Cabbage defense response provoked by Trichoderma Th-LAAO.

To investigate the molecular mechanism of Trichoderma L-amino acid oxidase (Th-LAAO) in protecting and in promoting growth of cabbage infected with Botrytis cinerea, a three-way interaction system was established. Cabbage leaves treated with purified Th-LAAO significantly constrained damaged leaf area caused by B. cinerea infection. In response to Th-LAAO treatment, the expression levels of genes involved in photosynthesis, such as ribulose-1,5-bisphosphate carboxylase oxygenase, Rubisco activase, and ATP synthase increased 2.54, 2.18, and 1.41 folds, respectively. The transcription levels of sucrose transport protein 1 increased 7.6 fold. As to the expression of defense-related genes, the transcription level of ascorbate peroxidase increased 1.46 fold. On the contrary, pathogenesis-related protein 1, chitinase, ß-1,3 glucanase, and glutathione S-transferase decreased significantly. Overall, the results indicated that Th-LAAO may stimulate CO2 fixation and sucrose transport and elicit host defense responses in cabbage against B. cinerea, and this elicitation of defense response is likely to contribute to induced systemic resistance of host plant.

Gene-to-Gene Network Analysis of the Mediation of Plant Innate Immunity by the Eliciting Plant Response-Like 1 (Epl1) Elicitor of Trichoderma formosa.

A new clade, Trichoderma formosa, secretes eliciting plant response-like 1 (Epl1), a small peptide elicitor that stimulates plant immunity. Nicotiana benthamiana pretreated with Epl1 for 3 days developed immunity against Tomato mosaic virus (ToMV) infection. The transcriptome profiles of T. formosa and N. benthamiana were obtained by deep sequencing; the transcript of Epl1 is 736 nt in length and encodes a 12-kDa peptide. Identifying critical genes in Epl1-mediated immunity was challenging due to high similarity between the transcriptome expression profiles of Epl1-treated and ToMV-infected N. benthamiana samples. Therefore, an efficient bioinformatics data mining approach was used for high-throughput transcriptomic assays in this study. We integrated gene-to-gene network analysis into the ContigViews transcriptome database, and genes related to jasmonic acid and ethylene signaling, salicylic acid signaling, leucine-rich repeats, transcription factors, and histone variants were hubs in the gene-to-gene networks. In this study, the Epl1 of T. formosa triggers plant immunity against various pathogen infections. Moreover, we demonstrated that high-throughput data mining and gene-to-gene network analysis can be used to identify critical candidate genes for further studies on the mechanisms of plant immunity.

Analysis of the structure and activity of the promoter regions of the metallothionein genes of the freshwater pearl mussel Hyriopsis schlegelii.

To investigate the regulation of metallothionein genes (HsMTs) of Hyriopsis schlegelii, 1,121-bp and 1,270-bp regions of the HsMT1 and HsMT2 promoters were cloned and analyzed, respectively. The two promoters shared partially conserved features and possessed distinct characteristics such as the number or position of metal response elements (MREs). Further analysis of the HsMT1 and HsMT2 promoters was performed by the reporter assay using the luciferase gene. Both promoters were activated by various metals, and presented different levels of metal ions inducibility in human hepatoblastoma cells. Deletion mutant assays demonstrated that both the longest promoter regions achieved the maximum inducibility, and the metal inducibility was dependent on the presence of the MRE in HsMT1 and the distal MRE in HsMT2. In addition, we cloned a putative metal responsive transcription factor (hereby designated as HsMTF-like) and studied its effect on HsMTs expression in human hepatoblastoma cells. An in vivo assay demonstrated that HsMTF-like activates basal HsMTs transcription level, and the MRE in the HsMTs promoter mediates this activation process. Moreover, this basal transcription level can be further boosted by zinc treatment. In conclusion, the regulation mechanism for MT activation in H. schlegelii should be evolutionarily conserved.

Comparative analysis of the complete mitochondrial genomes of three geographical topmouth culter (Culter alburnus) groups and implications for their phylogenetics.

Topmouth culter (C. alburnus) is an important commercial fish in China. We compared the nucleotide variations in the mtDNA genomes among three geographical groups of Culter alburnus: Liangzi Lake, Hubei Province (referred to as LZH); Taihu Lake, Jiangsu Province (TH); and Poyang Lake, Jiangxi Province (PYH). The similarity of whole mtDNA genomes ranged from 0.992 to 0.999. The similarity among 13 protein-coding genes, 2 rRNA genes, and the D-loop sequences was found to range from 0.982 to 0.996. This is useful data for future designing work for making specific molecular marker for distinguishing individuals of C. alburnus from the three geographical groups. An extended termination-associated sequence (ETAS) and several conserved blocks (CSB-F, CSB-E, CSB-D, CSB1, CSB2, and CSB3) were identified in the mtDNA control regions. A phylogenetic analysis shows a monophyletic relationship of the LZF-female and the LZF-male. However, the analysis also showed paraphyletic relationships for the other two geological groups. This result will be useful for the future breeding work of C. alburnus.

Molecular characterization and expression of metallothionein from freshwater pearl mussel, Hyriopsis schlegelii.

Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys-Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.

Anticancer activity of Kalanchoe tubiflora extract against human lung cancer cells in vitro and in vivo.

Uncontrolled cell proliferation is a common feature of human cancer. Some of herbal extract or plant-derived medicine had been shown as an important source of effective anticancer agents. We previously reported that an n-BuOH-soluble fraction of Kalanchoe tubiflora has antiproliferative activity by inducing mitotic catastrophe. In this study, we showed that the H2 O-soluble fraction of Kalanchoe tubiflora (KT-W) caused cell cycle arrest, and senescence-inducing activities in A549 cells. We used 2 dimensional PAGE to analyze the protein expression levels after KT-W treatment, and identified that the energy metabolism-related proteins and senescence-related proteins were disturbed. In vivo experiments showed that the tumor growths in A549-xenografted nude mice were effectively inhibited by KT-W. Our findings implied that KT-W is a putative antitumor agent by inducing cell cycle arrest and senescence. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1663-1673, 2016.

Transcriptional regulation of the iac locus from Acinetobacter baumannii by the phytohormone indole-3-acetic acid.

The iac locus is involved in indole-3-acetic acid (IAA) catabolism in Acinetobacter baumannii. Nine structural genes of iac are transcribed in the same direction, whereas iacR, which encodes a MarR-type transcriptional regulator, is transcribed in the opposite direction. The IacA protein, which is encoded by the second structural gene of the iac locus, is expressed in an IAA-dependent manner. Here, we characterized gene expression from this locus in wild type A. baumannii and in an iacR mutant; this revealed that the iacH promoter is negatively regulated by IacR. The transcriptional site of iacH was determined by using 5' rapid amplification of cDNA ends; one IacR-binding site was identified between positions -35 and +28 of the iacH promoter. Sequence analysis and an electrophoretic mobility shift assay indicated that recombinant IacR binds specifically to a sequence with dyad symmetry in the iacR-iacH overlapping promoters in the absence of IAA. In addition, a two-plasmid expression system in Escherichia coli showed that IAA probably serves as a ligand that binds to IacR and releases it from the iacH promoter, thereby allowing RNA polymerase to transcribe iac. Thus, iac is expressed in order to promote IAA degradation, whereas free IacR is required for iac repression. We conclude that IacR serves as a key regulator of IAA degradation in A. baumannii in the rhizosphere. These results provide new insights into the possible role of A. baumannii in the environment.

De novo transcriptome sequencing to identify the sex-determination genes in Hyriopsis schlegelii.

This study presents the first analysis of expressed transcripts in the spermary and ovary of Hyriopsis schlegelii (H. schlegelii). A total of 132,055 unigenes were obtained and 31,781 of these genes were annotated. In addition, 19,511 upregulated and 25,911 downregulated unigenes were identified in the spermary. Ten sex-determination genes were selected and further analyzed by real-time PCR. In addition, mammalian genes reported to govern sex-determination pathways, including Sry, Dmrt1, Dmrt2, Sox9, GATA4, and WT1 in males and Wnt4, Rspo1, Foxl2, and ß-catenin in females, were also identified in H. schlegelii. These results suggest that H. schlegelii and mammals use similar gene regulatory mechanisms to control sex determination. Moreover, genes associated with dosage compensation mechanisms, such as Msl1, Msl2, and Msl3, and hermaphrodite phenotypes, such as Tra-1, Tra-2α, Tra-2ß, Fem1A, Fem1B, and Fem1C, were also identified in H. schlegelii. The identification of these genes indicates that diverse regulatory mechanisms regulate sexual polymorphism in H. schlegelii.

Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel, Hyriopsis schlegelii.

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5'-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.

Euphorbia formosana root extract induces apoptosis by caspase-dependent cell death via Fas and mitochondrial pathway in THP-1 human leukemic cells.

Acute myeloid leukemia (AML), a very rare type of cancer, generally affects patients over 50 years old. While clinical drugs to treat advanced stages of AML exist, the disease becomes increasingly resistant to therapies. Euphorbia formosana Hayata (EF) is a native Taiwanese medicinal plant used to treat rheumatism, liver cirrhosis, herpes zoster, scabies, and photoaging, along with tumor suppression. However, the mechanisms by which it suppresses tumors have not been explored. Here, we provide molecular evidence that a hot-water extract of Euphorbia formosana (EFW) selectively inhibited the growth of human leukemic cancer cells more than other solid human cancer cell lines. Most importantly, the plant extract had limited toxicity toward healthy peripheral blood mononuclear cells (PBMCs). After THP-1 leukemic cells were treated with 50-100 µg/mL EFW for one day, the S phase DNA content of the cells increased, while treatment with 200-400 µg/mL caused the cells to accumulate in the G0/G1 phase. Notably, EFW did not affect A-549 lung cancer cells. The effectiveness of EFW against THP-1 cells may be through caspase-dependent apoptosis in leukemic cells, which is mediated through the Fas and mitochondrial pathways. The potent antileukemic activity of EFW in vitro warrants further investigation of this plant to treat leukemias and other malignancies.

L-amino acid oxidase-induced apoptosis in filamentous Botrytis cinerea.

As opposed to single-cell yeast and mammalian cell lines, apoptosis has not been greatly investigated in filamentous fungi because antibodies to the relevant fungal apoptosis-related proteins are not available commercially and because multicellular organisms cannot be studied using flow cytometry. Here we demonstrate how antibodies from a nonfungal source could be used to investigate this pathway. We show that apoptosis in the filamentous fungus Botrytis cinerea is triggered by the mitochondria-mediated caspase pathway, with release of the apoptotic factors cytochrome c, caspase 3, and caspase 9, on treatment with Trichoderma harzianum-derived L-amino acid oxidase.

Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea mycelium in culture conditions.

ABSTRACT Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative l-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an l-amino acid oxidase (Th-l-AAO) was identified from T. harzianum ETS 323. The secretion of Th-l-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-l-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-l-AAO also showed disease control against the development of B. cinerea on postharvest apple fruit and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-l-AAO, suggesting that Th-l-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.

The genetic architecture of phenotypic diversity in the Betta fish (Betta splendens).

The Betta fish displays a remarkable variety of phenotypes selected during domestication. However, the genetic basis underlying these traits remains largely unexplored. Here, we report a high-quality genome assembly and resequencing of 727 individuals representing diverse morphotypes of the Betta fish. We show that current breeds have a complex domestication history with extensive introgression with wild species. Using a genome-wide association study, we identify the genetic basis of multiple traits, including coloration patterns, the "Dumbo" phenotype with pectoral fin outgrowth, extraordinary enlargement of body size that we map to a major locus on chromosome 8, the sex determination locus that we map to dmrt1, and the long-fin phenotype that maps to the locus containing kcnj15. We also identify a polygenic signal related to aggression, involving multiple neural system-related genes such as esyt2, apbb2, and pank2. Our study provides a resource for developing the Betta fish as a genetic model for morphological and behavioral research in vertebrates.

Expression of L-amino acid oxidase of Trichoderma harzianum in tobacco confers resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a flavoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.

Plate assay for fungal enzymes using cellophane membranes.

Fungal mycelia mass and pigments are major obstacles to investigating the secretion of bioactive substances such as enzyme activities using a plate assay. In this study, we applied a cellophane membrane and demonstrated that it can block mycelia mass and conidia (especially pigmented spores that would likely interfere with any subsequent color development-based activity detection) while allowing secreting enzymes to pass through. Visual observation after lifting the cellophane membrane and the collected mycelia and conidia indicated that the bioactivities on specific plates were improved significantly, although some fungal growth hurdle was noted. This proved to be true whether the assays were color development based or not.

The preventive effect of oral EGCG in a fetal alcohol spectrum disorder mouse model.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is a challenging public health problem. Previous studies have found an association between FASD and oxidative stress. In the present study, we assessed the role of oxidative stress in ethanol-induced embryonic damage and the effect of (-)-epigallocatechin-3-gallate (EGCG), a powerful antioxidant extracted from green tea, on the development of FASD in a murine model. METHODS: Pregnant female mice were given intraperitoneal ethanol (25%, 0.005 to 0.02 ml/g) on gestational day 8 (G8) to establish the FASD model. On G10.25, mice were sacrificed and embryos were collected and photographed to determine head length (HL), head width (HW), and crown rump length (CRL). For mice given EGCG, administration was through a feeding tube on G7 and G8 (dose: 200, 300, or 400 mg/kg/d, the total amount for a day was divided into 2 equal portions). G10.25 embryos were evaluated morphologically. Brain tissues of G9.25 embryos were used for RT-PCR and western blotting of neural marker genes and proteins and detection of oxidative stress indicators. RESULTS: Administration of ethanol to pregnant mice on G8 led to the retardation of embryonic growth and down-regulation of neural marker genes. In addition, administration of ethanol (0.02 ml/g) led to the elevation of oxidative stress indicators [hydrogen peroxide (H2O2) and malondialdehyde (MDA)]. Administration of EGCG on G7 and G8 along with ethanol on G8 ameliorated the ethanol-induced growth retardation. Mice given EGCG (400 mg/kg/d) along with ethanol had embryo sizes and neural marker genes expression similar to the normal controls. Furthermore, EGCG (400 mg/kg on G7 and G8) inhibited the increase in H2O2 and MDA. CONCLUSIONS: In a murine model, oxidative stress appears to play an important role in ethanol-induced embryonic growth retardation. EGCG can prevent some of the embryonic injuries caused by ethanol.

Advancements on the zebrafish glioma model.

Glioma derived from the neural ectoderm is the most common brain tumor and is of great damage to human health among all lethal tumors. Scientists have been trying their best to find new methods and develop new drugs to treat glioma in recent years. The animal glioma model is of great importance to the research. Researchers have developed many animal glioma models, like the rat and mouse model. Now we are trying to develop a new zebrafish glioma model, which has much more advantages and fewer disadvantages than the traditional models in regard to gene mutation, chemical induction, and xenografts. Establishing a glioma model in zebrafish is feasible and would be of great use to patients with this common brain tumor.

Induced proteome of Trichoderma harzianum by Botrytis cinerea.

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including beta-1,3-glucanases, beta-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.

Molecular characterization of an inhibitor of apoptosis protein (IAPs) in freshwater pearl mussel, Hyriopsis schlegelii.

The inhibitor of apoptosis proteins (IAPs) played important roles in inhibiting the apoptosis of tumor cells by regulating caspase activity in mammals. In this study, we first cloned the full-length cDNA sequence of IAPs gene (designated as Hs-IAPs) in Hyriopsis schlegelii. The Hs-IAPs gene contained an open reading frame of 1719 nucleotides, encoding a predicted protein of 572 amino acids. qRT-PCR assay indicated that the Hs-IAPs gene was ubiquitously expressed in different tissues, and the highest expression level was in gills. Furthermore, we purified and obtained the recombinant protein of Hs-IAPs which showed a molecular weight of 82.5 kDa. We used H2O2 stimulation experiment to explore the possible function of Hs-IAPs. The results showed that the percentage of viable cells significantly increased following the Hs-IAPs concentration. These indicated that the Hs-IAPs may play a role in anti-oxidation causing by H2O2, and its anti-oxidative may be crucial in the process of apoptosis.

Efficient isolation of anthraquinone-derivatives from Trichoderma harzianum ETS 323.

Anthraquinone-derivatives, chrysophanol and pachybasin, were purified by a silica column chromatography with two different solvent systems from Trichoderma harzianum ETS 323. The fungus was incubated in sugarcane bagasse solid medium at room temperature without rotation. Structure of chrysophanol was solved by X-ray diffraction and pachybasin by NMR spectra. About 233+/-13 mg of pure chrysophanol and 773+/-40 mg of pure pachybasin were recovered per kg of solid cultural medium, with yields 1.7+/-0.2% and 5.6+/-0.5%, respectively.