Hydrogen sulfide, a novel small molecule signalling agent, participates in the regulation of ganoderic acids biosynthesis induced by heat stress in Ganoderma lucidum.

Hydrogen sulfide (H2S), an emerging small-molecule signalling agent, was recently shown to play a significant role in many physiological processes, but relatively few studies have been conducted on microorganisms compared with mammals and plants. By studying the pretreatment of H2S donor sodium hydrosulfide (NaHS) and the scavenger hypotaurine (HT) and Cystathionine ß-synthase silenced strains, we found that H2S could alleviate the HS-induced ganoderic acids (GAs) biosynthesis. Our transcriptome results also showed that many signaling pathways and metabolic pathways, such as the glycolysis, TCA, oxidative phosphorylation and pentose phosphate pathway, are influenced by H2S. Further experimental results indicated that H2S could affect the physiological process of Ganoderma lucidum by interacting with multiple signals, including ROS, NO, AMPK, sphingolipid, mTOR, phospholipase D and MAPK, and physiological and pharmacological analyses showed that H2S might alleviate the biosynthesis of GAs by inhibiting the intracellular calcium in G. lucidum.

The Slt2-MAPK pathway is involved in the mechanism by which target of rapamycin regulates cell wall components in Ganoderma lucidum.

The fungal cell wall is very important for cell growth and survival during stress, and the target of rapamycin (TOR) pathway plays a major role in regulating cell growth in response to environmental cues. Ganoderma lucidum is an important edible and medicinal fungus, and the function of TOR in this organism remains unclear. As shown in the present study, the TOR pathway regulates cell wall integrity (CWI) in G. lucidum. Inhibition of TOR signaling by RNA interference (RNAi) or rapamycin treatment reduced the growth of G. lucidum mycelia, increased contents of the cell wall components chitin and ß-1,3-glucan, and increased cell wall thickness. Furthermore, inhibition of TOR signaling enhanced the relative level of phosphorylated Slt2, a member of the MAPK cascade involved in CWI signaling. Moreover, when treated with rapamycin, significantly lower chitin and ß-1,3-glucan contents were observed in Slt2-silenced strains than in WT strains, indicating that TOR regulates the synthesis of these cell wall components through the Slt2-MAPK pathway. These results indicate a potential relationship between TOR signaling and CWI signaling. Additionally, participation of Slt2-MAPK in TOR-mediated regulation of cell wall component production has not previously been reported in a microorganism.

Conversion of phosphatidylinositol (PI) to PI4-phosphate (PI4P) and then to PI(4,5)P2 is essential for the cytosolic Ca2+ concentration under heat stress in Ganoderma lucidum.

How cells drive the phospholipid signal response to heat stress (HS) to maintain cellular homeostasis is a fundamental issue in biology, but the regulatory mechanism of this fundamental process is unclear. Previous quantitative analyses of lipids showed that phosphatidylinositol (PI) accumulates after HS in Ganoderma lucidum, implying the inositol phospholipid signal may be associated with HS signal transduction. Here, we found that the PI-4-kinase and PI-4-phosphate-5-kinase activities are activated and that their lipid products PI-4-phosphate and PI-4,5-bisphosphate are increased under HS. Further experimental results showed that the cytosolic Ca2+ ([Ca2+ ]c ) and ganoderic acid (GA) contents induced by HS were decreased when cells were pretreated with Li+ , an inhibitor of inositol monophosphatase, and this decrease could be rescued by PI and PI-4-phosphate. Furthermore, inhibition of PI-4-kinases resulted in a decrease in the Ca2+ and GA contents under HS that could be rescued by PI-4-phosphate but not PI. However, the decrease in the Ca2+ and GA contents by silencing of PI-4-phosphate-5-kinase could not be rescued by PI-4-phosphate. Taken together, our study reveals the essential role of the step converting PI to PI-4-phosphate and then to PI-4,5-bisphosphate in [Ca2+ ]c signalling and GA biosynthesis under HS.

Infection Function of Adhesin-Like Protein ALP609 from Spiroplasma melliferum CH-1.

Spiroplasma melliferum is the causative agent of spiroplasmosis in honeybees. During infection, adhesion of spiroplasmas to the host cells through adhesion factors is a crucial step. In this study, we identified an adhesin-like protein (ALP609) in S. melliferum CH-1 and investigated its role in the infection. To determine whether ALP609 is an adhesion factor, we performed indirect immunofluorescence microscopy to visualize its adhesion properties. Subsequently, an infection model of S. melliferum CH-1 was established using primary midgut cells of Apis mellifera to examine the adhesion and invasion of spiroplasma using anti-ALP609 antibodies inhibition assays and competition assays with recombinant ALP609 in vitro. We found that anti-ALP609 antibodies could inhibit the adhesion and invasion of spiroplasma to the midgut cells of A. mellifera and reduce midgut cell invasion on increased exposure to recombinant ALP609. To the best of our knowledge, this is the first report identifying adhesion-related factors in S. melliferum. Our results suggested that ALP609 is an adhesin-like protein critical for invasion of S. melliferum CH-1 into midgut cells of A. mellifera.

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.

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.

Survey of Fusarium spp. Causing Wheat Crown Rot in Major Winter Wheat Growing Regions of China.

Fusarium crown rot of wheat has become more prevalent in China. To investigate the phylogenetic structure of Fusarium causing wheat crown rot in China, wheat basal stems with symptoms of the disease were collected from 2009 to 2013 in Jiangsu, Anhui, Henan, Hebei, and Shandong provinces. In total, 175 Fusarium isolates were collected and their mycotoxin chemotypes and distribution were identified. Among the 175 isolates, 123 were Fusarium asiaticum; 95 of these were the chemotype 3-acetyl-deoxynivalenol (3-AcDON) and 28 were nivalenol (NIV). Thirty-seven isolates belonged to F. graminearum, which were all 15-AcDON. Smaller numbers of isolates consisted of F. acuminatum, F. pseudograminearum, and F. avenaceum. The virulence of F. asiaticum and F. graminearum isolates on wheat crowns and heads was comparable. The virulence of isolates of the DON and NIV chemotype were statistically similar, but DON tended to be more aggressive. The DON concentrations in grains from wheat heads inoculated with isolates causing either Fusarium head blight or crown rot were similar. In the five provinces, F. asiaticum of the 3-AcDON chemotype was the predominant pathogen causing crown rot, followed by F. graminearum. Recent changes in causal Fusarium species, chemotypes, and distribution in China are discussed.

GlSwi6 Positively Regulates Cellulase and Xylanase Activities through Intracellular Ca2+ Signaling in Ganoderma lucidum.

Ganoderma lucidum is a white-rot fungus that produces a range of lignocellulolytic enzymes to decompose lignin and cellulose. The mitogen-activated protein kinase (MAPK) pathway has been implicated in xylanases and cellulases production. As the downstream transcription factor of Slt2-MAPK, the function of Swi6 in G. lucidum has not been fully studied. In this study, the transcription factor GlSwi6 in G. lucidum was characterized and shown to significantly positively regulate cellulases and xylanases production. Knockdown of the GlSwi6 gene decreased the activities of cellulases and xylanases by approximately 31%~38% and 54%~60% compared with those of the wild-type (WT) strain, respectively. Besides, GlSwi6 can be alternatively spliced into two isoforms, GlSwi6A and GlSwi6B, and overexpression of GlSwi6B increased the activities of cellulase and xylanase by approximately 50% and 60%, respectively. Further study indicates that the existence of GlSwi6B significantly increased the concentration of cytosolic Ca2+. Our study indicated that GlSwi6 promotes the activities of cellulase and xylanase by regulating the Ca2+ signaling. These results connected the GlSwi6 and Ca2+ signaling in the regulation of cellulose degradation, and provide an insight for further improvement of cellulase or xylanase activities in G. lucidum as well as other fungi.

A new Neotyphodium species from Festuca parvigluma Steud. grown in China.

Neotyphodium species evolved from Epichloë species and are asexual, seedborne endophytes in many cool-season grasses. Here we propose a new species inhabiting Festuca parvigluma native to China. Morphology, host specificity and molecular phylogenetic evidence supported recognition of this new species. Sequences of beta-tubulin gene (tubB) introns and translation elongation factor 1-alpha gene (tefA) introns were present as two copies in all five isolates examined. In phylogenetic analyses copy 1 was closely related to E. yangzii in the EBY clade and copy 2 with E. typhina in the ETC clade, indicating this new species might have originated as a result of hybridization between members of these two clades. Referring to the distribution area of host plants, Neotyphodium sinofestucae is proposed for this new species.

A new stromata-producing Neotyphodium species symbiotic with clonal grass Calamagrostis epigeios (L.) Roth. grown in China.

We describe a new stromata-producing Neotyphodium species symbiotic with clonal Calamagrostis epigeios (L.) Roth. Stromata on the grass, 47.5-186 mm long, occurred frequently, but neither perithecium nor mature ascus was observed. Morphology of fungal isolates obtained from symptomatic and asymptomatic tillers were identical to each other and similar to those of epichloë endophytes. In phylogenetic analysis all selected five fungal isolates clustered into a significantly distinct clade based on sequences of beta-tubulin gene (tubB) introns and translation elongation factor 1-alpha gene (tefA) introns with bootstrap values of 99%, supporting erection of a new species. Concerning the production of extremely long stromata on the host plants and absence of sexual spores, we propose the name Neotyphodium stromatolongum Y. Ji, L. Zhan et Z. Wang, sp. nov.

Heat stress-induced reactive oxygen species participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in Ganoderma lucidum.

Heat stress (HS) is an important environmental factor that affects the growth and metabolism of edible fungi, but the molecular mechanism of the heat stress response (HSR) remains unclear. We previously reported that HS treatment increased the length between two hyphal branches and induced the accumulation of ganoderic acid biosynthesis and the gene expression of heat shock proteins (HSPs) in Ganoderma lucidum. In this study, we found that HS induced a significant increase in the cytosolic ROS concentration, and exogenously added ROS scavengers NAC, VC and NADPH oxidase (Nox) inhibitor DPI reduce the cytosolic ROS accumulation in G. lucidum. In addition, the phenomena of the increased gene expression and increased length between the two hyphal branches and the accumulation of GA biosynthesis induced by HS were mitigated. Furthermore, we investigated the effects of HS on Nox-silenced strains (NoxABi-10, NoxABi-11 and NoxRi-4, NoxRi-7) and found that the level of ROS concentration was lower than that in wild-type (WT) strains treated with HS. Additionally, Nox silenced strains reduced the HS-induced increase in HSP expression, the length between two hyphal branches and GA biosynthesis compared with the WT strain. These data indicate that HS-induced ROS participate in the regulation of HSP expression, hyphal branching and ganoderic acid biosynthesis in G. lucidum. In addition, these findings identified potential pathways linking ROS networks to HSR, physiological and metabolic processes in fungi and provide a valuable reference for studying the role of ROS in HSR, mycelium growth and secondary metabolites.

Identification of Reference Genes and Analysis of Heat Shock Protein Gene Expression in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, after Exposure to Heat Stress.

Ganoderma lucidum has been considered an emerging model species for studying how environmental factors regulate the growth, development, and secondary metabolism of Basidiomycetes. Heat stress, which is one of the most important environmental abiotic stresses, seriously affects the growth, development, and yield of microorganisms. Understanding the response to heat stress has gradually become a hotspot in microorganism research. But suitable reference genes for expression analysis under heat stress have not been reported in G. lucidum. In this study, we systematically identified 11 candidate reference genes that were measured using reverse transcriptase quantitative polymerase chain reaction, and the gene expression stability was analyzed under heat stress conditions using geNorm and NormFinder. The results show that 5 reference genes-CYP and TIF, followed by UCE2, ACTIN, and UBQ1-are the most stable genes under our experimental conditions. Moreover, the relative expression levels of 3 heat stress response genes (hsp17.4, hsp70, and hsp90) were analyzed under heat stress conditions with different normalization strategies. The results show that use of a gene with unstable expression (SAND) as the reference gene leads to biased data and misinterpretations of the target gene expression level under heat stress.

A new species of Epichloë symbiotic with Chinese grasses.

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.