Swi6B, an alternative splicing isoform of Swi6, mediates the cell wall integrity of Ganoderma lucidum.

The cell wall integrity (CWI) signaling activates the transcription factor Swi6 through a MAPK signaling cascade in response to cell wall stresses. In this study, we observed two different mRNA variants of swi6 (GlSwi6A and GlSwi6B) existed, due to alternative splicing. Besides, the expression level of GlSwi6B was higher than that of the GlSwi6A mRNA variant. The co-silencing of GlSwi6A and GlSwi6B was more sensitive to cell wall stress compared with WT, resulting in a decrease of 78% and 76% in chitin and ß-1,3-d-glucan content respectively. However, only the overexpression of GlSwi6B decreased the sensitivity to cell wall stress and increased the content of chitin and ß-1,3-d-glucan compared with the WT strain. Furthermore, Y1H, EMSA and BLI assays revealed that the GlSwi6B could bind to the promoters of chitin and glucan synthesis genes (GL24454 and GL18134). However, the binding phenome has not been observed in the isoform GlSwi6A. Taken together, our results found two different transcripts generated from Swi6, in which the alternative splice isoform of GlSwi6B participates in regulating the CWI of G. lucidum. This study provides the first insight into the alternative splicing isoform of GlSwi6B in the regulation of CWI signaling in fungi.

GCN4 Regulates Secondary Metabolism through Activation of Antioxidant Gene Expression under Nitrogen Limitation Conditions in Ganoderma lucidum.

Nitrogen limitation has been widely reported to affect the growth and development of fungi, and the transcription factor GCN4 (general control nonderepressible 4) is involved in nitrogen restriction. Here, we found that nitrogen limitation highly induced the expression of GCN4 and promoted the synthesis of ganoderic acid (GA), an important secondary metabolite in Ganoderma lucidum. The activated GCN4 is involved in regulating GA biosynthesis. In addition, the accumulation of reactive oxygen species (ROS) also affects the synthesis of GA under nitrogen restrictions. The silencing of the gcn4 gene led to further accumulation of ROS and increased the content of GA. Further studies found that GCN4 activated the transcription of antioxidant enzyme biosynthesis genes gr, gst2, and cat3 (encoding glutathione reductase, glutathione S-transferase, and catalase, respectively) through direct binding to the promoter of these genes to reduce the ROS accumulation. In conclusion, our study found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. This provides an essential insight into the understanding of GCN4 transcriptional regulation of the ROS signaling pathway and enriches the knowledge of nitrogen regulation mechanisms in fungal secondary metabolism of G. lucidum.IMPORTANCE Nitrogen has been widely reported to regulate secondary metabolism in fungi. Our study assessed the specific nitrogen regulatory mechanisms in Ganoderma lucidum. We found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. Our research highlights a novel insight that GCN4, the nitrogen utilization regulator, participates in secondary metabolism through ROS signal regulation. In addition, this also provides a theoretical foundation for exploring the regulation of other physiological processes by GCN4 through ROS in fungi.

Glsirt1-mediated deacetylation of GlCAT regulates intracellular ROS levels, affecting ganoderic acid biosynthesis in Ganoderma lucidum.

Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. However, in Basidiomycetes, the extent of lysine acetylation of nonhistone proteins remains largely unknown. Recently, we identified the deacetylase Glsirt1 as a key regulator of the biosynthesis of ganoderic acid (GA), a key secondary metabolite of Ganoderma lucidum. To gain insight into the characteristics, extent, and biological function of Glsirt1-mediated lysine acetylation in G. lucidum, we aimed to identify additional Glsirt1 substrates via comparison of acetylomes between wild-type (WT) and Glsirt1-silenced mutants. A large amount of Glsirt1-dependent lysine acetylation occurs in G. lucidum according to the results of this omics analysis, involving energy metabolism, protein synthesis, the stress response and other pathways. Our results suggest that GlCAT is a direct target of Glsirt1 and that the deacetylation of GlCAT by Glsirt1 reduces catalase activity, thereby leading to the accumulation of intracellular reactive oxygen species (ROS) and positively regulating the biosynthesis of GA. Our findings provide evidence for the involvement of nonhistone lysine acetylation in the biological processes of G. lucidum and help elucidate the involvement of important ROS signaling molecules in regulating physiological and biochemical processes in this organism. In conclusion, this proteomic analysis reveals a striking breadth of cellular processes affected by lysine acetylation and provides new nodes of intervention in the biosynthesis of secondary metabolites in G. lucidum.

Suaeda salsa Root-Associated Microorganisms Could Effectively Improve Maize Growth and Resistance under Salt Stress.

Root-associated microorganisms are widely recognized as playing an important role in mitigating stress-induced damage to plants, but the responses of rhizosphere microbial communities after inoculation and their relationship with plant responses remain unclear. In this study, the bacterium Providencia vermicola BR68 and the fungus Sarocladium kiliense FS18 were selected from among 91 strains isolated from the halophyte Suaeda salsa to interact with maize seedlings under salt stress. The results showed that compared with NaCl-only treatment, inoculation with strains BR68 and FS18 significantly improved the growth, net photosynthetic rate, and antioxidant enzyme activities of maize; significantly reduced proline content and generation rate of reactive oxygen species (ROS); and alleviated oxidative stress and osmotic stress. Moreover, inoculation with these two strains increased the activities of soil microbiome enzymes such as sucrase, catalase, and fluorescein diacetate hydrolase, which improved maize physiologies and promoted maize growth under salt stress. In addition, these inoculated strains significantly affected the abundance of certain genera, and the correlation trends for these genera with soil properties and maize physiologies were similar to those of these inoculated strains. Strain BR68 was indirectly associated with bacterial communities through BR-specific biomarkers, and bacterial communities and soil properties explained most of the variation in maize physiologies and growth. Inoculation of strain FS18 was directly associated with variations in soil properties and maize physiologies. The two strains improved maize growth under salt stress and alleviated stress damage in maize in different ways. The links among salt-tolerant microorganisms, soil, and plants established in this study can inform strategies for improving crop cultivation in salinized lands. IMPORTANCE This study demonstrates that halophyte root-associated microorganisms can promote crop tolerance to salt stress and clarify the mechanism by which the strains work in rhizosphere soil. The links among salt-tolerant microorganisms, soil, and plants established in this study can inform strategies for improving crop cultivation in salinized lands.

GlPP2C1 Silencing Increases the Content of Ganodermalingzhi Polysaccharide (GL-PS) and Enhances Slt2 Phosphorylation.

Polysaccharides have attracted much attention in the food industry due to their diverse biological activities. To date, research on the mechanism of polysaccharide synthesis has mainly focused on the role of crucial enzymes in the polysaccharide synthesis pathway, but other genes that regulate polysaccharide synthesis have not been well explored. In this study, the GlPP2C1 gene, encoding a phosphoprotein type 2C phosphatase, was cloned, and PP2C-silenced strains (PP2C1i-1 and PP2C1i-3) were screened. Measurements of the polysaccharide content and cell wall tolerance revealed that GlPP2C1 silencing increased the polysaccharide content and enhanced cell wall resistance in Ganoderma lingzhi. The contents of intracellular polysaccharides (IPS), extracellular polysaccharides (EPS) and ß-1,3-D-glucan in PP2C-silenced strains were increased by 25%, 33% and 36%, respectively, compared with those in the WT strains and strains transformed with an empty vector. Further mechanistic studies showed that GlPP2C1 silencing increased the content of Ganoderma lingzhi polysaccharides (GL-PS) through Slt2. In summary, this study revealed the mechanism through which protein phosphatase regulates GL-PS biosynthesis for the first time.

Lenthionine, a Key Flavor Substance in Lentinula edodes, Is Regulated by Cysteine under Drought Stress.

Due to its unique flavor profile, Lentinula edodes has become one of the most popular edible mushrooms in the world, but the regulatory mechanism of its flavor substances has not been revealed. To study the mechanism that regulates the anabolic metabolism of the important flavor substance lenthionine (LT), the effect of cysteine (Cys) synthesized by the cystathionine-γ-lyase (CSE-1) gene participating in the regulation of LT metabolism under drought stress was analyzed. Our results showed that drought stress promoted the accumulation of LT, and the key genes GTT and LECSL were activated. Furthermore, drought stress promoted the accumulation of intracellular Cys and activated the key gene for Cys synthesis, CSE-1. Both inhibition of the CSE enzyme activity by inhibitors and silencing of the CSE-1 gene under drought stress significantly reduced the intracellular contents of Cys and LT, but the inhibition of LT synthesis disappeared after the exogenous addition of Cys. These results indicate that LT synthesis in L. edodes under drought stress is dependent on Cys. In summary, the mechanism of the regulation of flavor substances in edible mushrooms by the environment was revealed for the first time.