Phosphatidate phosphatase Pah1 has a role in the hyphal growth and virulence of Candida albicans.

Phosphatidate phosphatases play essential roles in lipid metabolism by converting phosphatidic acid to diacylglycerol. Here, we have investigated the roles of a phosphatidate phosphatase, Pah1, in the fungal pathogen Candida albicans. Deleting PAH1 causes multiple phenotypes, especially severe hyphal defects, increased sensitivity to cell wall stress, and reduced virulence in mice. By qPCR, we detected a significant downregulation of hyphal-specific genes including two key hyphal-promoting genes UME6 and HGC1. Overexpression of UME6 in pah1Δ/Δ cells largely restored the hyphal growth, indicating that the reduced expression of UME6 is primarily responsible for the hyphal defects. We also detected decreased expression of three hyphal-promoting transcription factors EFG1, FLO8, and CPH1 in pah1 mutants, consistent with the reduced expression of UME6. Furthermore, the pah1Δ/Δ mutant exhibited increased sensitivity to cell wall stress. During systemic infection of mice, the mutant showed significantly impaired ability to colonize the kidney and to kill the host. Together, C. albicans PAH1 plays an important role in hyphal growth, adaptability to environmental stresses, and virulence. Thus, Pah1 could be targeted for the development of new antifungal drugs.

Artificial cultivation of true morels: current state, issues and perspectives.

Morels (Morchella, Ascomycota), which are some of the most highly prized edible and medicinal mushrooms, are of great economic and scientific value. Morel cultivation has been a research focus worldwide for more than 100 years, and the outdoor cultivation of morels has succeeded and expanded to a large scale in China in recent years. In this study, we review the progress in recent research regarding the life cycle and reproductive systems in the genus Morchella and the current state of outdoor cultivation. Sclerotia formation and conidia production are two important phases during the life cycle. The morel species cultivated commercially in America is M. rufobrunnea based on molecular phylogenetic analysis. The species currently cultivated in China are black morels, including M. importuna, M. sextalata and M. eximia. The field cultivation of morels expanded in the majority of the provinces in China with a yield of fresh morels of 0-7620 kg per ha. The key techniques include spawn production, land preparation and spawning, the addition of exogenous nutrition, fruiting management and harvesting. The application of exogenous nutrition is the most important breakthrough in the field of morel cultivation, but the mechanism remains unclear. It was estimated that the total amount of field cultivated fresh morels was ∼500 t in 2015-2016. We also discuss the potential issues remaining in the current literature and suggest directions for future studies.

Reactive oxygen species induce sclerotial formation in Morchella importuna.

Morels are some of the most highly prized edible and medicinal mushrooms, and the outdoor cultivation has been achieved in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and the number of sclerotia indicates the spawn quality during cultivation. However, the sclerotial formation and differentiation mechanisms are poorly understood. In this study, the sclerotial formation process of Morchella importuna and the effects of reactive oxygen species on scerotial formation were studied. Scerotial formation was defined as five distinctive phases, hypha early, hyphal growth, sclerotial initiation, development, and maturation. The mycelia in the sclerotium-forming area were swollen, darkened, and dense with sclerotial formation, but hydrogen peroxide accumulated in the region lacking sclerotial formation. The expression of all six genes for superoxide dismutases tested increased with sclerotial maturation. A difference in hydrogen peroxide concentration of 20 mM could promote the sclerotial initiation and induce expression of sod genes. The MAPK signaling pathway was activated, and they passed the signal from an area of high oxidative stress to a low area to initiate sclerotial formation. An understanding of the sclerotial formation mechanisms in M. importuna may help to understand the life cycle and facilitate the fruiting body cultivation.

Characterization of Pph3-mediated dephosphorylation of Rad53 during methyl methanesulfonate-induced DNA damage repair in Candida albicans.

Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus Candida albicans The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in C. albicans We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in pph3Δ/Δ cells. Phosphomimetic mutation of the three residues (rad53-3D) impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the rad53-3D mutant also occurred in the pph3Δ/Δ mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in C. albicans.

Tpd3-Pph21 phosphatase plays a direct role in Sep7 dephosphorylation in Candida albicans.

Septins are a component of the cytoskeleton and play important roles in diverse cellular processes including cell cycle control, cytokinesis and polarized growth. In fungi, septin organization, dynamics and function are regulated by phosphorylation, and several kinases responsible for the phosphorylation of several septins have been identified. However, little is known about the phosphatases that dephosphorylate septins. Here, we report the characterization of Tpd3, a structural subunit of the PP2A family of phosphatases, in the pathogenic fungus Candida albicans. We found that tpd3Δ/Δ cells are defective in hyphal growth and grow as pseudohyphae under yeast growth conditions with aberrant septin organization. Western blotting detected hyperphosphorylation of the septin Sep7 in cells lacking Tpd3. Tpd3 and Sep7 colocalize at the bud neck and can coimmunoprecipitate. Furthermore, we discovered similar defects in cells lacking Pph21, a catalytic subunit of the PP2A family, and its physical association with Tpd3. Importantly, purified Tpd3-Pph21 complexes can dephosphorylate Sep7 in vitro. Together, our findings strongly support the idea that the Tpd3-Pph21 complex dephosphorylates Sep7 and regulates morphogenesis and cytokinesis. The tpd3Δ/Δ mutant is greatly reduced in virulence in mice, providing a potential antifungal target.

Sds22 participates in Glc7 mediated Rad53 dephosphorylation in MMS-induced DNA damage in Candida albicans.

The protein kinase Rad53 and its orthologs play a fundamental role in regulating the DNA damage checkpoint in eukaryotes. Rad53 is activated by phosphorylation in response to DNA damage and deactivated by dephosphorylation after the damage is repaired. However, the phosphatases involved in Rad53 deactivation are not entirely understood. In this study, by investigating the consequences of overexpressing SDS22, a gene encoding a regulatory subunit of the PP1 phosphatase Glc7, in the human fungal pathogen Candida albicans, we discovered that Sds22 plays an important role in Rad53 dephosphorylation and thus the deactivation of the DNA damage checkpoint. Sds22 cellular levels increase when cells are exposed to DNA damaging agents and decrease after removing the genotoxins. Depletion of Glc7 has similar phenotypes. We provide evidence that Sds2 acts through inhibitory physical association with Glc7. Our findings provide novel insights into the mechanisms for the control of DNA damage checkpoint. Furthermore, SDS22 overexpression reduces C. albicans virulence in a mouse model of systemic infection, suggesting potential targets for developing antifungal drugs.

Effects of Six Natural Compounds and Their Derivatives on the Control of Coccidiosis in Chickens.

Chicken coccidiosis costs the poultry industry over GBP 10 billion per year. The main method of preventing and controlling coccidiosis in chickens continues to be the use of drugs. Unfortunately, the prevalence of drug resistance in the field reduces or even eliminates the effectiveness of drugs, and drug residues in the food supply chain can also can be harmful to humans. Therefore, safe and effective anticoccidial drugs are urgently needed. Natural products have many advantages such as being safe, effective and inexpensive and are a sustainable way to control coccidiosis. In this study, the anticoccidial effects of six natural compounds were tested by Eimeria tenella infection. Oocyst production, cecum lesion, body weight gain, feed conversion ratio, and intestinal microbiota were measured. The results showed that nerolidol had a moderate effect on maintaining both body weight gain and feed conversion ratio. Silymarin and dihydroartemisinin showed significant anticoccidial effects by reducing total oocyst output. Dihydroartemisinin also significantly reduced the cecum lesion caused by Eimeria infection, but this compound may be toxic to the host at such informed doses because it decreases growth and survival rates. In addition, both silymarin and dihydroartemisinin partly restored the microbiota after challenge. This indicates that silymarin, dihydroartemisinin, and nerolidol are effective in the control of chicken coccidiosis. Our data provide basic knowledge about the anticoccidial effects of such natural compounds/derivates.

Mating-Type Genes Play an Important Role in Fruiting Body Development in Morchella sextelata.

True morels (Morchella spp.) are edible mushrooms that are commercially important worldwide due to their rich nutrition and unique appearance. In recent years, outdoor cultivation has been achieved and expanded on a large scale in China. However, the mechanisms of fruiting body development in morels are poorly understood. In this study, the role of mating-type genes in fruiting body development was researched. Fruiting bodies cultivated with different mating-type strains showed no difference in appearance, but the ascus and ascospores were slightly malformed in fruiting bodies obtained from the MAT1-1 strains. The transcript levels of mating-type genes and their target genes revealed that the regulatory mechanisms were conserved in ascomycetes fungi. The silencing of mat1-2-1 by RNA interference verified the direct regulatory effect of mat1-2-1 on its target genes at the asexual stage. When cultivated with the spawn of single mating-type strains of MAT1-1 or MAT1-2, only one corresponding mating-type gene was detected in the mycelial and conidial samples, but both mat1-1-1 and mat1-2-1 were detected in the samples of primordium, pileus, and stipe. An understanding of the mating-type genes' role in fruiting body development in M. sextelata may help to understand the life cycle and facilitate artificial cultivation.

Efficient CRISPR/Cas9 system based on autonomously replicating plasmid with an AMA1 sequence and precisely targeted gene deletion in the edible fungus, Cordyceps militaris.

Cordyceps militaris is a popular edible fungus with important economic value worldwide. In this study, an efficient CRISPR/Cas9 genome-editing system based on an autonomously replicating plasmid with an AMA1 sequence was constructed. Further, a precisely targeted gene deletion via homology-directed repair was effectively introduced in C. militaris. Gene editing was successful, with efficiencies of 55.1% and 89% for Cmwc-1 and Cmvvd, respectively. Precisely targeted gene deletion was achieved at an efficiency of 73.9% by a single guide RNA supplementation with donor DNAs. Double genes, Cmwc-1 and Cmvvd, were edited simultaneously with an efficiency of 10%. Plasmid loss was observed under non-selective culture conditions, which could permit recycling of the selectable marker and avoid the adverse effects of the CRISPR/Cas9 system on the fungus, which is beneficial for the generation of new cultivars. RNA Pol III promoters, endogenous tRNAPro of C. militaris, and chimeric AfU6-tRNAGly can be used to improve the efficiency. Polyethylene glycol-mediated protoplast transformation was markedly more efficient than Agrobacterium tumefaciens-mediated transformation of C. militaris. To our knowledge, this is the first description of genome editing and precisely targeted gene deletion in mushrooms based on AMA1 plasmids. Our findings will enable the modification of multiple genes in both functional genomics research and strain breeding.

Preparation of Wax-Based Warm Mixture Additives from Waste Polypropylene (PP) Plastic and Their Effects on the Properties of Modified Asphalt.

The production of high-performance, low-cost warm mix additives (WMa) for matrix asphalt remains a challenge. The pyrolysis method was employed to prepare wax-based WMa using waste polypropylene plastic (WPP) as the raw material in this study. Penetration, softening point, ductility, rotational viscosity, and dynamic shear rheological tests were performed to determine the physical and rheological properties of the modified asphalt. The adhesion properties were characterized using the surface free energy (SFE) method. We proved that the pyrolysis temperature and pressure play a synergistic role in the production of wax-based WMa from WPPs. The product prepared at 380 °C and 1.0 MPa (380-1.0) can improve the penetration of matrix asphalt by 61% and reduce the viscosity (135 °C) of matrix asphalt by 48.6%. Furthermore, the modified asphalt shows favorable elasticity, rutting resistance, and adhesion properties; thus, it serves as a promising WMa for asphalt binders.

Comparative transcriptome analysis of cells from different areas reveals ROS responsive mechanism at sclerotial initiation stage in Morchella importuna.

Morels are some of the most highly prized edible and medicinal mushrooms, with great economic and scientific value. Outdoor cultivation has been achieved and expanded on a large scale in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and previous reports indicated that reactive oxygen species (ROS) play an important role. However, ROS response mechanisms at sclerotial initiation (SI) stage are poorly understood. In this study, comparative transcriptome analyses were performed with sclerotial and hyphal cells at different areas in the same plate at SI stage. Gene expression was significantly different at SI stage between sclerotial formation and mycelia growth areas. GO and KEGG analyses indicated more vigorous metabolic characteristics in the hyphae area, while transcription process, DNA repair, and protein processing were enriched in sclerotial cells. Gene expression related to H2O2 production was high in the hyphae area, while expression of H2O2-scavenging genes was high in sclerotial cells, leading to a higher H2O2 concentration in the hyphal region than in the sclerotium. Minor differences were observed in gene expression of H2O2-induced signaling pathway in sclerotial and hyphal cells; however, expression levels of the target genes of transcription factor MSN2, important in the H2O2-induced signaling pathways, were significantly different. MSN2 enhanced stress response regulation in sclerotia by regulating these target genes. Small molecular HSPs were also found upregulated in sclerotial cells. This study indicated that sclerotial cells are more resistant to ROS stress than hyphal cells through transcriptional regulation of related genes.

Encapsulation of fluazinam to extend efficacy duration in controlling Botrytis cinerea on cucumber.

BACKGROUND: Fluazinam is an effective fungicide in controlling gray mold, but has short duration of efficacy. Increasing application dosage may cause phytotoxicity. To overcome this shortage, a controlled-release technology was studied by encapsulating fluazinam. Ethyl cellulose polymer microcapsules were loaded with fluazinam to formulate a fluazinam capsule suspension (FCS). The efficacy for inhibition of B. cinerea and persistency of the FCS were examined by comparing with fluazinam technical concentrate (FTC) and aqueous fluazinam suspension concentrate (FSC) using microscopic observation and high-performance liquid chromatography analysis. RESULTS: FCS formed capsules, with median size of 3.17 µm in diameter, had 82.3% encapsulation efficiency. It had a stronger inhibitory activity against B. cinerea than FTC and FSC measured 7 days after the treatments. The half-life of FCS on cucumber leaves was 3.4 days, longer than the 2.3 days of FSC. CONCLUSION: FCS formulation significantly improved the inhibition of B. cinerea and resulted in prolonged and sustained release. Moreover, microencapsulation increased the duration of the efficacy of fluazinam on target crops. This formulation could help to sustain pesticides and protect the environment. © 2021 Society of Chemical Industry.

Inhibition of starch digestion: The role of hydrophobic domain of both α-amylase and substrates.

The physicochemical mechanism of starch digestion is very complicated since it may be affected by the non-valence interactions of the amylase inhibitor with the substrate or the enzyme. The role of hydrophobic interaction in the process of starch digestion is not clear. In this study, pluronics (PLs) with different hydrophobicity were used as model amphiphilic compounds to study their inhibition on starch digestion using multi-spectroscopic methods. The results showed that the hydrophobic nature of PLs changed starch structure, but it had a greater effect on the structure of α-amylase by exposing more tryptophan residues and increasing α-helix and ß-sheet contents. Further investigation by using different chain-length fatty acids confirmed the results. The finding in this study is informative to design and fabricate α-amylase inhibitors for controlling starch digestion at the molecular level.

Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium.

A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its activity in reducing postharvest decay of peanut kernels caused by Aspergillus flavus in in vitro and in vivo tests. The results showed that the concentrations of antagonist had a significant effect on biocontrol effectiveness in vivo: when the concentration of the washed bacteria cell suspension was used at 1x10(9)CFU/ml, the percentage rate of rot of peanut kernels was 31.67%+/-2.89%, which was markedly lower than that treated with water (the control) after 7days of incubation at 28 degrees C. The results also showed that unwashed cell culture of B. megaterium was as effective as the washed cell suspension, and better biocontrol was obtained when longer incubation time of B. megaterium was applied. When the incubation time of B. megaterium was 60-h, the rate of decay declined to 41.67%+/-2.89%. Furthermore, relative to the expression of 18S rRNA, the mRNA abundances of aflR gene and aflS gene in the experiment group were 0.28+/-0.03 and 0.024+/-0.005 respectively, indicating that this strain of B. megaterium could significantly reduce the biosynthesis of aflatoxins and expression of aflR gene and aflS gene (p<0.01). To the best of our knowledge, this is a first report demonstrating that the marine bacterium B. megaterium could be used as a biocontrol agent against postharvest fungal disease caused by A. flavus.