A conserved clathrin-coated vesicle component, OsSCYL2, regulates plant innate immunity in rice.

Clathrin-mediated vesicle trafficking (CMVT) is a fundamental process in all eukaryotic species, and indispensable to organism's growth and development. Recently, it has been suggested that CMVT also plays important roles in the regulation of plant immunity. However, the molecular link between CMVT and plant immunity is largely unknown. SCY1-LIKE2 (SCYL2) is evolutionally conserved among the eukaryote species. Loss-of-function of SCYL2 in Arabidopsis led to severe growth defects. Here, we show that mutation of OsSCYL2 in rice gave rise to a novel phenotype-hypersensitive response-like (HR) cell death in a light-dependent manner. Although mutants of OsSCYL2 showed additional defects in the photosynthetic system, they exhibited enhanced resistance to bacterial pathogens. Subcellular localisation showed that OsSCYL2 localized at Golgi, trans-Golgi network and prevacuolar compartment. OsSCYL2 interacted with OsSPL28, subunit of a clathrin-associated adaptor protein that is known to regulate HR-like cell death in rice. We further showed that OsSCYL2-OsSPL28 interaction is mediated by OsCHC1. Collectively, we characterized a novel component of the CMVT pathway in the regulation of plant immunity. Our work also revealed unidentified new functions of the very conserved SCYL2. It thus may provide new breeding targets to achieve both high yield and enhanced resistance in crops.

Semianalytical model for the electromagnetic enhancement by a rectangular nanowire optical antenna on metallic substrate.

The electromagnetic enhancement by a metallic nanowire optical antenna on metallic substrate is investigated theoretically. By considering the excitation and multiple scattering of surface plasmon polaritons in the nanogap between the antenna and the substrate, we build up an intuitive and comprehensive model that provides semianalytical expressions for the electromagnetic field in the nanogap to achieve an understanding of the mechanism of electromagnetic enhancement. Our results show that antennas with short lengths that support the lowest order of resonance can achieve a high electric-field enhancement factor over a large range of incidence angles. Two phase-matching conditions are derived from the model for predicting the antenna lengths at resonance. Excitation of symmetric or antisymmetric localized surface plasmon resonance is further explained with the model. The model also shows superior computational efficiency compared to the full-wave numerical method when scanning the antenna length, the incidence angle, or the wavelength.

Establishment of uracil auxotrophic dikaryotic strains of Lentinula edodes by crossbreeding.

The uracil auxotrophic monokaryotic strain 423-9 of Lentinula edodes was crossed with nine monokaryons (cro2-2-9, W66-1, xd2-3-2, QingKe 20A, 241-1-1, 9015-1, L66-2, 241-1-2, and Qing 23A) derived from wild type strains of L. edodes. Nine dikaryotic hybrids were established from these crosses. These hybrids were fruited and 496 single spore isolates were obtained. Among these single spore isolates, 166 were identified as monokaryons under a microscope. We screened these monokaryons on selective medium and obtained 19 uracil auxotrophic monokaryons. By using the Monkaryon-monkaryon crossing method among the uracil auxotrophic monokaryons, 56 uracil auxotrophic dikaryotic strains were established on selective medium. These dikaryotic strains were unable to grow on minimal medium without uracil and exhibited slow growth rates on PDA plates compared to the wild type strain. The uracil auxotrophic dikaryotic strains also showed more vigorous growth on sawdust cultivation medium containing uracil than that without uracil. The fruiting tests showed that they formed normal fruiting bodies on the sawdust medium containing uracil. The results show that the uracil auxotrophic dikaryotic strain of L. edodes could be produced by mating, and will provide a valuable resource for future genetic studies and for spawn protection and identification.

Discovery and Characterization of the Highly Active Fungal Immunomodulatory Protein Fip-vvo82.

Volvaria volvacea (Bull. ex Fr.) Sing, an important edible and medicinal macro-fungus, has been used to remedy various diseases for hundreds of years in East Asia. To identify key proteins with the unique therapeutic activity in V. volvacea, we conducted a genomewide comparison of V. volvacea protein families and those of other edible fungi that lack therapeutic functions and identified seven fungal immunomodulatory proteins (FIPs) in V. volvacea. On the basis of the predicted physiological and biochemical characteristics of the seven FIPs, the novel Fip-vvo82 was inferred to have high immunomodulatory activity; this was confirmed by molecular and immunological experiments and further characterized by modeling the three-dimensional structure and protein-protein docking. This is the first study to show that V. volvacea has more than one FIP.

Structural elucidation and anti-inflammatory activity of a proteoglycan from spent substrate of Lentinula edodes.

A proteoglycan LEPS1 was firstly isolated and purified from the spent substrate of Lentinula edodes, an agricultural waste that may cause environmental pollution. The average molecular weight of LEPS1 was 1.18 × 104 g/mol, and carbohydrate moiety (88.9 %) was composed of glucose, arabinose, galactose, xylose and mannose at a molar ratio of 1.2:1.2:1.0:2.3:1.1. The protein moiety (8.5 %) of LEPS1 was bonded to the polysaccharide chain via O-glycosidic linkage. LEPS1 could significantly improve the inflammatory injury of LPS stimulated RAW264.7 macrophages by inhibiting the secretion of NO and decreasing the levels of pro-inflammatory factors (TNF-α, IL-1ß and IL-6). LEPS1 inhibited JAK-STAT1 and p38 MAPK signaling pathway via modulating JAK expression, phosphorylation of STAT1 and phosphorylation of p38, respectively. Moreover, LEPS1 could promote the expression of CD 206 and IL-10 which were the markers for repairing macrophages. Overall, LEPS1 had anti-inflammatory activity and can potentially treat as a novel anti-inflammation agent. This work could provide scientific basis and valuable information for the highly efficient utilization of spent L. edodes substrates as the by-product in mushroom industries.

Transformation of Compatible Mating-Type Genes in Monokaryons Triggers Fruiting Body Development by Activating Mating Pathways in Pleurotus eryngii.

Fruiting body formation is the most important developmental event in the edible mushroom life cycle; however, the genetic regulation of this process is not well understood. Pleurotus eryngii is a widely cultivated mushroom with high economic value. The mating of two monokaryons carrying compatible A and B mating-type genes is required for the development of fruiting bodies in P. eryngii. In this study, we showed that the monokaryons of P. eryngii transformed with compatible homeodomain (A mating type) and pheromone (B mating type) genes can complete fruiting body development but cannot form basidiospores. Transcriptional analyses revealed that expression of endogenous homeodomain and pheromone receptor genes and mating signaling pathways were activated by transferred homeodomain and pheromone genes in the transformants. Our findings provide a novel model for studying fruiting body development, which may accelerate the genetic breeding of edible mushrooms in the future. IMPORTANCE Fruiting bodies of edible mushrooms have high nutritional value. However, the fruiting body development of mushrooms is not well understood, and thus, many wild edible mushrooms of economic importance cannot be cultivated artificially. Moreover, variety among cultivatable mushrooms has improved marginally. Under natural conditions, fruiting body development can be initiated only in a dikaryon, the sexual mycelium obtained from mating two compatible monokaryons. The present work showed induction of fruiting body development in Pleurotus eryngii monokaryons by genetic manipulation. Gene expression analyses revealed key genes and signaling pathways involved in the fruiting body development of P. eryngii.

Selection and validation of internal control genes for quantitative real-time RT‒qPCR normalization of Phlebopus portentosus gene expression under different conditions.

Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable RT‒qPCR gene expression analysis results. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on their expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder, and RefFinder under different conditions and developmental stages. The results revealed that MSF1 domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA-binding protein 1 (TBP1), and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study on the identification of reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.

The complete mitochondrial genome of the edible mushroom Grifola frondosa.

The culinary-medicinal mushroom Grifola frondosa is widely cultivated in East Asia. In this study, the complete mitochondrial genome of G. frondosa was determined using Illumina sequencing. The circular molecule was 197,486 bp in length with a content of 25.01% GC, which was one of the largest mitochondrial genomes in the order Polyporales. A total of 39 known genes encoding 13 common mitochondrial genes, 24 tRNA genes, 1 ribosomal protein s3 gene (rps3), and 1 DNA polymerase gene (dpo) were predicted in this genome. The phylogenetic analysis showed that G. frondosa clustered together with Sparassis crispa, Laetiporus sulphureus, Wolfiporia cocos, and Taiwanofungus camphoratus. The complete mitochondrial genome reported here may provide new insight into genetic information and evolution for further studies.

Whole-Genome and Transcriptome Sequencing of Phlebopus portentosus Reveals Its Associated Ectomycorrhizal Niche and Conserved Pathways Involved in Fruiting Body Development.

Phlebopus portentosus (Berk. and Broome) Boedijin, a widely consumed mushroom in China and Thailand, is the first species in the order Boletaceae to have been industrially cultivated on a large scale. However, to date, the lignocellulose degradation system and molecular basis of fruiting body development in P. portentosus have remained cryptic. In the present study, genome and transcriptome sequencing of P. portentosus was performed during the mycelium (S), primordium (P), and fruiting body (F) stages. A genome of 32.74 Mb with a 48.92% GC content across 62 scaffolds was obtained. A total of 9,464 putative genes were predicted from the genome, of which the number of genes related to plant cell wall-degrading enzymes was much lower than that of some saprophytic mushrooms with specific ectomycorrhizal niches. Principal component analysis of RNA-Seq data revealed that the gene expression profiles at all three stages were different. The low expression of plant cell wall-degrading genes also confirmed the limited ability to degrade lignocellulose. The expression profiles also revealed that some conserved and specific pathways were enriched in the different developmental stages of P. portentosus. Starch and sucrose metabolic pathways were enriched in the mycelium stage, while DNA replication, the proteasome and MAPK signaling pathways may be associated with maturation. These results provide a new perspective for understanding the key pathways and hub genes involved in P. portentosus development.

Chilling stress reduced protein translation by the ubiquitination of ribosomal proteins in Volvariella volvacea.

In Volvariella volvacea, an important edible mushroom species, cryogenic autolysis is a typical part of abnormal metabolism; however, the underlying mechanisms remain unclear. Ubiquitylome analysis revealed that chilling stress (CS) affected protein translation and degradation by ubiquitination. Comparative proteomics analysis showed that CS downregulated protein expression in V. volvacea V23 instead of VH3 (improved chilling stress resistance strain). The integrative ubiquitylome, proteomics, and transcriptome analyses indicated that CS reduced protein translation by the ubiquitination of ribosomal proteins. An activity assay of the 20S proteasome showed that CS decreased the degradation efficiency of the ubiquitin-proteasome system. UBEV2, one type of ubiquitin-conjugating enzyme E2 (UBE2) in V. volvacea, was upregulated after cold stress treatment using western blot analysis. GST pull-down experiments of UBEV2 provided evidence that CS affected protein translation by the ubiquitination of ribosomal proteins. Co-IP experiments confirmed that UBEV2 bound to the ubiquitinated SSB2, a ribosome-associated molecular chaperone. An anti-freezing experiment demonstrated that the UBE2 inhibitor could improve the cold stress resistance of V. volvacea. Our observations revealed that CS triggered ubiquitination-mediated autolysis associated with a decrease in protein translation and highlighted the mechanistic role of UBEV2 in facilitating cryogenic autolysis in V. volvacea. SIGNIFICANCE: Volvariella volvacea, the edible straw mushroom, is a highly nutritious food source widely cultivated on a commercial scale in tropical and subtropical regions. The challenges associated with the cryogenic autolysis preservation of V. volvacea have limited its marketability. This issue of cryogenic autolysis is both an interesting scientific problem to solve and a practical economic matter. Integrative ubiquitylome, proteomics, and transcriptome analyses, together with GST pulldown and Co-IP experiments, indicated that chilling stress reduced protein translation by the ubiquitination of ribosomal proteins in V. volvacea. This study significantly contributes to our understanding of ubiquitination-mediated autolysis associated with a decrease in protein translation in V. volvacea. Our data highlight the mechanistic role of UBEV2 in facilitating the cryogenic autolysis of V. volvacea. We provided a new idea for the preservation of V. volvacea by inhibiting UBEV2 to increase its marketability.

Chilling Stress Triggers VvAgo1-Mediated miRNA-Like RNA Biogenesis in Volvariella volvacea.

In Volvariella volvacea, an important species of edible mushroom, cryogenic autolysis is a typical phenomenon that occurs during abnormal metabolism. Analysis of gene expression profiling and qPCR showed that chilling stress (CS) significantly and continuously upregulated only one type of Argonaute in V. volvacea, i.e., VvAgo1. Structural and evolutionary analysis revealed that VvAgo1 belongs to the Ago-like family, and its evolution has involved gene duplication, subsequent gene loss, and purifying selection. Analysis of its interaction network and expression suggested that CS triggers VvAgo1-mediated miRNA-like RNA (milRNA) biogenesis in V. volvacea V23 but not in VH3 (a composite mutant strain from V23 with improved CS resistance). Small RNA sequencing and qPCR analysis confirmed that CS triggered the increased milRNA expression in V23 and not in VH3. The predicted target genes of the increased milRNAs were enriched in several pathways, such as signal transduction and ubiquitination. Heatmap analysis showed that CS altered the expression profile of milRNAs with their target genes related to signal transduction and ubiquitination in V23. Combined analysis of transcriptome and proteome data confirmed that most of the target genes of the increased milRNAs were not translated into proteins. Our observations indicate that CS might trigger VvAgo1-mediated RNAi to facilitate the cryogenic autolysis of V. volvacea.

Agrobacterium-mediated transformation of arthroconidia obtained from the edible mushroom Hypsizygus marmoreus.

Using the carboxin resistance gene from Pleurotus eryngii as a selective marker, we introduced foreign DNA into the arthroconidia of Hypsizygus marmoreus through Agrobacterium-mediated transformation. The function of the exogenous GUS (ß-glucuronidase) gene driven by the CaMV35S promoter was detected in the transformants.

Three complete mitochondrial genomes of straw-rotting edible fungus Volvariella volvacea using next generation sequencing.

The straw-rotting edible fungus Volvariella volvacea is a widely cultivated edible fungus across China and Southeast Asian countries. Three complete mitochondrial genomes of V. volvacea from China, Thailand, and India were determined using the next-generation sequencing technology. The genome sizes of the three strains (China, Thailand, and India) were 62,541 bp, 64,531 bp, and 65,668 bp with GC contents of 38.46%, 38.56%, and 38.52%, respectively. All the genomes encoded 14 conserved protein-coding genes, the small ribosomal RNA subunits (rns), large ribosomal RNA subunits (rnl), and 23 tRNAs were located on the same strand. In the putative protein-coding genes, four introns were distributed in cox1 in the genomes of V23-1 and V8. 5 introns (four introns invaded into cox1and one intron invaded into cob) were detected in Tai8. The phylogenetic analysis confirmed that V. volvacea was a number of Agaricales. This mitochondrial genome may open new avenues for understanding the phylogeny and evolution of Pluteaceae and Agaricales.

The genome of Pleurotus eryngii provides insights into the mechanisms of wood decay.

Pleurotus eryngii (DC.) Quél. is widely used for bioconverting lignocellulosic byproducts into biofuel and value added products. Sequencing and annotating the genome of a monokaryon strain P. eryngii 183 allows us to gain a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases for degradation of lignocellulose in white-rot fungi. The genomic data provides insights into genomic basis of degradation mechanisms of lignin and cellulose and may pave new avenues for lignocellulose bioconversion.

[Effect of vascular endothelial growth factor on bone marrow-derived mesenchymal stem cell proliferation and the signaling mechanism].

OBJECTIVE: To observe the effect of vascular endothelial growth factor (VEGF) on bone marrow-derived mesenchymal stem cell (MSC) proliferation and explore the signaling mechanism involved. METHODS: MSC culture was performed following the classical whole bone marrow adhering method. The characteristics of MSC were identified by induction of multi-lineage differentiation and flow cytometry for surface marker analysis (CD34, CD45, CD29, and CD90). Following the addition of 50 nmol/L wortmannin, 50 µmol/L PD98059, 30 µmol/L SB203580, 10 µmol/L H89, 20 µmol/L Y27632, 1 µmol/L rapamycin, 10 µmol/L straurosporine, 6 nmol/L Go6976, or 50 µmol/L Pseudo Z inhibitors in the cell culture, the MSC were treated with 20 ng/ml VEGF and the changes of the cell proliferation rate was measured with MTT assay. RESULTS: Cultured MSC were capable of multi-linage differentiation and did not express VEGF-R, CD29 or CD90. Treatment with 20 ng/ml VEGF obviously promoted MSC proliferation, and this effect was inhibited partially by p38 mitogen-activated protein kinase (MAPK) inhibitor rapamycin, PD98059, SB203580, Go6976, and straurosporine. CONCLUSIONS: VEGF promotes MSC proliferation in close relation to the AKT-PKC pathway, in which PKC signal pathway may play the central role.