstrate that the treatment of 5% HRW significantly decreased the ROS content, maintained biomass and polar growth morphology of mycelium, and decreased secondary metabolism under HAc-induced oxidative stress. Furthermore, the roles of HRW were largely dependent on restoring the glutathione system under HAc stress in G. lucidum. To provide further evidence, we used two glutathione peroxidase (GPX)-defective strains, the gpxi strain, the mercaptosuccinic acid (MS, a GPX inhibitor)-treated wide-type (WT) strain, and gpx overexpression strains for further research. The results show that HRW was unable to relieve the HAc-induced ROS overproduction, decreased biomass, mycelium morphology change and increased secondary metabolism biosynthesis in the absence of GPX function. The gpx overexpression strains exhibited resistance to HAc-induced oxidative stress. Thus, we propose that HRW regulates morphology, growth and secondary metabolism via glutathione peroxidase under HAc stress in the fungus G. lucidum. Furthermore, our research also provides a method to study the ROS system in other fungi.

UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose.

UDP-glucose pyrophosphorylase (UGP) is a key enzyme involved in carbohydrate metabolism, but there are few studies on the functions of this enzyme in fungi. The ugp gene of Ganoderma lucidum was cloned, and enzyme kinetic parameters of the UGP recombinant protein were determined in vitro, revealing that this protein was functional and catalyzed the reversible conversion between Glc-1-P and UDP-Glc. ugp silencing by RNA interference resulted in changes in the levels of the intermediate metabolites Glc-1-P and UDP-Glc. The compounds and structure of the cell wall in the silenced strains were also altered compared with those in the wild-type strains. Moreover, the number of hyphal branches was also changed in the silenced strains. To verify the role of UGP in hyphal branching, a ugp-overexpressing strain was constructed. The results showed that the number of hyphal branches was influenced by UGP. The mechanism underlying hyphal branching was further investigated by adding exogenous Glc-1-P. Our results showed that hyphal branching was regulated by a change in the cytosolic Ca(2+) concentration, which was affected by the level of the intermediate metabolite Glc-1-P, in G. lucidum. Our findings indicate the existence of an interaction between carbon metabolism and Ca(2+) signaling in this fungus.

Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes)

We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 μmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 μg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 μmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 μmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

The pH-responsive transcription factor PacC regulates mycelial growth, fruiting body development, and ganoderic acid biosynthesis in Ganoderma lucidum.

Abstract Ganoderma lucidum is a medicinal macrofungus that is widely used in traditional Chinese medicine. Nonetheless, the scarcity of basic biological studies of this organism has hindered the further development of its commercial value. The pH-responsive transcription factor PacC/Rim101 governs the adaptation to environmental pH, the development and the secondary metabolism of many fungi. In this study, a homologue of PacC/Rim101 that encodes GlPacC was identified in the higher basidiomycete G. lucidum. GlPacC is composed of 807 amino acids and contains three typical C2H2 zinc-finger domains, two potential PEST domains, a putative PKA phosphorylation site, and a putative nuclear localization signal (NLS). GlPacC was transcribed at a high level when the fungus was under neutral and alkaline conditions, and silencing of GlPacC impaired the fungal response to ambient pH. The distance between the hyphal branches (of vegetative hyphae and aerial hyphae) was significantly increased in the GlPacC-silenced strains. The GlPacC-silenced strains grew abnormally or became sickly on solid culture medium and were unable to form primordia and fruiting bodies. The ganoderic acid content, levels of the sqs and ls transcripts, and contents of the metabolic intermediates squalene and lanosterol were all up-regulated in the GlPacC-silenced strains. Our results indicate that GlPacC is functional and plays complex roles in mycelial growth, fruiting body development and ganoderic acid biosynthesis in G. lucidum.

Phospholipase D and Phosphatidic Acid Mediate Heat Stress Induced Secondary Metabolism in Ganoderma lucidum

Phospholipid-mediated signal transduction plays a key role in responses to environmental changes, but little is known about the role of phospholipid signalling in microorganisms. Heat stress (HS) is one of the most important environmental factors. Our previous study found that HS could induce the biosynthesis of the secondary metabolites, ganoderic acids (GA). Here, we performed a comprehensive mass spectrometry-based analysis to investigate HS-induced lipid remodelling in Ganoderma lucidum. In particular, we observed a significant accumulation of phosphatidic acid (PA) on HS. Further genetic tests in which pld-silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS-activated phospholipase D (PLD) hydrolysing phosphatidylethanolamine. Furthermore, we determined the role of PLD and PA in HS-induced secondary metabolism in G. lucidum. Exogenous 1-butanol, which decreased PLD-mediated formation of PA, reverses the increased GA biosynthesis that was elicited by HS. The pld-silenced strains partly blocked HS-induced GA biosynthesis, and this block can be reversed by adding PA. Taken together, our results suggest that PLD and PA are involved in the regulation of HS-induced secondary metabolism in G. lucidum. Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.

Membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in Ganoderma lucidum

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

Functional analysis of an APSES transcription factor (GlSwi6) involved in fungal growth, fruiting body development and ganoderic-acid biosynthesis in Ganoderma lucidum

The APSES transcription factors have been identified as key regulators of fungal development and other biological processes in fungi. In the present study, the function of Ganoderma lucidum GlSwi6, a homolog of Saccharomyces cerevisiae Swi6, was characterized. RNAi was used to examine the function of GlSwi6 in G. lucidum. Silencing GlSwi6 resulted in multiple developmental defects, including reduced fungal growth and increased hyphal branching, and the GlSwi6-silenced strains did not exhibit primordium or fruiting body formation. In addition, the H2O2 and ganoderic-acid (GA) levels of the GlSwi6-silenced strains decreased approximately 50% and 25%, respectively, compared with those of the WT strain. Furthermore, the addition of H2O2 led to the recovery of the GA levels of GlSwi6-silenced strains, implying that GlSwi6 might regulate GA biosynthesis by regulating the intracellular ROS levels. Taken together, these results indicate that GlSwi6 is involved in fungal growth, development and GA biosynthesis in G. lucidum.