Intracellular Nitric Oxide and cAMP Are Involved in Cellulolytic Enzyme Production in Neurospora crassa.

Although molecular regulation of cellulolytic enzyme production in filamentous fungi has been actively explored, the underlying signaling processes in fungal cells are still not clearly understood. In this study, the molecular signaling mechanism regulating cellulase production in Neurospora crassa was investigated. We found that the transcription and extracellular cellulolytic activity of four cellulolytic enzymes (cbh1, gh6-2, gh5-1, and gh3-4) increased in Avicel (microcrystalline cellulose) medium. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) detected by fluorescent dyes were observed in larger areas of fungal hyphae grown in Avicel medium compared to those grown in glucose medium. The transcription of the four cellulolytic enzyme genes in fungal hyphae grown in Avicel medium was significantly decreased and increased after NO was intracellularly removed and extracellularly added, respectively. Furthermore, we found that the cyclic AMP (cAMP) level in fungal cells was significantly decreased after intracellular NO removal, and the addition of cAMP could enhance cellulolytic enzyme activity. Taken together, our data suggest that the increase in intracellular NO in response to cellulose in media may have promoted the transcription of cellulolytic enzymes and participated in the elevation of intracellular cAMP, eventually leading to improved extracellular cellulolytic enzyme activity.

Characteristics of a Rollable Dielectric Barrier Discharge Plasma and Its Effects on Spinach-Seed Germination.

We investigated the characteristics of a rollable dielectric barrier discharge (RDBD) and evaluate its effects on seed germination rate and water uptake. The RDBD source was composed of a polyimide substrate and copper electrode, and it was mounted in a rolled-up structure for omnidirectional and uniform treatment of seeds with flowing synthetic air gas. The rotational and vibrational temperatures were measured to be 342 K and 2860 K, respectively, using optical emission spectroscopy. The chemical species analysis via Fourier-transform infrared spectroscopy and 0D chemical simulation showed that O3 production was dominant and NOx production was restrained at the given temperatures. The water uptake and germination rate of spinach seeds by 5 min treatment of RDBD was increased by 10% and 15%, respectively, and the standard error of germination was reduced by 4% in comparison with the controls. RDBD enables an important step forward in non-thermal atmospheric-pressure plasma agriculture for omnidirectional seed treatment.

Plasma Promotes Fungal Cellulase Production by Regulating the Levels of Intracellular NO and Ca2.

For the industrial-scale production of useful enzymes by microorganisms, technological development is required for overcoming a technical bottleneck represented by poor efficiency in the induction of enzyme gene expression and secretion. In this study, we evaluated the potential of a non-thermal atmospheric pressure plasma jet to improve the production efficiency of cellulolytic enzymes in Neurospora crassa, a filamentous fungus. The total activity of cellulolytic enzymes and protein concentration were significantly increased (1.1~1.2 times) in media containing Avicel 24-72 h after 2 and 5 min of plasma treatment. The mRNA levels of four cellulolytic enzymes in fungal hyphae grown in media with Avicel were significantly increased (1.3~17 times) 2-4 h after a 5 min of plasma treatment. The levels of intracellular NO and Ca2+ were increased in plasma-treated fungal hyphae grown in Avicel media after 48 h, and the removal of intracellular NO decreased the activity of cellulolytic enzymes in media and the level of vesicles in fungal hyphae. Our data suggest that plasma treatment can promote the transcription and secretion of cellulolytic enzymes into the culture media in the presence of Avicel (induction condition) by enhancing the intracellular level of NO and Ca2+.

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.

MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum.

MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

Effects of high voltage nanosecond pulsed plasma and micro DBD plasma on seed germination, growth development and physiological activities in spinach.

In this study, we analyzed seed germination, seedling growth, and physiological aspects after treatment with high voltage nanosecond pulsed plasma and micro DBD plasma in spinach (Spinacia oleracea L.), a green leafy vegetable known to have low germination rate. Both germination and dry weight of seedlings increased after high voltage pulse shots were applied to spinach seeds. However seeds treated with many shots (10 shots) showed a decrease in germination rate and seedling growth. Seeds treated with air DBD plasma exhibited slightly higher germination and subsequent seedling growth than those treated with N2 plasma. Seed surface was degenerated after treated with high voltage pulsed plasma and micro DBD plasma but no significant difference in the degree of degeneration was observed among micro DBD plasma treatment time. Level of GA3 hormone and mRNA expression of an amylolytic enzyme-related gene in seeds were elevated 1 day after treatment with high voltage pulsed plasma. The relative amount of chlorophyll and total polyphenols in spinach seedlings grown from seeds treated with air DBD plasma was increased in 30 s, 1 min, and 3 min treatments. Taken together, our results suggest a possibility that plasma can enhance seed germination by triggering biochemical processes in seeds.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Nitric Oxide in Fungi: Production and Function.

Nitric oxide (NO) is synthesized in all kingdoms of life, where it plays a role in the regulation of various physiological and developmental processes. In terms of endogenous NO biology, fungi have been less well researched than mammals, plants, and bacteria. In this review, we summarize and discuss the studies to date on intracellular NO biosynthesis and function in fungi. Two mechanisms for NO biosynthesis, NO synthase (NOS)-mediated arginine oxidation and nitrate- and nitrite-reductase-mediated nitrite reduction, are the most frequently reported. Furthermore, we summarize the multifaceted functions of NO in fungi as well as its role as a signaling molecule in fungal growth regulation, development, abiotic stress, virulence regulation, and metabolism. Finally, we present potential directions for future research on fungal NO biology.

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.

Global analysis of serine-threonine protein kinase genes in Neurospora crassa.

Serine/threonine (S/T) protein kinases are crucial components of diverse signaling pathways in eukaryotes, including the model filamentous fungus Neurospora crassa. In order to assess the importance of S/T kinases to Neurospora biology, we embarked on a global analysis of 86 S/T kinase genes in Neurospora. We were able to isolate viable mutants for 77 of the 86 kinase genes. Of these, 57% exhibited at least one growth or developmental phenotype, with a relatively large fraction (40%) possessing a defect in more than one trait. S/T kinase knockouts were subjected to chemical screening using a panel of eight chemical treatments, with 25 mutants exhibiting sensitivity or resistance to at least one chemical. This brought the total percentage of S/T mutants with phenotypes in our study to 71%. Mutants lacking apg-1, an S/T kinase required for autophagy in other organisms, possessed the greatest number of phenotypes, with defects in asexual and sexual growth and development and in altered sensitivity to five chemical treatments. We showed that NCU02245/stk-19 is required for chemotropic interactions between female and male cells during mating. Finally, we demonstrated allelism between the S/T kinase gene NCU00406 and velvet (vel), encoding a p21-activated protein kinase (PAK) gene important for asexual and sexual growth and development in Neurospora.

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.

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+.

Small RNA Isolation and Library Construction for Expression Profiling of Small RNAs from Neurospora crassa and Fusarium oxysporum and Analysis of Small RNAs in Fusarium oxysporum-Infected Plant Root Tissue.

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30 nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300 µg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.

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).

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.

Cold plasma seed priming modulates growth, redox homeostasis and stress response by inducing reactive species in tomato (Solanum lycopersicum).

Seed germination and vegetative growth are two important plant growing stages that are vulnerable to physical and biological stress. Improvement in crop germination potential and seedling growth rate generally leads to high crop productivity. Cold plasma is a promising technology used to improve seed germination and growth. Structural changes on tomato seed surface exposed with cold air plasma jet for a different time period (1 min, 5 min, 10 min) was examined by SEM. For in-depth study, different physiological parameter such as seed germination and seedling growth, biochemical parameter such as reactive species status, antioxidants and phytohormone, and molecular analysis of various gene expression was also evaluated. Drought stress tolerance potential of cold plasma primed tomato seedling was also examined under 30% PEG stress. Cold plasma seed priming modulates tomato seed coat and improves the germination efficiency. It also induces growth, antioxidants, phytohormone, defense gene expression, and drought stress tolerance potential of tomato seedling. Cold plasma seeds priming augment the reactive species at a molecular level within seedlings, which changes the biochemistry and physiological parameters of plants by inducing different cellular signaling cascades.

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.

Mitogen-activated protein kinase cascade required for regulation of development and secondary metabolism in Neurospora crassa.

Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hyphae and the formation of asexual macroconidia was reduced in Deltamik-1 mutants and almost abolished in Deltamek-1 and Deltamak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. Deltamik-1, Deltamek-1, and Deltamak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAPK was not phosphorylated in Deltamik-1 and Deltamek-1 mutants, consistent with the involvement of MIK-1, MEK-1, and MAK-1 in the same signaling cascade. Interestingly, we observed increased levels of mRNA and protein for tyrosinase in the mutants under nitrogen starvation, a condition favoring sexual differentiation. Tyrosinase is an enzyme that catalyzes production of the secondary metabolite l-DOPA melanin. These results implicate the MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi.

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.