Genome sequencing and de novo assembly of Trichoderma longibrachiatum isolate collected from Florida agricultural soils.

We report a draft genome assembly of Trichoderma longibrachiatum isolate GEV 3550, obtained from Florida, United States of America.

Next-generation fungal identification using target enrichment and Nanopore sequencing.

BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

Advancing the automation of plant nucleic acid extraction for rapid diagnosis of plant diseases in space.

Human space exploration missions will continue the development of sustainable plant cultivation in what are obviously novel habitat settings. Effective pathology mitigation strategies are needed to cope with plant disease outbreaks in any space-based plant growth system. However, few technologies currently exist for space-based diagnosis of plant pathogens. Therefore, we developed a method of extracting plant nucleic acid that will facilitate the rapid diagnosis of plant diseases for future spaceflight applications. The microHomogenizer™ from Claremont BioSolutions, originally designed for bacterial and animal tissue samples, was evaluated for plant-microbial nucleic acid extractions. The microHomogenizer™ is an appealing device in that it provides automation and containment capabilities that would be required in spaceflight applications. Three different plant pathosystems were used to assess the versatility of the extraction process. Tomato, lettuce, and pepper plants were respectively inoculated with a fungal plant pathogen, an oomycete pathogen, and a plant viral pathogen. The microHomogenizer™, along with the developed protocols, proved to be an effective mechanism for producing DNA from all three pathosystems, in that PCR and sequencing of the resulting samples demonstrated clear DNA-based diagnoses. Thus, this investigation advances the efforts to automate nucleic acid extraction for future plant disease diagnosis in space.

Comparative Genomics of Fusarium circinatum Isolates Used to Screen Southern Pines for Pitch Canker Resistance.

Pitch canker, caused by the fungal pathogen Fusarium circinatum, is a global disease affecting many Pinus spp. Often fatal, this disease causes significant mortality in both commercially grown and natural pine forests and is an issue of current and growing concern. F. circinatum isolates collected from three locations in the U.S. state of Florida were shown to be virulent on both slash and loblolly pine, with two of the isolates causing equivalent and significantly larger lesions than those caused by the third isolate during pathogenicity trials. In addition, significant genetic variation in lesion length in the pedigreed slash pine population was evident and rankings of parents for lesion length were similar across isolates. Experimental data demonstrate that both host and pathogen genetics contribute to disease severity. High-quality genomic assemblies of all three isolates were created and compared for structural differences and gene content. No major structural differences were observed among the isolates; however, missing or altered genes do contribute to genomic variation in the pathogen population. This work evaluates in planta virulence among three isolates of F. circinatum, provides genomic resources to facilitate study of this organism, and details comparative genomic methods that may be used to explore the pathogen's contribution to disease development.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Role of Sirtuin 1 in Palmitic Acid-Induced Endoplasmic Reticulum Stress in Cardiac Myoblasts.

BACKGROUND: Lipotoxicity causes endoplasmic reticulum (ER) stress, leading to cell apoptosis. Sirtuin 1 (Sirt1) regulates gene transcription and cellular metabolism. In this study, we investigated the role of Sirt1 in palmitate-induced ER stress. METHODS: Both H9c2 myoblasts and heart-specific Sirt1 knockout mice fed a palmitate-enriched high-fat diet were used. RESULTS: The high-fat diet induced C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) expression in both Sirt1 knockout mice and controls. The Sirt1 knockout mice showed higher CHOP and ATF4 expression compared to those in the control. Palmitic acid (PA) induced ATF4 and CHOP expression in H9c2 cells. PA-treated H9c2 cells showed decreased cytosolic NAD+/NADH alongside reduced Sirt1's activity. The H9c2 cells showed increased ATF4 and CHOP expression when transfected with plasmid encoding dominant negative mutant Sirt1. Sirt1 activator SRT1720 did not affect CHOP and ATF4 expression. Although SRT1720 enhanced the nuclear translocation of ATF4, the extent of the binding of ATF4 to the CHOP promoter did not increase in PA treated-H9c2 cells. CONCLUSION: PA-induced ER stress is mediated through the upregulation of ATF4 and CHOP. Cytosolic NAD+ concentration is diminished by PA-induced ER stress, leading to decreased Sirt1 activity. The Sirt1 activator SRT1720 promotes the nuclear translocation of ATF4 in PA-treated H9c2 cells.

Draft Genome Sequence and De Novo Assembly of a Fusarium oxysporum f. sp. lycopersici Isolate Collected from the Andean Region in Colombia.

We report a draft genome assembly of the causal agent of tomato vascular wilt, Fusarium oxysporum f. sp. lycopersici isolate 59, obtained from the Andean region in Colombia.

The cAMP-dependent protein kinase A pathway perturbs autophagy and plays important roles in development and virulence of Sclerotinia sclerotiorum.

Previous research has demonstrated that sclerotia production is suppressed by exogenous cyclic AMP (cAMP) in Sclerotinia sclerotiorum and enhanced upon deletion of the adenylate cyclase gene. This study focuses on further functionally characterizing the cAMP-dependent protein kinase A (PKA) signaling pathway in S. sclerotiorum. Here, we demonstrate functions for two components of cAMP signaling: the catalytic, SsPKA, and the regulatory, SsPKAR, subunits of cAMP-dependent PKA. Growth and virulence were greatly reduced by disruption of either Sspka2 or SspkaR in addition to deficiencies in appressorium development. Surprisingly, disruption of both Sspka2 (dSspka2) and SspkaR (dSspkaR) display an up-regulation of autophagy without nutrient starvation suggesting that properly regulated PKA activity is required for control of autophagy. SsPKAR is demonstrated to be required for carbohydrate metabolism and mobilization, which are required for appressorium development and sclerotium initiation. A closer examination of dSspkaR during Nicotiana benthamiana infection revealed that an oxalic acid (OA)-independent necrosis protein(s) or metabolite(s) may be involved in the lesion development in dSspkaR-N. benthamiana interaction. In summary, these data demonstrate that the cAMP-dependent PKA signaling is essential for multiple forms of S. sclerotiorum development as well as virulence which rely on optimal regulation of autophagy.

Phosphodiesterase-1 inhibitor modulates Ca2+ regulation in sirtuin 1-deficient mouse cardiomyocytes.

BACKGROUND: Phosphodiesterase inhibitors can be used to enhance second messenger signaling to regulate intracellular Ca2+ cycling. This study investigated whether ITI-214, a selective phosphodiesterase-1 inhibitor, modulates intracellular Ca2+ regulation, resulting in a positive inotropic effect in sirtuin 1 (Sirt1)-deficient cardiomyocytes. METHODS: Mice with cardiac-specific Sirt1 knockout (Sirt1-/-) were used, with Sirt1flox/flox mice serving as controls. Electromechanical analyses of ventricular tissues were conducted, and we monitored intracellular Ca2+ using Fluo-3 as well as reactive oxygen species production in isolated cardiomyocytes. RESULTS: Sirt1-/- ventricles showed prolonged action potential duration at 90% repolarization and increased contractile force after treatment with ITI-214. The rates and sustained durations of burst firing in ventricles were higher and longer, respectively, in Sirt1-/- ventricles than in controls. ITI-214 treatment decreased the rates and shortened the durations of burst firing in Sirt1-/- mice. Sirt1-/- cardiomyocytes showed reduced Ca2+ transient amplitudes and sarcoplasmic reticulum (SR) Ca2+ stores compared to those in control cardiac myocytes, which was reversed after ITI-214 treatment. SR Ca2+ leakage was larger in Sirt1-/- cardiac myocytes than in control myocytes. ITI-214 reduced SR Ca2+ leakage in Sirt1-/- cardiac myocytes. Increased levels of reactive oxygen species in Sirt1-/- cardiomyocytes compared to those in controls were reduced after ITI-214 treatment. Levels of Ca2+ regulatory proteins, including ryanodine receptor 2, phospholamban, and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a were not affected by ITI-214 administration. CONCLUSIONS: Our results suggest that ITI-214 improves intracellular Ca2+ regulation, which in turn exerts inotropic effects and suppresses arrhythmic events in Sirt1-deficient ventricular myocytes.

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes.

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca2+ and Na+ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac-specific Sirt1 knockout (Sirt1-/- ). Sirt1flox/flox mice were served as control. Sirt1-/- mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca2+ transient amplitudes and sarcoplasmic reticulum (SR) Ca2+ stores, and increased SR Ca2+ leak were shown in the Sirt1-/- mice. Electrophysiological measurements were performed using patch-clamp method. While L-type Ca2+ current (ICa, L ) was smaller in Sirt1-/- myocytes, reverse-mode Na+ /Ca2+ exchanger (NCX) current was larger compared with those in control myocytes. Late Na+ current (INa, L ) was enhanced in the Sirt1-/- mice, alongside with elevated cytosolic Na+ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1-/- mice. Sirt1-/- cardiomyocytes showed down-regulation of L-type Ca2+ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca2+  ATPase 2a (SERCA2a), but up-regulation of Ca2+ /calmodulin-dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca2+ and Na+ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.

Mangosteen pericarp components alleviate progression of prostatic hyperplasia and mitochondrial dysfunction in rats.

Prostatic hyperplasia, characterized by progressive hyperplasia of glandular and stromal tissues, is the most common proliferative abnormality of the prostate in aging men. A high-fat diet (HFD) usually is a major factor inducing oxidative stress, inflammation, and an abnormal state of the prostate. Mangosteen pericarp powder (MPP) has abundant xanthones which can be antioxidant, anti-inflammatory, and antiproliferative agents. Therefore, the purpose of this study was to research whether MPP supplementation can affect the progression of prostatic hyperplasia. Twenty-four male F344 rats were randomly divided into four groups, including a control group (C), prostatic hyperplasia-induced group (P), prostatic hyperplasia-induced with low-dose MPP group (PL), and induced with high-dose MPP group (PH). The P, PL, and PH groups were given weekly intraperitoneal injections of 3,2'-dimethyl-4-aminobiphenyl (DMAB) at 25 mg/kg body weight for 10 weeks, and simultaneously fed an HFD for 24 weeks. Our findings first demonstrated that MPP consumption significantly decreased the prostate weight, serum testosterone and dihydrotestosterone concentrations, protein expression of proliferating cell nuclear antigen, and malondialdehyde levels and ameliorated mitochondrial function in prostatic tissues. These results suggest that MPP supplementation could be used to attenuate the progression of prostatic hyperplasia.

A Major Facilitator Superfamily Transporter Regulated by the Stress-Responsive Transcription Factor Yap1 Is Required for Resistance to Fungicides, Xenobiotics, and Oxidants and Full Virulence in Alternaria alternata.

Alternaria alternata relies on the ability to produce a host-selective toxin and to detoxify reactive oxygen species to successfully colonize the host. An A. alternata major facilitator superfamily transporter designated AaMFS54 was functionally characterized by analysis of loss- and gain-of-function mutations to better understand the factors required for fungal pathogenesis. AaMFS54 was originally identified from a wild-type expression library after being subtracted with that of a mutant impaired for the oxidative stress-responsive transcription regulator Yap1. AaMFS54 contains 14 transmembrane helixes. Fungal mutant lacking AaMFS54 produced fewer conidia and increased sensitivity to many potent oxidants (potassium superoxide and singlet-oxygen generating compounds), xenobiotics (2,3,5-triiodobenzoic acid and 2-chloro-5-hydroxypyridine), and fungicides (clotrimazole, fludioxonil, vinclozolin, and iprodione). AaMFS54 mutant induced necrotic lesion sizes similar to those induced by wild-type on leaves of susceptible citrus cultivars after point inoculation with spore suspensions. However, the mutant produced smaller colonies and less fluffy hyphae on the affected leaves. Virulence assays on citrus leaves inoculated by spraying with spores revealed that AaMFS54 mutant induced less severe lesions than wild-type, indicating the requirement of AaMFS54 in pathogenesis. All defective phenotypes were restored in a strain re-acquiring a functional copy of AaMFS54. Northern blotting analysis revealed that the expression of AaMFS54 was suppressed by xenobiotics. The current studies indicate that the Yap1-mediated transporter plays a role in resistance to toxic oxidants and fungicides in A. alternata. The relationships of MFS transporters with other regulatory components conferring stress resistance and A. alternata pathogenesis are also discussed.

Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen Sclerotinia sclerotiorum.

The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete S. sclerotiorum genome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of S. sclerotiorum and in a second independent gene. Targeting the previously characterized Ssoah1 gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function.IMPORTANCE Fungi that cause plant diseases by rotting or blighting host tissue with limited specificity remain among the most difficult to control. This is largely due to the quantitative nature of host resistance and a limited understanding of fungal pathogenicity. A mechanistic understanding of pathogenicity requires the ability to manipulate candidate virulence genes to test hypotheses regarding their roles in disease development. Sclerotinia sclerotiorum is among the most notorious of these so-called broad-host-range necrotrophic plant pathogens. The work described here provides a new method for rapidly constructing gene disruption vectors to create gene mutations with high efficiency compared with existing methods. Applying this method to characterize gene functions in S. sclerotiorum, we confirm the requirement for oxalic acid production as a virulence factor in multiple isolates of the fungus and demonstrate that melanin accumulation is not required for infection. Using this approach, the pace of functional gene characterization and the understanding of pathogenicity and related disease resistance will increase.

A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata.

Major Facilitator Superfamily (MFS) transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin), and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA). AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP) kinases, the 'two component' histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.

YAP1 homologue-mediated redox sensing is crucial for a successful infection by Monilinia fructicola.

Monilinia fructicola (G. Winter) Honey is a devastating pathogen on Rosaceae which causes blossom blight and fruit rot. Only a few studies related to the plant-pathogen interaction have been published and there is limited knowledge on the relationship between oxidative stress and successful infection in M. fructicola. In this study, we cloned and characterized a redox-responsive transcription factor MFAP1, a YAP1 homologue. MfAP1-silenced strains were generated by polyethylene glycol-mediated protoplast transformation or Agrobacterium T-DNA-mediated transformation. Pathogenicity assay demonstrated that MfAP1-silenced strains caused smaller lesions on rose and peach petals. Transformants carrying extra copies of MfAP1, driven by the native promoter, were generated for MfAP1 overexpression. Interestingly, MfAP1-overexpressing strains also caused smaller lesions on rose petals. Strains carrying two copies of MfAP1 accumulated reactive oxygen species (ROS) at higher levels and exhibited delayed accumulation of MfAP1 transcripts compared with the wild-type during pathogenesis. By the analysis of ROS production and the expression patterns of redox- and virulence-related genes in the wild-type strain and an MfAP1-overexpressing strain, we found that the M. fructicola wild-type strain responded to oxidative stress at the infection site, activated the expression of MfAP1 and up-regulated the genes required for ROS detoxification and fungal virulence. In contrast, MfAP1 expression in the MfAP1-overexpressing strain was suppressed after the induction of a strong oxidative burst at the infection site, altering the expression of ROS detoxification and virulence-related genes. Our results highlight the importance of MfAP1 and ROS accumulation in the successful infection of M. fructicola.

Hydrogen Sulfide Modulates the S-Nitrosoproteome and the Mitochondrial Morphology in Endothelial Cells.

BACKGROUND: Hydrogen sulfide (H2S) is one of the endogenous gaseous molecules promoting the production of nitric oxide (NO) which has cardioprotective functions. However, the role of the H2S-mediated protein S-nitrosoproteome and its subsequent physiological response remains unclear. METHODS: Endothelial cells EAhy 926 were treated with 50 µM of H2S for 2 hours. The NO bound S-nitrosoproteins were purified by a biotin-switch and then digested by trypsin. Resulting peptides from control and H2S treatment were separately labeled by isobaric tag for relative and absolute quantitation 114/115, quantified by liquid chromatography tandem-mass spectrometry and analyzed by ingenuity pathway analysis (IPA) software. The microP software was applied to analyze the morphological changes of mitochondria. RESULTS: With the treatment of H2S, 416 S-nitrosylated proteins were identified. IPA analysis showed that these proteins were involved in five signaling pathways. The NO-bound cysteine residues and the S-nitrosylation levels (115/114) were shown for ten S-nitrosoproteins. Western blot further verified the S-nitrosylation of thioredoxin-dependant peroxide reductase, cytochrome c oxidase and cytochrome b-c1 complex that are involved in the mitochondrial signaling pathway. H2O2-induced mitochondrial swelling can be reduced by the pretreatment of H2S. CONCLUSIONS: The H2S-mediated endothelial S-nitrosoproteome has been confirmed. In the present study, we have proposed the cardioprotective role of H2S via maintaining mitochondrial homeostasis.

How the Pathogenic Fungus Alternaria alternata Copes with Stress via the Response Regulators SSK1 and SHO1.

The tangerine pathotype of Alternaria alternata is a necrotrophic fungal pathogen causing brown spot disease on a number of citrus cultivars. To better understand the dynamics of signal regulation leading to oxidative and osmotic stress response and fungal infection on citrus, phenotypic characterization of the yeast SSK1 response regulator homolog was performed. It was determined that SSK1 responds to diverse environmental stimuli and plays a critical role in fungal pathogenesis. Experiments to determine the phenotypes resulting from the loss of SSK1 reveal that the SSK1 gene product may be fulfilling similar regulatory roles in signaling pathways involving a HOG1 MAP kinase during ROS resistance, osmotic resistance, fungicide sensitivity and fungal virulence. The SSK1 mutants display elevated sensitivity to oxidants, fail to detoxify H2O2 effectively, induce minor necrosis on susceptible citrus leaves, and displays resistance to dicarboximide and phenylpyrrole fungicides. Unlike the SKN7 response regulator, SSK1 and HOG1 confer resistance to salt-induced osmotic stress via an unknown kinase sensor rather than the "two component" histidine kinase HSK1. SSK1 and HOG1 play a moderate role in sugar-induced osmotic stress. We also show that SSK1 mutants are impaired in their ability to produce germ tubes from conidia, indicating a role for the gene product in cell differentiation. SSK1 also is involved in multi-drug resistance. However, deletion of the yeast SHO1 (synthetic high osmolarity) homolog resulted in no noticeable phenotypes. Nonetheless, our results show that A. alternata can sense and react to different types of stress via SSK1, HOG1 and SKN7 in a cooperative manner leading to proper physiological and pathological functions.

The glutathione peroxidase-mediated reactive oxygen species resistance, fungicide sensitivity and cell wall construction in the citrus fungal pathogen Alternaria alternata.

The ability to detoxify reactive oxygen species (ROS) is critical for pathogenicity in the necrotrophic fungus Alternaria alternata. We report a glutathione peroxidase 3 (AaGPx3) involved in the complex signalling network that is essential for the detoxification of cellular stresses induced by ROS and for A. alternata pathogenesis in citrus. AaGPx3 deletion mutants displayed increased sensitivity to H2 O2 and many ROS-generating compounds. AaGPx3 is required for correct fungal development as the AaGPx3 mutant strains showed a severe reduction in conidiation. AaGPx3 mutants accumulated higher chitin content than the wild-type and were less sensitive to the cell wall-targeting compounds calcofluor white and Congo red, as well as the fungicides fludioxonil and vinclozolin, suggesting a role of the glutathione systems in fungal cell wall construction. Virulence assays revealed that AaGPx3 is required for full virulence. The expression of AaGPx3 was downregulated in fungal strains carrying defective NADPH oxidase (Nox) or the oxidative stress responsive regulators YAP1 and HOG1, all implicated in ROS resistance. These results further support the important role of ROS detoxification during A. alternata pathogenesis in citrus. Overall, our study provides genetic evidence to define the central role of AaGPx3 in the biological and pathological functions of A. alternata.

Expression of Five Endopolygalacturonase Genes and Demonstration that MfPG1 Overexpression Diminishes Virulence in the Brown Rot Pathogen Monilinia fructicola.

Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG) genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR) revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the ß-glucuronidase (GUS) reporter gene fused with MfPG1 (MfPG1-GUS). The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS) on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus-M. fructicola interactions.

Improvements in white matter micro-structural integrity of right uncinate fasciculus and left fronto-occipital fasciculus of remitted first-episode medication-naïve panic disorder patients.

OBJECTIVE: We designed this study to investigate neural correlates of white matter micro-structural integrity of remitted patients with first-episode, medication-naïve and very late-onset panic disorder. METHOD: Twenty-one remitted patients with panic disorder completed treatment course with treatment of escitalopram (dose range around 10-15 mg/d). Twenty-one healthy controls were also enrolled into this study. Patients and controls all received 3-Tesla magnetic resonance imaging diffusion tensor imaging scanning at baseline and 6th week. We utilized FDT (FMRIB's Diffusion Toolbox v2.0) function of FSL (FMRIB Software Library) to calculate fractional anisotropy (FA). We compared FA values of patients and controls at baseline and 6th week to estimate the changes of FA of remitted patient group and inter-scan bias of controls. FA outputs of remitted patients and controls were compared by independent t test. RESULTS: We found increased FA in some regions of right uncinate fasciculus and left fronoto-occipital fasciculus after remission in patient group (corrected p<0.05). Reduced FA of other regions of right uncinate fasciculus was still observed in remitted patients when they were compared to the control group. CONCLUSION: Subtle changes of white matter micro-structural integrity after remission might represent neural correlates of treatment effects for first-episode, medication-naïve and very late-onset panic disorder.

An efficient and anonymous buyer-seller watermarking protocol.

For the purpose of deterring unauthorized duplication and distribution of multimedia contents, a seller may insert a unique digital watermark into each copy of the multimedia contents to be sold. When an illegal replica is found in the market sometime later, the seller can determine the responsible distributor by examining the watermark embedded. However, the accusation against the charged distributor, who was the buyer in some earlier transaction, is objectionable because the seller also has access to the watermarked copies and, hence, is able to release such a replica on her own. In this paper, a watermarking protocol is proposed to avoid such difficulties, known as the customer's right problem, in the phase of arbitration. The proposed watermarking protocol also provides a fix to Memon and Wong's scheme by solving the unbinding problem. In addition, the buyer is no longer required to contact the watermark certification authority during transactions, and the anonymity of the buyer can be retained through a trusted third party. The result is an efficient and anonymous buyer-seller watermarking protocol.