RNA-Seq-Based Transcriptome Analysis of Nitric Oxide Scavenging Response in Neurospora crassa.

While the biological role of naturally occurring nitric oxide (NO) in filamentous fungi has been uncovered, the underlying molecular regulatory networks remain unclear. In this study, we conducted an analysis of transcriptome profiles to investigate the initial stages of understanding these NO regulatory networks in Neurospora crassa, a well-established model filamentous fungus. Utilizing RNA sequencing, differential gene expression screening, and various functional analyses, our findings revealed that the removal of intracellular NO resulted in the differential transcription of 424 genes. Notably, the majority of these differentially expressed genes were functionally linked to processes associated with carbohydrate and amino acid metabolism. Furthermore, our analysis highlighted the prevalence of four specific protein domains (zinc finger C2H2, PLCYc, PLCXc, and SH3) in the encoded proteins of these differentially expressed genes. Through protein-protein interaction network analysis, we identified eight hub genes with substantial interaction connectivity, with mss-4 and gel-3 emerging as possibly major responsive genes during NO scavenging, particularly influencing vegetative growth. Additionally, our study unveiled that NO scavenging led to the inhibition of gene transcription related to a protein complex associated with ribosome biogenesis. Overall, our investigation suggests that endogenously produced NO in N. crassa likely governs the transcription of genes responsible for protein complexes involved in carbohydrate and amino acid metabolism, as well as ribosomal biogenesis, ultimately impacting the growth and development of hyphae.

Different Effects of Reactive Species Generated from Chemical Donors on Seed Germination, Growth, and Chemical Contents of Oryza sativa L.

Reactive oxygen and nitrogen species (RONS) play an important role as signaling molecules in redox reactions throughout a plant life cycle. The purpose of this study was to assess how hydrogen peroxide (H2O2), a reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated from sodium nitroprusside (SNP) and sodium nitrite, affects the germination, growth, and chemical contents of two rice cultivars (Pathum Tani and Sanpatong). The results showed that RNS generated from chemical donors and, especially, H2O2, enhanced the germination of the studied rice cultivars. Among the three chemical donors, H2O2 showed the best efficacy of the reactive species for activating early seed germination, followed by sodium nitrite and SNP. The highest percentage of seed germination rose to 99% at 6 h germination time after treatment with 25 mM of H2O2 for 24 h. Moreover, H2O2 produced a significant increase in the α-amylase activity and total soluble proteins. It was observed that a treatment with H2O2 on germinated seeds produced radicles with a dark blue color for longer than treatments with sodium nitrite and SNP. Our findings imply that H2O2 had a critical role in improving the germination and altering the chemical contents of rice seeds.

Enhancement of Fungal Enzyme Production by Radio-Frequency Electromagnetic Fields.

Enzyme production by microorganisms on an industrial scale has demonstrated technical bottlenecks, such as low efficiency in enzyme expression and extracellular secretion. In this study, as a potential tool for overcoming these technical limits, radio-frequency electromagnetic field (RF-EMF) exposure was examined for its possibility to enhance production of an enzyme, α-amylase, in a filamentous fungus, Aspergillus oryzae. The RF-EMF perfectly resonated at 2 GHz with directivity radiation pattern and peak gain of 0.5 dB (0.01 Watt). Total protein concentration and activity of α-amylase measured in media were about 1.5-3-fold higher in the RF-EMF exposed (10 min) sample than control (no RF-EMF) during incubation (the highest increase after 16 h). The level of α-amylase mRNA in cells was approximately 2-8-fold increased 16 and 24 h after RF-EMF exposure for 10 min. An increase in vesicle accumulation within fungal hyphae and the transcription of some genes involved in protein cellular trafficking was observed in RF-EMF-exposed samples. Membrane potential was not changed, but the intracellular Ca2+ level was elevated after RF-EMF exposure. Our results suggest that RF-EMF can increase the extracellular level of fungal total proteins and α-amylase activity and the intracellular level of Ca2+.

Application of Non-Thermal Plasma to Fungal Resources.

In addition to being key pathogens in plants, animals, and humans, fungi are also valuable resources in agriculture, food, medicine, industry, and the environment. The elimination of pathogenic fungi and the functional enhancement of beneficial fungi have been the major topics investigated by researchers. Non-thermal plasma (NTP) is a potential tool to inactivate pathogenic and food-spoiling fungi and functionally enhance beneficial fungi. In this review, we summarize and discuss research performed over the last decade on the use of NTP to treat both harmful and beneficial yeast- and filamentous-type fungi. NTP can efficiently inactivate fungal spores and eliminate fungal contaminants from seeds, fresh agricultural produce, food, and human skin. Studies have also demonstrated that NTP can improve the production of valuable enzymes and metabolites in fungi. Further studies are still needed to establish NTP as a method that can be used as an alternative to the conventional methods of fungal inactivation and activation.

Effects of Pre-Treatment Using Plasma on the Antibacterial Activity of Mushroom Surfaces.

Although non-thermal atmospheric pressure plasma is an efficient tool for preventing post-harvest microbial contamination, many studies have focused on the post-treatment of infected or contaminated foods. In this study, we examined the antimicrobial quality of mushrooms pre-treated with a non-thermal atmospheric pressure plasma jet (NTAPPJ) or plasma-treated water (PTW). The CFU (Colony Forming Unit) number of Escherichia coli inoculated on surfaces of mushrooms pre-treated with NTAPPJ or PTW was significantly reduced (about 60-75% for NTAPPJ and about 35-85% for PTW), and the reduction rate was proportional to the treatment time. Bacterial attachment and viability of the attached bacteria were decreased on NTAPPJ-treated mushroom surfaces. This may be caused by the increased hydrophilicity and oxidizing capacity observed on NTAPPJ-treated mushroom surfaces. In PTW-treated mushrooms, bacterial attachment was not significantly changed, but death and lipid peroxidation of the attached bacteria were significantly increased. Analysis of mushroom quality showed that loss of water content was greater in mushrooms treated with NTAPPJ compared to that in those with no treatment (control) and PTW treatment during storage. Our results suggest that pre-treatment with NTAPPJ or PTW can improve the antibacterial quality of mushroom surfaces by decreasing bacterial attachment (for NTAPPJ) and increasing bacterial lipid peroxidation (for both NTAPPJ and PTW).

Low-Temperature Plasma-Assisted Nitrogen Fixation for Corn Plant Growth and Development.

Nitrogen fixation is crucial for plants as it is utilized for the biosynthesis of almost all biomolecules. Most of our atmosphere consists of nitrogen, but plants cannot straightforwardly assimilate this from the air, and natural nitrogen fixation is inadequate to meet the extreme necessities of global nutrition. In this study, nitrogen fixation in water was achieved by an AC-driven non-thermal atmospheric pressure nitrogen plasma jet. In addition, Mg, Al, or Zn was immersed in the water, which neutralized the plasma-treated water and increased the rate of nitrogen reduction to ammonia due to the additional hydrogen generated by the reaction between the plasma-generated acid and metal. The effect of the plasma-activated water, with and without metal ions, on germination and growth in corn plants (Zea Mays) was investigated. The germination rate was found to be higher with plasma-treated water and more efficient in the presence of metal ions. Stem lengths and germination rates were significantly increased with respect to those produced by DI water irrigation. The plants responded to the abundance of nitrogen by producing intensely green leaves because of their increased chlorophyll and protein contents. Based on this report, non-thermal plasma reactors could be used to substantially enhance seed germination and seedling growth.

Plasma-mediated enhancement of enzyme secretion in Aspergillus oryzae.

Technical bottlenecks in protein production and secretion often limit the efficient and robust industrial use of microbial enzymes. The potential of non-thermal atmospheric pressure plasma to overcome these technical barriers was examined. Spores of the fermenting fungus Aspergillus oryzae (A. oryzae) were submerged in potato dextrose broth (PDB) (5 × 106 per ml) and treated with micro dielectric barrier discharge plasma at an input voltage of 1.2 kV and current of 50 to 63 mA using nitrogen as the feed gas. The specific activity of α-amylase in the broth was increased by 7.4 to 9.3% after 24 and 48 h of plasma treatment. Long-lived species, such as NO2 - and NO3 - , generated in PDB after plasma treatment may have contributed to the elevated secretion of α-amylase. Observations after 24 h of plasma treatment also included increased accumulation of vesicles at the hyphal tip, hyphal membrane depolarization and higher intracellular Ca2+ levels. These results suggest that long-lived nitrogen species generated in PDB after plasma treatment can enhance the secretion of α-amylase from fungal hyphae by depolarizing the cell membrane and activating Ca2+ influx into hyphal cells, eventually leading to the accumulation of secretory vesicles near the hyphal tips.

Plant Disease Control by Non-Thermal Atmospheric-Pressure Plasma.

Disease stresses caused by pathogenic microorganisms are increasing, probably because of global warming. Conventional technologies for plant disease control have often revealed their limitations in efficiency, environmental safety, and economic costs. There is high demand for improvements in efficiency and safety. Non-thermal atmospheric-pressure plasma has demonstrated its potential as an alternative tool for efficient and environmentally safe control of plant pathogenic microorganisms in many studies, which are overviewed in this review. Efficient inactivation of phytopathogenic bacterial and fungal cells by various plasma sources under laboratory conditions has been frequently reported. In addition, plasma-treated water shows antimicrobial activity. Plasma and plasma-treated water exhibit a broad spectrum of efficiency in the decontamination and disinfection of plants, fruits, and seeds, indicating that the outcomes of plasma treatment can be significantly influenced by the microenvironments between plasma and plant tissues, such as the surface structures and properties, antioxidant systems, and surface chemistry of plants. More intense studies are required on the efficiency of decontamination and disinfection and underlying mechanisms. Recently, the induction of plant tolerance or resistance to pathogens by plasma (so-called "plasma vaccination") is emerging as a new area of study, with active research ongoing in this field.

Analysis of the effects of Cu-MOFs on fungal cell inactivation.

Three dimensional (3D) copper metal organic frameworks (Cu-MOFs) containing glutarates and bipyridyl ligands (bpa = 1,2-bis(4-pyridyl)ethane, bpe = 1,2-bis(4-pyridyl)ethylene, or bpp = 1,3-bis(4-pyridyl)propane) were synthesized by using previously reported hydrothermal reactions or a layering method. All three Cu-MOFs contained well-defined one dimensional (1D) channels with very similar pore shapes and different pore dimensions. The bulk purities of the Cu-MOFs were confirmed using powder X-ray diffraction (PXRD) and infrared spectroscopy (IR) spectra. When the three types of Cu-MOFs were applied to Candida albicans cells and Aspergillus niger spores, an average of about 50-80% inactivation was observed at the highest concentration of Cu-MOFs (2 mg mL-1). The efficiency of the fungal inactivation was not significantly different among the three different types (bpa, bpe, bpp). Treatment of the fungi using Cu-MOFs induced an apoptosis-like death and this was more severe in A. niger than C. albicans. This may be due to elevation of the intracellular level of reactive oxygen species (ROS) in A. niger. Generation of the reactive species in solution by Cu-MOFs was observed. However, there was a dramatic variation in the levels observed among the three types. Our results suggest that Cu-MOFs can produce antifungal effects and induce apoptosis-like death of the fungi, which was probably caused by the elevated level of intracellular reactive species.

Cobalt(II)-coordination polymers containing glutarates and bipyridyl ligands and their antifungal potential.

Three new CoII-coordination polymers (Co-CPs) containing glutarates and bipyridyl ligands, formulated as [Co2(Glu)2(µ-bpa)2]·(H2O)4 (1), [Co4(Glu)4(µ-bpp)2] (2), and [Co2(Glu)2(µ-bpe)2]·(H2O)0.5 (3), were prepared, and their structures were determined by X-ray crystallography. Glutarates bridge CoII ions to form 2D sheets, and the sheets are connected either by bpa or by bpp ligands to form 3D networks 1 and 2, respectively. Both frameworks 1 and 2 are two-fold interpenetrated, and there is no significant void volume in either network. Four glutarates bridge two CoII ions to form chains, and these chains are connected by bpe ligands to form the 2D sheet 3. The antifungal properties of these new Co-CPs were tested against two model fungal pathogens, Candida albicans and Aspergillus niger. Under the maximum concentration of Co-CPs, 2.0 mg mL-1, the inhibition rates of Co-CPs against A. niger were much lower (44-62%) than those (90-99.98%) observed in C. albicans. The results indicate that 1-3 can inactivate C. albicans cells more efficiently than A. niger spores in the same treatment time, and the greater inactivation of C. albicans can be explained by dramatic changes in the morphology of C. albicans cells. We also found that Co-CPs could generate the reactive species NO and H2O2, and these species might play a role in inactivating fungal cells. Additionally, degradation tests confirmed that the leaching of CoII ions from Co-CPs was not significant. The small amount of leached CoII ions and the robust Co-CPs themselves as well as the reactive species generated by Co-CPs can actively participate in fungal inactivation.

Aspergillus oryzae spore germination is enhanced by non-thermal atmospheric pressure plasma.

Poor and unstable culture growth following isolation presents a technical barrier to the efficient application of beneficial microorganisms in the food industry. Non-thermal atmospheric pressure plasma is an effective tool that could overcome this barrier. The objective of this study was to investigate the potential of plasma to enhance spore germination, the initial step in fungal colonization, using Aspergillus oryzae, a beneficial filamentous fungus used in the fermentation industry. Treating fungal spores in background solutions of phosphate buffered saline (PBS) and potato dextrose broth (PDB) with micro dielectric barrier discharge plasma using nitrogen gas for 2 and 5 min, respectively, significantly increased the germination percentage. Spore swelling, the first step in germination, was accelerated following plasma treatment, indicating that plasma may be involved in loosening the spore surface. Plasma treatment depolarized spore membranes, elevated intracellular Ca2+ levels, and activated mpkA, a MAP kinase, and the transcription of several germination-associated genes. Our results suggest that plasma enhances fungal spore germination by stimulating spore swelling, depolarizing the cell membrane, and activating calcium and MAPK signaling.

Dynamics of nitric oxide level in liquids treated with microwave plasma-generated gas and their effects on spinach development.

In this study, we generated water and phosphate buffer treated with microwave plasma-generated gas in which the major component was nitric oxide (PGNO), and investigated the efficiency of the treated water and buffer in fertilization and sanitation. Real time NO level monitored by an electrode sensor was linearly increased over PGNO injection time, and removal of O2 from liquid before PGNO injection accelerated NO assimilation into liquids. Residual NO was still present 16 h after PGNO injection was stopped. H2O2, NO2-, and NO3- were also detected in PGNO-treated liquids. Spinach plants applied with 10 and 30 times diluted PGNO-treated water and 0.5 mM phosphate buffer showed slightly higher height and dry weight than control after 5 weeks. Plants grown with 10 and 30 times diluted PGNO-treated water exhibited the increased tolerance to water deficiency. Significant anti-microbial activity within 1 h was observed in un-diluted and in half-diluted PGNO-treated water and 0.5 mM phosphate buffer. Our results suggest that water or phosphate buffer containing NO, H2O2, NO2-, and NO3- can be produced by PGNO treatment, and that PGNO-treated water or buffer can be used as a potential fertilizer enhancing plant vitality with sanitation effect.

Biological and medical applications of plasma-activated media, water and solutions.

Non-thermal atmospheric pressure plasma has been proposed as a new tool for various biological and medical applications. Plasma in close proximity to cell culture media or water creates reactive oxygen and nitrogen species containing solutions known as plasma-activated media (PAM) or plasma-activated water (PAW) - the latter even displays acidification. These plasma-treated solutions remain stable for several days with respect to the storage temperature. Recently, PAM and PAW have been widely studied for many biomedical applications. Here, we reviewed promising reports demonstrating plasma-liquid interaction chemistry and the application of PAM or PAW as an anti-cancer, anti-metastatic, antimicrobial, regenerative medicine for blood coagulation and even as a dental treatment agent. We also discuss the role of PAM on cancer initiation cells (spheroids or cancer stem cells), on the epithelial mesenchymal transition (EMT), and when used for metastasis inhibition considering its anticancer effects. The roles of PAW in controlling plant disease, seed decontamination, seed germination and plant growth are also considered in this review. Finally, we emphasize the future prospects of PAM, PAW or plasma-activated solutions in biomedical applications with a discussion of the mechanisms and the stability and safety issues in relation to humans.

Diacylglycerol acyltransferase 2 of Mortierella alpina with specificity on long-chain polyunsaturated fatty acids: A potential tool for reconstituting lipids with nutritional value.

Based on available genome sequences and bioinformatics tools, we searched for an uncharacterized open reading frame of Mortierella alpina (MaDGAT2) using diacylglycerol acyltransferase sequence (fungal DGAT type 2B) as a query. Functional characterization of the identified native and codon-optimized M. alpina genes were then performed by heterologous expression in Saccharomyces cerevisiae strain defective in synthesis of neutral lipid (NL). Lipid analysis of the yeast tranformant carrying MaDGAT2 showed that the NL biosynthesis and lipid particle formation were restored by the gene complementation. Substrate specificity study of the fungal enzyme by fatty acid supplementation in the transformant cultures showed that it had a broad specificity on saturated and unsaturated fatty acid substrates for esterification into triacylglycerol (TAG). The n-6 polyunsaturated fatty acids (PUFAs) with 18 and 20 carbon atoms, including linoleic acid, γ-linolenic acid, dihomo γ-linolenic and arachidonic acid could be incorporated into TAG fraction in the yeast cells. Interestingly, among n-3 PUFAs tested, the MaDGAT2 enzyme preferred eicosapentaenoic acid (EPA) substrate as its highly proportional constituent found in TAG fraction. This study provides a potential genetic tool for reconstituting oils rich in long-chain PUFAs with nutritional value.

Metabolic engineering of long chain-polyunsaturated fatty acid biosynthetic pathway in oleaginous fungus for dihomo-gamma linolenic acid production.

Microbial lipids are promising alternative sources of long chain-polyunsaturated fatty acids (LC-PUFAs) for food, feed, nutraceutical and pharmaceutical sectors. Dihomo-γ-linolenic acid (C20:3Δ(8,11,14); DGLA) is an important LC-PUFAs with anti-inflammatory and anti-proliferative effects. To generate a DGLA-producing strain, fatty acid reconstitution in Aspergillus oryzae was performed by metabolic engineering through co-expression of codon-optimized Pythium Δ(6)-desaturase and Δ(6)-elongase, which had high conversion rates of substrates to respective products as compared to the native enzymes. The Δ(6)-desaturated and Δ(6)-elongated products, γ-linolenic acid (C18:3Δ(6,9,12); GLA) and DGLA, were accumulated in phospholipids rather than triacylglycerol. Interestingly, the manipulation of lipid quality in the oleaginous fungus did not affect growth and lipid phenotypes. This strategy might expand to development of the oleaginous fungal strain for producing other tailor-made oils with industrial applications.

Analysis of the Vitellogenin gene of rice moth, Corcyra cephalonica Stainton.

Vitellogenin (Vg) is a precursor of the major yolk protein, an essential nutrient for the embryonic development of oviparous animals including insects. Here, the gene(CceVg [Corcyra cephalonica Vg] ) encoding the Vg (CceVg of moth, C. cephalonica, was cloned and sequenced. The gene sequence was 6,721-bp long and contained 5five introns and six exons that together formed a 5,382-bp open reading frame. The deduced protein (CceVg) consisted of 1,793 amino acid residues, including a 16-amino-acid signal peptide. The putative molecular weight of the primary Vg protein was 202.46 kDa. The CceVg contained all conserved domains and motifs that were commonly found in most insect Vgs except the presence of a polyserine tract at the C-terminal region, which had not been reported in other lepidopteran Vgs. The expression pattern showed that CceVg was first transcribed at a very low level in the early larval stage but disappeared in later stage larva. In female, the CceVg mRNA was detected in early pupal stage and throughout adult stage. Interestingly, the CceVg mRNA was detected only in mated males at low levels, not in the virgin ones. Injection of CceVg double-stranded RNA into early-emergent females caused severely abnormal ovaries.