Transcriptomic and metabolomic analysis unveils a negative effect of glutathione metabolism on laccase activity in Cerrena unicolor 87613.

The white rot fungus Cerrena unicolor 87613 has been previously shown to be a promising resource in laccase production, an enzyme with significant biotechnological applications. Conventional methods face technical challenges in improving laccase activity. Attempts are still being made to develop novel approaches for further enhancing laccase activity. This study aimed to understand the regulation of laccase activity in C. unicolor 87613 for a better exploration of the novel approach. Transcriptomic and metabolomic analyses were performed to identify key genes and metabolites involved in extracellular laccase activity. The findings indicated a strong correlation between the glutathione metabolism pathway and laccase activity. Subsequently, experimental verifications were conducted by manipulating the pathway using chemical approaches. The additive reduced glutathione (GSH) dose-dependently repressed laccase activity, while the GSH inhibitors (APR-246) and reactive oxygen species (ROS) inducer (H2O2) enhanced laccase activity. Changes in GSH levels could determine the intracellular redox homeostasis in interaction with ROS and partially affect the expression level of laccase genes in C. unicolor 87613 in turn. In addition, GSH synthetase was found to mediate GSH abundance in a feedback loop. This study suggests that laccase activity is negatively influenced by GSH metabolism and provides a theoretical basis for a novel strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.IMPORTANCEThe production of laccase activity is limited by various conventional approaches, such as heterologous expression, strain screening, and optimization of incubation conditions. There is an urgent need for a new strategy to meet industrial requirements more effectively. In this study, we conducted a comprehensive analysis of the transcriptome and metabolome of Cerrena unicolor 87613. For the first time, we discovered a negative role played by reduced glutathione (GSH) and its metabolic pathway in influencing extracellular laccase activity. Furthermore, we identified a feedback loop involving GSH, GSH synthetase gene, and GSH synthetase within this metabolic pathway. These deductions were confirmed through experimental investigations. These findings not only advanced our understanding of laccase activity regulation in its natural producer but also provide a theoretical foundation for a strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.

Genome-wide study of Cerrena unicolor 87613 laccase gene family and their mode prediction in association with substrate oxidation.

BACKGROUND: Laccases are green biocatalysts with wide industrial applications. The study of efficient and specific laccase producers remains a priority. Cerrena species have been shown to be promising basidiomycete candidates for laccase production. Although two sets of Cerrena genome data have been publicly published, no comprehensive bioinformatics study of laccase gene family in C. unicolor has been reported, particularly concerning the analysis of their three-dimensional (3D) structures and molecular docking to substrates, like ABTS and aflatoxin B1 (AFB1). RESULTS: In this study, we conducted a comprehensive genome-wide analysis of laccase gene family in C. unicolor 87613. We identified eighteen laccase genes (CuLacs) and classified them into three clades using phylogenetic analysis. We characterized these laccases, including their location in contig 5,6,9,12,15,19,26,27, gene structures of different exon-intron arrangements, molecular weight ranging from 47.89 to 141.41 kDa, acidic pI value, 5-15 conserved protein motifs, signaling peptide of extracellular secretion (harbored by 13 CuLacs) and others. In addition, the analysis of cis-acting element in laccase promoters indicated that the transcription response of CuLac gene family was regulatable and complex under different environmental cues. Furthermore, analysis of transcription pattern revealed that CuLac8, 12 and CuLac2, 13 were the predominant laccases in response to copper ions or oxidative stress, respectively. Finally, we focused on the 3D structure analysis of CuLac proteins. Seven laccases with extra transmembrane domains or special sequences were particularly interesting. Predicted structures of each CuLac protein with or without these extra sequences showed altered interacting amino acid residues and binding sites, leading to varied affinities to both ABTS and AFB1. As far as we know, it is the first time to discuss the influence of the extra sequence on laccase's affinity to substrates. CONCLUSIONS: Our findings provide robust genetic data for a better understanding of the laccase gene family in C. unicolor 87613, and create a foundation for the molecular redesign of CuLac proteins to enhance their industrial applications.

Characterisation of a laccase isolated from Trametes hirsuta and its application in the oligomerisation of phenolic compounds.

Phenolic compounds are widely distributed in nature and industrial environment, and their detoxification or bioactive enhancement is of great value to environmental protection and industrial development. Laccases are multicopper oxidases that catalyse the oligo- or polymerisation of phenolic compounds. Identifying new laccase producers and investigating their application potential are of great importance. In this study, a white-rot fungus, Trametes hirsuta EZ1, with significantly high laccase productivity was isolated. The optimum conditions were studied for the maximum fermentation of extracellular laccase, which was achieved at 150 U/mL with a medium containing 10% strain EZ1, 7% maltodextrin, 1.5% peptone, and 0.5 mM Cu2+, and incubation at initial pH 6.0, 32 °C, and 180 rpm for nine days. Subsequently, a 70-kDa laccase was purified that showed activity over a wide range of temperature and pH, sensitivity to many metal ions and sodium dodecyl sulphate, and high tolerance to organic solvents. Purified laccase showed a significant unreported effect by catalysing catechol or ferulic acid into dimers, trimers, and tetramers or caffeic acid into dimers, trimers, tetramers, and pentamers. The oligomeric mixtures exhibited increased antioxidative capacity compared to that of each parent monomer, except for caffeic acid derivatives. Our study offers a novel strain source for laccase production and broadens its application in the enhancement of bioactive compounds.

Development of a monitoring system for disassembled towers with internal suspension poles.

The traditional construction monitoring methods of suspended pole-mounted decomposed towers are mostly manual monitoring. The monitoring personnel has multiple blind spots, and the possibility of misjudgment based on personal experience is relatively large. It is difficult to ensure the construction safety of the suspended pole decomposing tower. For this reason, combined with the current power Internet of Things technology, this paper develops an intelligent monitoring system for suspended pole-mounted decomposing towers. According to the construction technology and its safety requirements of inner suspension derrick for transmission tower erection in sections, this system is classified into intellisense layer, wireless transport layer and information integration layer. According to the physical characteristics of the seven major risk points of the inner suspension pole group tower, the intellisense layer developed corresponding sensing equipment to obtain risk information. In the wireless transport layer, the ZigBee and 4G communication technologies are selected to interconnect self-constituted LAN and 4G wide area networks, to complete on-site data interaction and long-distance transmission. In the information integration layer, the force of cable, the inclination and height of derrick, and the distance between derrick and tower are determined. The system has been verified by the 500 kV delivery project of Fujian Zhouning Pumped Storage Power Station. The average error of critical monitoring point data is 4.14%, and the average data transmission delays in the far and near fields of the system are 18 ms and 176 ms.

Bioinformatic Analysis Reveals the Distinct Role of 5'UTR-Specific m6A RNA Modification in Mice Developing Cerebral Cortices.

N6-methyladenosine (m6A) abundantly exists in the cerebral cortex and is emerging as an essential factor in cortical development and function. As the m6A-binding site appears to be dynamically methylated in different RNA regions at the temporal-specific developing stage, it is of value to distinguish the unique character of region- and temporal-specific m6A. Herein, we analyzed the status of temporal-specific m6A within RNA 5' untranslated region (5'UTR) using m6A-methylated sequencing data and transcriptomic sequencing data from 12.5- to 13-day embryonic cerebral cortices and 14-day postnatal ones. We identified sorts of RNAs that are uniquely m6A-methylated in the 5'UTR and sorted them into specific neurological processes. Compared with 3'UTR-m6A-methylated RNAs, 5'UTR-m6A-methylated RNAs showed unique functions and mechanisms in regulating cortical development, especially through the pathway of mRNA transport and surveillance. Moreover, the 5'UTR-specific m6A was associated with neurological disorders as well. The FoxO signaling pathway was then focused by these pathogenic 5'UTR-m6A-methylated RNAs and explored to be involved in the determination of neurological disorders. Additionally, the 5'UTR-m6A modification patterns and transcriptional patterns play independent but cohesive roles in the developing cortices. Our study emphasizes the importance of 5'UTR-specific m6A in the developing cortex and provides an informative reference for future studies of 5'UTR-specific m6A in normal cortical development and neurological disorders.

Analyses of transcriptomics and metabolomics reveal pathway of vacuolar Sur7 contributed to biocontrol potential of entomopathogenic Beauveria bassiana.

Beauveria bassiana is a critical entomopathogenic fungus for pest biocontrol, whose efficiency depends on fungal development and stress resistance. Unlike its revealed location in plasma membrane patches in other organisms, B. bassiana Sur7 specifically localized in vacuoles. This vacuolar Sur7 was previously demonstrated to affect stress tolerance, hyphal development and virulence. There, however, remain more mechanistic details to be explored. In this study, transcriptomics and metabolomics were applied to investigate the mechanism of vacuolar Sur7. Analyses of transcriptomics and metabolomics displayed many differentially expressed genes and abundant metabolites in response to Sur7 loss, respectively. Together with genes associated with vacuolar biofunction (including transportation and hydrolysis), the altered metabolites contributed to cell wall construction and stress resistance. Particularly, an N-acetylglucosamine-associated Brg1/Nrg1 pathway was enriched and partially affected by Sur7. Absence of Sur7 changed the expression level of Brg1/Nrg1 pathway-related transcript factors, which interfered with downstream phenotype of sporulation. In addition, Sur7 was involved in the accumulation of sphingoid bases, which may affect sphingolipid-related signaling pathway. Although experimental evidence is further required, our studies provide a preliminary framework for future exploring the regulatory mechanism of Sur7, and give a new version of metabolic agency connecting Sur7 and downstream signaling pathway.

Transcriptomic analysis of Sur7-mediated response of Beauveria bassiana to different nutritional conditions.

Integrity of the cell wall is requisite for fungal growth and function. Sur7 governs cell wall composition, and affects conidial sporulation and germination in Beauveria bassiana, a filamentous entomopathogenic fungus. The role of Sur7 in fungal growth on various nutrients remains unclear. We have previously reported that Sur7 deletion results in the attenuation of B. bassiana growth on supplemented Sabouraud dextrose agar (SDAY) and minimal Czapek-Dox agar (CDA) compared to wild type (WT). Here, we used transcriptomic analysis to compare WT and Sur7 mutant (ΔSur7) responses to CDA and SDAY. Growth on CDA, compared with that on SDAY, affected the expression of more genes in the WT than in the mutant. Differentially expressed genes were enriched for transportation process terms in the ΔSur7 mutant and metabolic process terms in the WT. Different processes were repressed in the ΔSur7 (metabolic process) and WT (ribosome synthesis) cells. Despite the shared enrichment of nitrogen metabolism genes, differentially expressed genes were enriched in distinct saccharide-energy metabolism terms in each strain. We conclude that Sur7 ensures the growth of B. bassiana in a minimal medium by influencing the expression of genes involved in the consumption of sucrose via specific energy metabolism pathways.

Subcellular localization of Sur7 and its pleiotropic effect on cell wall integrity, multiple stress responses, and virulence of Beauveria bassiana.

Sur7 is one of multiple proteins constituting MCC (membrane compartment of Can1 acting as an arginine/H+ symporter), a crucial membrane domain that can form punctuate eisosome spots on the plasma membrane and execute diverse functions in model yeast but remains poorly understood in filamentous fungi. Here, a Sur7 homolog bearing a typical SUR7 domain and four transmembrane domains was shown to localize in the conidial vesicles and enter vacuoles and appear sporadically on the periphery membrane during hyphal growth in the insect-pathogenic fungus Beauveria bassiana, implicating an involvement of Sur7 in cellular events linked to both plasma membrane and vacuoles. Deletion of sur7 resulted in reduced conidiation capacity and impaired conidial quality, which was featured by slower germination, attenuated virulence, and reduced carbohydrate epitopes (ß-N-acetylglucosamine and sialic acids). Also, the hyphal cell walls of the deletion mutant were severely impaired due to ~ 70% reductions in chitin and neutral carbohydrate contents and a moderate increase in alkali-soluble carbohydrate content. Consequently, the deletion mutant became more sensitive to three cell wall perturbing chemicals (Congo red, calcofluor white, and SDS) and an antifungal drug (caspofungin) and surprisingly showed a hypersensitivity to oxidative stress of H2O2 and an increased sensitivity to osmotic stress of NaCl or sorbitol. Its hypersensitivity to H2O2 was associated with transcriptional repression of critical catalase genes required for H2O2 decomposition. These findings unveil that Sur7 takes part in both MCC/eisosome and vacuolar events and hence acts as a sustainer of conidiation capacity, cell wall integrity, multiple stress tolerance, and virulence in B. bassiana. Key points • Sur7 is a component of the crucial membrane domain MCC in Beauveria bassiana. • Sur7 localizes mainly in the vacuoles and sporadically on the periphery membrane. • Sur7 is required for cell wall integrity and has a pleiotropic effect on B. bassiana.

Dynamic observation and analysis of metabolic response to moxibustion stimulation on ethanol-induced gastric mucosal lesions (GML) rats.

BACKGROUND: Gastric mucosal lesion (GML) is the initiating pathological process in many refractory gastric diseases. And moxibustion is an increasingly popular alternative therapy that prevents and treats diseases. However, there are few published reports about developing pathology of GML and therapeutic mechanism of moxibustion treatment on GML. In this study, we investigated pathology of GML and therapeutic mechanism of moxibustion treatment on GML. METHODS: The male Sprague-Dawley (SD) rats were induced by intragastric administration of 75% ethanol after fasting for 24 h and treated by moxibustion at Zusanli (ST36) and Liangmen (ST21) for 1 day, 4 days or 7 days. Then we applied 1H NMR-based metabolomics to dynamic analysis of metabolic profiles in biological samples (stomach, cerebral cortex and medulla). And the conventional histopathological examinations as well as metabolic pathways assays were also performed. RESULTS: Moxibustion intervention showed a beneficial effect on GML by modulating comprehensive metabolic alterations caused by GML, including energy metabolism, membrane metabolism, cellular active and neurotransmitters function. CONCLUSIONS: Moxibustion can effectively treat gastric mucosal damage and effectively regulate the concentration of some related differential metabolites to maintain the stability of the metabolic pathway.

Dynamic Analysis of Metabolic Response in Gastric Ulcer (GU) Rats with Electroacupuncture Treatment Using 1H NMR-Based Metabolomics.

Gastric ulcer (GU), a common digestive disease, has a high incidence and seriously endangers health of human. According to the previous studies, it has been proved that electroacupuncture at acupoints of stomach meridian had a good effect on GU. However, there are few published studies on metabolic response in gastric ulcer (GU) rats with electroacupuncture treatment. Herein, we observed the metabolic profiles in biological samples (stomach, liver, and kidney) of GU rats with electroacupuncture treatment by 1H NMR metabolomics combined with pathological examination. The male SD rats were induced by intragastric administration of 70% ethanol after fasting for 24 hours and treated by electroacupuncture at Zusanli (ST36) and Liangmen (ST21) for 1 day, 4 days, or 7 days, respectively. And the conventional histopathological examinations as well as metabolic pathways assays were also performed. We found that GU rats were basically cured after electroacupuncture treatment for 4 days and had a complete recovery after electroacupuncture treatment for 7 days by being modulated comprehensive metabolic changes, involved in the function of neurotransmitters, energy metabolism, cells metabolism, antioxidation, tissue repairing, and other metabolic pathways. These findings may be helpful to facilitate the mechanism elucidating of electroacupuncture treatment on GU.

Gas chromatography-mass spectrometry based metabolomics profile of hippocampus and cerebellum in mice after chronic arsenic exposure.

Intake of arsenic (As) via drinking water has been a serious threat to global public health. Though there are numerous reports of As neurotoxicity, its pathogenesis mechanisms remain vague especially its chronic effects on metabolic network. Hippocampus is a renowned area in relation to learning and memory, whilst recently, cerebellum is argued to be involved with process of cognition. Therefore, the study aimed to explore metabolomics alternations in these two areas after chronic As exposure, with the purpose of further illustrating details of As neurotoxicity. Twelve 3-week-old male C57BL/6J mice were divided into two groups, receiving deionized drinking water (control group) or 50 mg/L of sodium arsenite (via drinking water) for 24 weeks. Learning and memory abilities were tested by Morris water maze (MWM) test. Pathological and morphological changes of hippocampus and cerebellum were captured via transmission electron microscopy (TEM). Metabolic alterations were analyzed by gas chromatography-mass spectrometry (GC-MS). MWM test confirmed impairments of learning and memory abilities of mice after chronic As exposure. Metabolomics identifications indicated that tyrosine increased and aspartic acid (Asp) decreased simultaneously in both hippocampus and cerebellum. Intermediates (succinic acid) and indirect involved components of tricarboxylic acid cycle (proline, cysteine, and alanine) were found declined in cerebellum, indicating disordered energy metabolism. Our findings suggest that these metabolite alterations are related to As-induced disorders of amino acids and energy metabolism, which might therefore, play an important part in mechanisms of As neurotoxicity.

Effect of Moxibustion on Intestinal Microbiome in Acute Gastric Ulcer Rats.

In Traditional Chinese Medicine (TCM), moxibustion had been used for thousands of years. Many clinical case reports and scientific studies had proved that moxibustion had a good effect in treating acute gastric ulcer (AGU). Some studies had shown that the relative content and species of bacteria in the intestinal would be changed when gastric mucosal injury happened. However, there was little research on the effect of intestinal microbiome with AGU rats that were treating by moxibustion. This study is aimed at analyzing the effect of fecal microbiome in rats with AGU by the 16S rDNA sequencing technology. Male SD rats were established by orally feeding once with 70% ethanol at 4 ml/kg except the control group, then treated by moxibustion in the stomach meridian group ("Liangmen," "Zusanli") and the gallbladder meridian group ("Riyue," "Yanglingquan") for 5 days. The 16S rDNA sequencing technology analysis of feces combined with histopathological methods and molecular biological detection methods was used to evaluate the therapeutic mechanism of moxibustion on AGU. AGU brought cause changes in the number and species of intestinal bacteria. Moxibustion on stomach meridian group could reduce the area of gastric mucosal injury and regulate the relative content of GAS and EGF. Moreover, moxibustion on the stomach meridian group could increase the relative content and species of beneficial bacteria in the intestine of rats with AGU. The relative abundance of intestinal probiotics was significantly upregulated in Alphaproteobacteria, Actinomycetales, and Bacillales. In addition, moxibustion might promote the repair of gastric mucosal injury by increasing the number and species of beneficial bacteria in the intestine.

Difference of Liver and Kidney Metabolic Profiling in Chronic Atrophic Gastritis Rats between Acupuncture and Moxibustion Treatment.

Acupuncture and moxibustion proved to be very effective in chronic atrophic gastritis (CAG). According to the Chinese traditional medicine theory, chronic diseases have an influence on the function of liver and kidney. However, there is little research to demonstrate this theory. This study is aimed at assessing the 1H NMR-based metabolic profiling in liver and kidney of CAG rats and comparing the difference between electroacupuncture and moxibustion treatment. Male SD rats were subjected to CAG modeling by intragastric administration of mixture of 2% sodium salicylate and 30% alcohol coupled with compulsive sporting and irregular fasting for 12 weeks and then treated by electroacupuncture or moxibustion at Liangmen (ST 21) and Zusanli (ST 36) acupoints for 2 weeks. A 1H NMR analysis of liver and kidney samples along with histopathological examination and molecular biological assay was employed to assess and compare the therapeutic effects of electroacupuncture and moxibustion. CAG brought characterization of metabolomic signatures in liver and kidney of rats. Both electroacupuncture and moxibustion treatment were found to normalize the CAG-induced changes by restoring energy metabolism, neurotransmitter metabolism, antioxidation metabolism, and other metabolism, while the moxibustion treatment reversed more metabolites related to energy metabolism in liver than electroacupuncture treatment. CAG did have influence on liver and kidney of rats. Both of these treatments had good effects on CAG by reversing the CAG-induced perturbation in liver and kidney. For regulating the energy metabolism in liver, the moxibustion played more important role than electroacupuncture treatment.

Antioxidant enzymes and their contributions to biological control potential of fungal insect pathogens.

Filamentous fungal insect pathogens represent a source of biological insecticides and acaricides formulated using intact cells, such as conidia or other spores. These mycoinsecticides infect arthropod pests through cuticular penetration. In field application, formulated fungal cells are exposed to environmental stresses, including solar UV irradiation, high temperature, and applied chemical herbicides and fungicides, as well as stress from host immune defenses. These stresses often result in accumulation of toxic reactive oxygen species (ROS), generating oxidative stress to the fungal cells and hence affecting the efficacy and persistency of fungi formulated for pest control. In response, fungi have evolved effective antioxidant mechanisms that include enzyme families that act as ROS scavengers, e.g., superoxide dismutases, catalases, peroxidases, thioredoxins /thioredoxin reductases, and glutaredoxins/glutathione reductases. Over two dozen antioxidant enzymes dispersed in different families have been characterized in Beauveria bassiana in recent years. This mini-review focuses on the progress detailed in the studies of these enzymes and provides an overview of their antioxidant activities and contributions to conidial thermotolerance, UV resistance and virulence. These activities are crucial for the biological control potential of mycoinsecticide formulation and have significantly advanced our understanding of how these organisms work. Several potent antioxidant genes have been exploited for successful genetic engineering of entomopathogenic fungi aimed at enhancing their potential against arthropod pests.

Two eisosome proteins play opposite roles in autophagic control and sustain cell integrity, function and pathogenicity in Beauveria bassiana.

Pil1A and Pil1B are two core eisosome proteins that are homologous to yeast Pil1/Lsp1 or filamentous fungal Pil1A/Pil1B but have been unexplored in entomopathogenic fungi. Here we examined subcellular localization and functions of Pil1A and Pil1B in Beauveria bassiana, a fungal insect pathogen. Either localization or co-localization experiments of the two proteins demonstrated that Pil1A and Pil1B were simultaneously localized at the periphery of hyphal cells for formation of stable, punctuate spots in B. bassiana. This is different from a reliance of proper Lsp1/Pil1B localization upon Pil1/Pil1A in other fungi. Deletions of pil1A and pil1B caused opposite changes in expression of many autophagy-related genes and formation of intravacuolar autophagosomes. Such opposite changes were restored to nearly normal status by exogenous rapamycin, implicating a link of Pil1A/B to the target of rapamycin signalling pathway. All single/double deletion mutants of pil1A and pil1B lost almost all pathogenicity due to reduced ability to secrete Pr1 proteases for cuticle degradation. They also showed differential changes in cell wall integrity and multiple stress responses. These findings unveil opposite roles for Pil1A and Pil1B in autophagic regulation and an essentiality of both for cell integrity, function and pathogenicity of the fungal entomopathogen.

Distinct roles of two cytoplasmic thioredoxin reductases (Trr1/2) in the redox system involving cysteine synthesis and host infection of Beauveria bassiana.

Two thioredoxin (Trx) reductases (Trr1/2) are known to play overlapping roles in the yeast Trx-Trr redox system but are generally unexplored in filamentous fungi, which possess multiple Trx homologues. This study seeks to characterize the functions of Trr1 and Trr2 in Beauveria bassiana, a filamentous fungal insect pathogen, and to probe their Trx partners. Both Trr1 and Trr2 were evidently localized in the cytoplasm of B. bassiana, unlike the two yeast homologues that have been reported to localize in the cytoplasm and mitochondria, respectively. Most of the six trx genes were greatly upregulated at the transcriptional level in the absence of trr1 instead of trr2 in B. bassiana, in which the trr1/2 double deletion failed in many attempts. Deletion of trr1 resulted in increased Trx activity, severe cysteine auxotrophy, and drastically reduced activities of peroxidases and superoxide dismutases under normal or oxidative conditions despite little change in catalase activity. Such changes disappeared in the absence of trr2 and were completely restored by complementation of trr1/2 or overexpression of trx1/6 in the Δtrr1 mutant, but were not restored at all by overexpression of trx2/3/4/5 or trr2 in the same mutant. All of these mutants exhibited similar trends of changes in the antioxidant response, conidiation, germination, thermotolerance, UV-B resistance, and virulence. Taken together, the findings indicate that Trr1 could reduce Trx2-5 and hence dominate the intracellular redox state, profoundly affecting the potential of B. bassiana against arthropod pests. Trr2 could reduce Trx1/6 but function only in the absence of Trr1.

Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen.

Subcellular localization of six thioredoxins and their antioxidant activity and contributions to biological control potential in Beauveria bassiana.

Thioredoxins (Trx) can detoxify sulfide or act as electron donors in the reduction of disulfide and dithiol to protect yeast cells from ROS damage but remain poorly explored in filamentous fungi. Here we show more Trx homologs in Beauveria bassiana than in many other fungi and examine their functions. This filamentous entomopathogen has six Trx homologs, including four (Txr1-4) evidently localized in cytoplasm, one (Trx5) in nuclear membrane and another (Trx6) in mitochondria. Deletion of each trx had no effect on radial growth on rich or minimal medium but resulted in remarkable transcriptional up-regulation of other partners for compensation. Compared with wild-type, only Δtrx2 was significantly more sensitive to menadione whereas none of six Δtrx mutants was responsive to other oxidants including H2O2. Intriguingly, Δtrx2 showed uniquely a significant increase in total Trx activity in normal cultures but a remarkable decrease in total SOD activity in the cultures grown normally or co-cultivated with menadione. The ratio of reduced/oxidized glutathione accumulated in hyphal cells stressed with menadione decreased to only 0.4 in Δtrx2 from ∼1.0 observed in wild-type and other mutants. The six Δtrx mutants displayed one or more phenotypic changes associated with the fungal biocontrol potential, including conidiation, and germination, thermotolerance, UV-B resistance and virulence of their conidia. All the changes were restored by trx complementation. Taken together, the greater Trx diversity evolutionarily gained by B. bassiana could help it to maintain cellular redox homeostasis and infect insect hosts in diverse habitats.

The role of three calcineurin subunits and a related transcription factor (Crz1) in conidiation, multistress tolerance and virulence in Beauveria bassiana.

The eukaryotic calcineurin (CN) pathway comprising catalytic A (CnA) and regulatory B subunits (CnB) is crucial for many biological processes but functionally unexplored in entomopathogenic fungi. Here, we characterise three CN subunits (CnA1, CnA2 and CnB) and a downstream CN-responsive zinc finger transcription factor (Crz1) in Beauveria bassiana. CN-mediated phosphatase activity decreased by 16-38 % in all deletion mutants compared with wild type. Growth and conidiation were most defective in ΔcnB, which showed a large proportion of abnormally branched germlings but were less defective in ΔcnA1 and ΔcnA2. Conidiation defects also occurred in Δcrz1, uniquely accompanied with slower germination. Compared with wild type, the four deletion mutants became, to varying degrees, more sensitive to Ca(2+), Mn(2+), Zn(2+), Mg(2+), two oxidants, three cell wall stressors, carbendazim, heat shock and ultraviolet (UV)-B irradiation. They were also less virulent to Spodoptera litura larvae. Only ΔcnB and Δcrz1 were less tolerant to high osmolarity. The altered phenotypes of the deletion mutants were associated with lower intracellular mannitol and trehalose levels, reduced overall activity of superoxide dismutases and catalases, altered cell wall composition and down-regulation of numerous phenotype-influencing genes. Additionally, the transcription of six cascaded genes in two stress-responsive mitogen-activated protein kinase (MAPK) pathways and the phosphorylation of hallmarking Hog1 and Slt2 were largely down-regulated in all the deletion mutants under osmotic and cell wall stresses, respectively. All the changes were restored by gene complementation. Taken together, three calcineurin subunits and Crz1 play vital, but variable, roles in B. bassiana responses to environmental stresses during development and host signals during infection.

Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses.

The catalase family of Beauveria bassiana (fungal entomopathogen) consists of catA (spore-specific), catB (secreted), catP (peroxisomal), catC (cytoplasmic) and catD (secreted peroxidase/catalase), which were distinguished in phylogeny and structure and functionally characterized by constructing single-gene disrupted and rescued mutants for enzymatic and multi-phenotypic analyses. Total catalase activity decreased 89% and 56% in ΔcatB and ΔcatP, corresponding to the losses of upper and lower active bands gel-profiled for all catalases respectively, but only 9-12% in other knockout mutants. Compared with wild type and complement mutants sharing similar enzymatic and phenotypic parameters, all knockout mutants showed significant (9-56%) decreases in the antioxidant capability of their conidia (active ingredients of mycoinsecticides), followed by remarkable phenotypic defects associated with the fungal biocontrol potential. These defects included mainly the losses of 40% thermotolerance (45°C) in ΔcatA, 46-48% UV-B resistance in ΔcatA and ΔcatD, and 33-47% virulence to Spodoptera litura larvae in ΔcatA, ΔcatP and ΔcatD respectively. Moreover, the drastic transcript upregulation of some other catalase genes observed in the normal culture of each knockout mutant revealed functionally complimentary effects among some of the catalase genes, particularly between catB and catC whose knockout mutants displayed little or minor phenotypic changes. However, the five catalase genes functioned redundantly in mediating the fungal tolerance to either hyperosmotic or fungicidal stress. The differentiated roles of five catalases in regulating the B. bassiana virulence and tolerances to oxidative stress, high temperature and UV-B irradiation provide new insights into fungal adaptation to stressful environment and host invasion.