The Hansenula polymorpha mitochondrial carrier family protein Mir1 is dually localized at peroxisomes and mitochondria.

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324.

Glucose and HODEs regulate Aspergillus ochraceus quorum sensing through the GprC-AcyA pathway.

Aspergillus ochraceus is the traditional ochratoxin A (OTA)-producing fungus with density-dependent behaviors, which is known as quorum sensing (QS) that is mediated by signaling molecules. Individual cells trend to adapt environmental changes in a "whole" flora through communications, allowing fungus to occupy an important ecological niche. Signals perception, transmission, and feedback are all rely on a signal network that constituted by membrane receptors and intracellular effectors. However, the interference of density information in signal transduction, which regulates most life activities of Aspergillus, have yet to be elucidated. Here we show that the G protein-coupled receptor (GPCR) to cAMP pathway is responsible for transmitting density information, and regulates the key point in life cycle of A. ochraceus. Firstly, the quorum sensing phenomenon of A. ochraceus is confirmed, and identified the density threshold is 103 spores/mL, which represents the low density that produces the most OTA in a series quorum density. Moreover, the GprC that classified as sugar sensor, and intracellular adenylate cyclase (AcyA)-cAMP-PKA pathway that in response to ligands glucose and HODEs are verified. Furthermore, GprC and AcyA regulate the primary metabolism as well as secondary metabolism, and further affects the growth of A. ochraceus during the entire life cycle. These studies highlight a crucial G protein signaling pathway for cell communication that is mediated by carbohydrate and oxylipins, and clarified a comprehensive effect of fungal development, which include the direct gene regulation and indirect substrate or energy supply. Our work revealed more signal molecules that mediated density information and connected effects on important adaptive behaviors of Aspergillus ochraceus, hoping to achieve comprehensive prevention and control of mycotoxin pollution from interrupting cell communication.

Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing.

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3' splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

Red and far-red light improve the antagonistic ability of Trichoderma guizhouense against phytopathogenic fungi by promoting phytochrome-dependent aerial hyphal growth.

Light as a source of information regulates morphological and physiological processes of fungi, including development, primary and secondary metabolism, or the circadian rhythm. Light signaling in fungi depends on photoreceptors and downstream components that amplify the signal to govern the expression of an array of genes. Here, we investigated the effects of red and far-red light in the mycoparasite Trichoderma guizhouense on its mycoparasitic potential. We show that the invasion strategy of T. guizhouense depends on the attacked species and that red and far-red light increased aerial hyphal growth and led to faster overgrowth or invasion of the colonies. Molecular experiments and transcriptome analyses revealed that red and far-red light are sensed by phytochrome FPH1 and further transmitted by the downstream MAPK HOG pathway and the bZIP transcription factor ATF1. Overexpression of the red- and far-red light-induced fluffy gene fluG in the dark resulted in abundant aerial hyphae formation and thereby improvement of its antagonistic ability against phytopathogenic fungi. Hence, light-induced fluG expression is important for the mycoparasitic interaction. The increased aggressiveness of fluG-overexpressing strains was phenocopied by four random mutants obtained after UV mutagenesis. Therefore, aerial hyphae formation appears to be a trait for the antagonistic potential of T. guizhouense.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Measuring Urease and Phospholipase Secretion in Cryptococcus neoformans.

Urease and phospholipase are enzymes that are important virulence factors for Cryptococcus neoformans. These are two of the most studied enzymes involved in how C. neoformans breaches the blood-brain barrier. Additionally, phospholipase secretion also supports dissemination from the lungs. This chapter describes the methods used to measure the secretion of these enzymes, which may be used to characterize strain invasiveness and virulence.

Conservation of the Pal/Rim Pathway in Ustilaginomycetes.

The ability of fungi to effectively sense and internalize signals related to extracellular changing environments is essential for survival. This adaptability is particularly important for fungal pathogens of humans and plants that must sense and respond to drastic environmental changes when colonizing their hosts. One of the most important physicochemical factors affecting fungal growth and development is the pH. Ascomycota fungal species possess mechanisms such as the Pal/Rim pathway for external pH sensing and adaptation. However, the conservation of this mechanism in other fungi, such as Ustilaginomycetes is still little studied. To overcome this knowledge gap, we used a comparative genomic approach to explore the conservation of the Pal/Rim pathway in the 13 best sequenced and annotated Ustilaginomycetes. Our findings reveal that the Rim proteins and the Endosomal Sorting Complex Required for Transport (ESCRT) proteins are conserved in Ustilaginomycetes. They conserve the canonical domains present in Pal/Rim and ESCRT proteins of Ascomycota. This study sheds light on the molecular mechanisms used by these fungi for responding to extracellular stresses such as the pH, and open the door to further experimentations for understanding the molecular bases of the signaling in Ustilaginomycetes.

Rational modification of melatonin for broad-spectrum antifungal agents discovery.

Natural products, known for their environmental safety, are regarded as a significant basis for the modification and advancement of fungicides. Melatonin, as a low-cost natural indole, exhibits diverse biological functions, including antifungal activity. However, its potential as an antifungal agent has not been fully explored. In this study, a series of melatonin derivatives targeting the mitogen-activated protein kinase (Mps1) protein of fungal pathogens were synthesized based on properties of melatonin, among which the trifluoromethyl-substituted derivative Mt-23 exhibited antifungal activity against seven plant pathogenic fungi, and effectively reduced the severity of crop diseases, including rice blast, Fusarium head blight of wheat and gray mold of tomato. In particular, its EC50 (5.4 µM) against the rice blast fungus Magnaporthe oryzae is only one-fourth that of isoprothiolane (22 µM), a commercial fungicide. Comparative analyzes revealed that Mt-23 simultaneously targets the conserved protein kinase Mps1 and lipid protein Cap20. Surface plasmon resonance assays showed that Mt-23 directly binds to Mps1 and Cap20. In this study, we provide a strategy for developing antifungal agents by modifying melatonin, and the resultant melatonin derivative Mt-23 is a commercially valuable, eco-friendly and broad-spectrum antifungal agent to combat crop disease.

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

FgPfn participates in vegetative growth, sexual reproduction, pathogenicity, and fungicides sensitivity via affecting both microtubules and actin in the filamentous fungus Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum species complexes (FGSG), is an epidemic disease in wheat and poses a serious threat to wheat production and security worldwide. Profilins are a class of actin-binding proteins that participate in actin depolymerization. However, the roles of profilins in plant fungal pathogens remain largely unexplored. Here, we identified FgPfn, a homolog to profilins in F. graminearum, and the deletion of FgPfn resulted in severe defects in mycelial growth, conidia production, and pathogenicity, accompanied by marked disruptions in toxisomes formation and deoxynivalenol (DON) transport, while sexual development was aborted. Additionally, FgPfn interacted with Fgα1 and Fgß2, the significant components of microtubules. The organization of microtubules in the ΔFgPfn was strongly inhibited under the treatment of 0.4 µg/mL carbendazim, a well-known group of tubulin interferers, resulting in increased sensitivity to carbendazim. Moreover, FgPfn interacted with both myosin-5 (FgMyo5) and actin (FgAct), the targets of the fungicide phenamacril, and these interactions were reduced after phenamacril treatment. The deletion of FgPfn disrupted the normal organization of FgMyo5 and FgAct cytoskeleton, weakened the interaction between FgMyo5 and FgAct, and resulting in increased sensitivity to phenamacril. The core region of the interaction between FgPfn and FgAct was investigated, revealing that the integrity of both proteins was necessary for their interaction. Furthermore, mutations in R72, R77, R86, G91, I101, A112, G113, and D124 caused the non-interaction between FgPfn and FgAct. The R86K, I101E, and D124E mutants in FgPfn resulted in severe defects in actin organization, development, and pathogenicity. Taken together, this study revealed the role of FgPfn-dependent cytoskeleton in development, DON production and transport, fungicides sensitivity in F. graminearum.

An oxylipin signal confers protection against antifungal echinocandins in pathogenic aspergilli.

Aspergillus fumigatus is the leading causative agent of life-threatening invasive aspergillosis in immunocompromised individuals. One antifungal class used to treat Aspergillus infections is the fungistatic echinocandins, semisynthetic drugs derived from naturally occurring fungal lipopeptides. By inhibiting beta-1,3-glucan synthesis, echinocandins cause both fungistatic stunting of hyphal growth and repeated fungicidal lysis of apical tip compartments. Here, we uncover an endogenous mechanism of echinocandin tolerance in A. fumigatus whereby the inducible oxylipin signal 5,8-diHODE confers protection against tip lysis via the transcription factor ZfpA. Treatment of A. fumigatus with echinocandins induces 5,8-diHODE synthesis by the fungal oxygenase PpoA in a ZfpA dependent manner resulting in a positive feedback loop. This protective 5,8-diHODE/ZfpA signaling relay is conserved among diverse isolates of A. fumigatus and in two other Aspergillus pathogens. Our findings reveal an oxylipin-directed growth program-possibly arisen through natural encounters with native echinocandin producing fungi-that enables echinocandin tolerance in pathogenic aspergilli.

Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley.

The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide-binding leucine-rich-repeat (NLR) receptor MLA1 and therefore is designated AVRA1. Here, we show that AVRA1 and the sequence-unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two-hybrid next-generation interaction screens (Y2H-NGIS), followed by binary Y2H and in planta protein-protein interactions studies, and identified a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein HvERdj3B. Silencing of this ER quality control (ERQC) protein increased Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVRA1 and BEC1016 translocate into the ER signal peptide-independently. Overexpression of the two effectors impeded trafficking of a vacuolar marker through the ER; silencing of HvERdj3B also exhibited this same cellular phenotype, coinciding with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, requires ERQC. Here, the J-domain protein HvERdj3B appears to be essential and can be regulated by AVRA1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVRA1 J-domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition.

Lignocellulolytic Enzymes Production by Four Wild Filamentous Fungi for Olive Stones Valorization: Comparing Three Fermentation Regimens.

Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum, Fusarium nygamai, Trichoderma capillare, and Aspergillus calidoustus, are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of ß-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare, SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 µg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus. Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.

A fungal protein organizes both glycogen and cell wall glucans.

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and ß-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

Coupling thermotolerance and high production of recombinant protein by CYR1N1546K mutation via cAMP signaling cascades.

In recombinant protein-producing yeast strains, cells experience high production-related stresses similar to high temperatures. It is possible to increase recombinant protein production by enhancing thermotolerance, but few studies have focused on this topic. Here we aim to identify cellular regulators that can simultaneously activate thermotolerance and high yield of recombinant protein. Through screening at 46 °C, a heat-resistant Kluyveromyces marxianus (K. marxianus) strain FDHY23 is isolated. It also exhibits enhanced recombinant protein productivity at both 30 °C and high temperatures. The CYR1N1546K mutation is identified as responsible for FDHY23's improved phenotype, characterized by weakened adenylate cyclase activity and reduced cAMP production. Introducing this mutation into the wild-type strain greatly enhances both thermotolerance and recombinant protein yields. RNA-seq analysis reveals that under high temperature and recombinant protein production conditions, CYR1 mutation-induced reduction in cAMP levels can stimulate cells to improve its energy supply system and optimize material synthesis, meanwhile enhance stress resistance, based on the altered cAMP signaling cascades. Our study provides CYR1 mutation as a novel target to overcome the bottleneck in achieving high production of recombinant proteins under high temperature conditions, and also offers a convenient approach for high-throughput screening of recombinant proteins with high yields.

Kinetic studies on optimized extracellular laccase from Trichoderma harzianum PP389612 and its capabilities for azo dye removal.

BACKGROUND: Azo dyes represent a common textile dye preferred for its high stability on fabrics in various harsh conditions. Although these dyes pose high-risk levels for all biological forms, fungal laccase is known as a green catalyst for its ability to oxidize numerous dyes. METHODS: Trichoderma isolates were identified and tested for laccase production. Laccase production was optimized using Plackett-Burman Design. Laccase molecular weight and the kinetic properties of the enzyme, including Km and Vmax, pH, temperature, and ionic strength, were detected. Azo dye removal efficiency by laccase enzyme was detected for Congo red, methylene blue, and methyl orange. RESULTS: Eight out of nine Trichoderma isolates were laccase producers. Laccase production efficiency was optimized by the superior strain T. harzianum PP389612, increasing production from 1.6 to 2.89 U/ml. In SDS-PAGE, purified laccases appear as a single protein band with a molecular weight of 41.00 kDa. Km and Vmax values were 146.12 µmol guaiacol and 3.82 µmol guaiacol/min. Its activity was stable in the pH range of 5-7, with an optimum temperature range of 40 to 50 °C, optimum ionic strength of 50 mM NaCl, and thermostability properties up to 90 °C. The decolorization efficiency of laccase was increased by increasing the time and reached its maximum after 72 h. The highest efficiency was achieved in Congo red decolorization, which reached 99% after 72 h, followed by methylene blue at 72%, while methyl orange decolorization efficiency was 68.5%. CONCLUSION: Trichoderma laccase can be used as an effective natural bio-agent for dye removal because it is stable and removes colors very well.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Unravelling the Function of the Sesquiterpene Cyclase STC3 in the Lifecycle of Botrytis cinerea.

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant ΔBcstc3 and an overexpressed-Bcstc3 transformant (OvBcstc3) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5 and Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described are identified.

Characterization, Antioxidant Capacity and Protective Effect of Peptides from Cordyceps militaris Cultivated with Tussah Pupa on Oxidative Injured HepG2 Cells.

The antioxidant capacity and protective effect of peptides from protein hydrolysate of Cordyceps militaris cultivated with tussah pupa (ECPs) on H2O2-injured HepG2 cells were studied. Results indicated ECP1 (<3 kDa) presented the strongest antioxidant activity compared with other molecular weight peptides. Pretreated with ECPs observably enhanced survival rates and reduced apoptosis rates of HepG2 cells. ECPs treatment decreased the ROS level, MDA content and increased CAT and GSH-Px activities of HepG2 cells. Besides, the morphologies of natural peptides from C. militaris cultivated with tussah pupa (NCP1) and ECP1 were observed by scanning electron microscopy (SEM). Characterization results suggested the structure of NCP1 was changed by enzymatic hydrolysis treatment. Most of hydrophobic and acidic amino acids contents (ACC) in ECP1 were also observably improved by enzymatic hydrolysis. In conclusion, low molecular weight peptides had potential value in the development of cosmetics and health food.

Secreted in Xylem (SIX) genes in Fusarium oxysporum f.sp. cubense (Foc) unravels the potential biomarkers for early detection of Fusarium wilt disease.

Secreted in Xylem (SIX) are small effector proteins released by Fusarium oxysporum f.sp. cubense (Foc) into the plant's xylem sap disrupting the host's defence responses causing Fusarium wilt disease resulting in a significant decline in banana crop yields and economic losses. Notably, different races of Foc possess unique sets of SIX genes responsible for their virulence, however, these genes remain underutilized, despite their potential as biomarkers for early disease detection. Herein, we identified seven SIX genes i.e. SIX1, SIX2, SIX4, SIX6, SIX8a, SIX9a and SIX13 present in Foc Tropical Race 4 (FocTR4), while only SIX9b in Foc Race 1 (Foc1). Analysis of SIX gene expression in infected banana roots revealed differential patterns during infection providing valuable insights into host-pathogen interactions, virulence level, and early detection time points. Additionally, a comprehensive analysis of virulent Foc1_C2HIR and FocTR4_C1HIR isolates yielded informative genomic insights. Hence, these discoveries contribute to our comprehension of potential disease control targets in these plants, as well as enhancing plant diagnostics and breeding programs.