Cloning and sequence analysis of the nmnat gene
Based on the amino acid similarity to Dichomitus squalens nmnat (NCBI Reference Sequence: XP_007369986.1), the gene Glnmnat (GenBank Accession Number: MH394247) was screened from the G. lucidum genome database (http://www.herbalgenomics.org/galu/). The length of the nmnat cDNA was 891 bp, and predictions indicated that this cDNA encodes a 297-amino acid protein with a molecular mass of 32.89 kDa and an isoelectric point of 6.05. Analysis of the predicted amino acid sequence of Glnmnat identified the two highly conserved ATP-binding domains with the motifs SxTxxR and GxxxPx[T/H]xxH, which can be easily detected in multiple sequence alignments and are commonly present in nmnat-like proteins  (Fig. 1). These results suggested that the Glnmnat gene that was cloned may belong to the nmnat family.
Construction of G. lucidum nmnat overexpression strains and measurements of NAD+ content
To explore the function of NAD+ in G. lucidum, an OE::nmnat vector was constructed (Fig. S1A), and Agrobacterium tumefaciens-mediated transformation (ATMT) was performed to construct OEnmnat mutant strains. The PCR results showed that the putative transformants harboured the hygromycin B phosphotransferase (hph) gene and the gpd promoter-nmnat gene (Fig. S1B). Quantitative reverse-transcription PCR (qRT-PCR) analysis was performed to detect the transcription level of nmnat in the selected mutant strains. Thirty positive transformants were selected, seven of which had a high transcript levels, with relative nmnat expression that was approximately 3-6-fold higher than that of the WT strain (Fig. S1C). The G. lucidum strains OE::nmnat4 and OE::nmnat19 were randomly selected for further analysis. (Fig. 2A). Because the nmnat gene is a key gene in the synthesis of NAD+, the NAD+ content in the two nmnat overexpression strains was measured. Compared with the WT strain, the NAD+ content in G. lucidum the G. lucidum OE::nmnat4 and OE::nmnat19 strains was increased by 1.35- and 1.38-fold, respectively (Fig. 2B). This result was consistent with the observed trend of nmnat gene expression at the transcript level. The increase in the NAD+ content further indicated that the cloned gene was a functional nmnat gene.
G. lucidum nmnat overexpression strains display better growth characteristics
Previous studies have shown that the NAD+ content in cells may play an important role in the response to nutritional conditions . To investigate the influence of NAD+ on the mycelial growth of G. lucidum on different carbon sources, all tested strains were inoculated on minimal medium (MM) agar plates with supplemented with 1% carbon sources, namely, glucose, sucrose, lactose, glycerol, xylan, CMC-Na or lignin. The Glnmnat overexpression strains showed dramatically increased colony growth on these carbon sources than the WT strains (Fig. 3A and B), indicating that NAD+ may be involved in the process of nutrition utilization.
Effects of NAD+ on cellulase production in G. lucidum
To further investigate the role of NAD+ in nutrient utilization in G. lucidum, the same weight of precultured G. lucidum mycelia in CYM liquid medium were transferred to liquid MM containing 1% Avicel as the sole carbon source and for the determination of cellulase activity. As shown in Fig. 4A and B, the CMCase activity (representing endo-β-glucanase activity) in the G. lucidum OE::nmnat4 and OE::nmnat19 strains increased by approximately 2.8- and 3-fold compared with that observed in the WT strain, respectively. In addition, pNPGase activity (representing β-glucosidase activity) in the G. lucidum OE::nmnat4 and OE::nmnat19 strains increased by approximately 1.9- and 2.1-fold compared with that of the WT strain, respectively. Furthermore, the total protein concentrations of all strains was also determined using a BCA Protein Assay kit. As shown in Fig. 4C, no significant differences in total protein concentrations were observed between the WT and overexpression strains. Protein secretion is not directly correlated with mycelial growth and may be affected by complex inducing factors.
In addition, the transcription levels of the genes encoding major cellulases and transcription factors in G. lucidum were evaluated. Three putative endoglucanase-encoding genes (GL24196, GL29421, and GL28282), three putative cellobiohydrolase-encoding genes (GL18725, GL 29727, and GL30351) and three putative beta-glucosidase-encoding genes (GL27550, GL20743, and GL24911) were selected from the genome of G. lucidum. As shown in Fig. 4D, the gene expression of the major cellulases was significantly upregulated in the Glnmnat overexpression strains compared with that observed in the WT strain. Subsequently, two negative transcriptional regulators for cellulase production, creA (GL19424) and ace1 (GL15296), and two positive transcriptional regulators for cellulase production, clr-1 (GL26482) and clr-2 (GL15667), were evaluated. These results showed that the expression of clr-1 and clr-2 was significantly upregulated in the Glnmnat overexpression strains compared with that observed in the WT strains, whereas that of creA and ace1 was significantly downregulated (Fig. 4E). These results were consistent with the observed increase in cellulase activity and indicated that NAD+ may influence cellulase production in G. lucidum.
Effect of NAD+ on the accumulation of Ca2+ in G. lucidum
Previous studies have demonstrated that the calcium signal transduction pathway can upregulate cellulase gene expression . Therefore, fluo-3-pentaacetoxymethyl ester (Fluo-3AM), an acetoxymethyl ester of a Ca2+-specific probe, was used to detect relative amounts of free intracellular Ca2+. As shown in Fig. 5A and B, the fluorescence intensity of the G. lucidum OE::nmnat4 and OE::nmnat19 strains was significantly higher than that observed in the WT strain, and the Ca2+ fluorescence value was upregulated by approximately 3.95- and 2.10-fold compared with the WT strain, respectively. To investigate whether increased levels of cytosolic Ca2+ can trigger calcium signal transduction pathways in G. lucidum, qRT-PCR was performed to analyse the transcriptional levels of calcium signalling-related genes in G. lucidum, including calmodulin (cam), Ca2+ and cam-dependent protein kinase genes (camk1, camk2 and camk3), the calreticulin [regulatory] gene, the calcineurin [catalytic] genes (cna1 and cna2), and the calcineurin-responsive zinc finger transcription factor gene (crz). As shown in Fig. 5C, the expression of most of these genes was increased to varying degrees in the G. lucidum nmnat overexpression strains compared with that observed in the WT strain. These results suggest that NAD+ may increase the concentration of cytosolic Ca2+, thereby stimulating the calcium signal transduction pathway in G. lucidum.
NAD+ regulates cellulase activity via intracellular Ca2+
LaCl3, a plasma membrane Ca2+ channel blocker, was used to prevent the influx of external Ca2+. As shown in Fig. 6A and B, the cytosolic Ca2+ concentration could be effectively attenuated in the G. lucidum nmnat overexpression strains after the addition of LaCl3. Furthermore, CMCase and pNPGase activities and the transcription of related genes were analysed. As expected, after the addition of 5 mM LaCl3 to the G. lucidum nmnat overexpression mutants, both the CMCase and pNPGase activities significantly decreased (Fig. 7A and B), and the transcription of genes encoding the major cellulases also exhibited a similar tendency (Fig. 7C~K). Similarly, the transcription of clr-1 and clr-2 was markedly reduced in the G. lucidum nmnat overexpression strains treated with 5 mM LaCl3, while that of creA and ace1 was markedly improved (Fig. 7L~O). Taken together, these data indicate that NAD+ may induce cellulase induction by altering the Ca2+ concentration in G. lucidum.