0000000000991963
AUTHOR
Stefan Jacob
Evaluating ancient Egyptian prescriptions today: Anti-inflammatory activity of Ziziphus spina-christi.
Abstract Background Ziziphus spina-christi (L.) Desf. (Christ's Thorn Jujube) is a wild tree today found in Jordan, Israel, Egypt, and some parts of Africa, which was already in use as a medicinal plant in Ancient Egypt. In ancient Egyptian prescriptions, it was used in remedies against swellings, pain, and heat, and thus should have anti-inflammatory effects. Nowadays, Z. spina-christi, is used in Egypt (by Bedouins, and Nubians), the Arabian Peninsula, Jordan, Iraq, and Morocco against a wide range of illnesses, most of them associated with inflammation. Pharmacological research undertaken to date suggests that it possesses anti-inflammatory, hypoglycemic, hypotensive and anti-microbial e…
Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.
Abstract The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast‐growing and unlike yeast, posttranslational modifications like N‐glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non‐activated carbon‐hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for pr…
Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae.
The aim of this study is a functional characterization of 10 putative histidine kinases (HIKs)-encoding genes in the phytopathogenic fungus Magnaporthe oryzae. Two HIKs were found to be required for pathogenicity in the fungus. It was found that the mutant strains ΔMohik5 and ΔMohik8 show abnormal conidial morphology and furthermore ΔMohik5 is unable to form appressoria. Both HIKs MoHik5p and MoHik8p appear to be essential for pathogenicity since the mutants fail to infect rice plants. MoSln1p and MoHik1p were previously reported to be components of the HOG pathway in M. oryzae. The ΔMosln1 mutant is more susceptible to salt stress compared to ΔMohik1, whereas ΔMohik1 appears to be stronger…
Rapid adaptation of signaling networks in the fungal pathogen Magnaporthe oryzae
Abstract Background One fundamental question in biology is how the evolution of eukaryotic signaling networks has taken place. “Loss of function” (lof) mutants from components of the high osmolarity glycerol (HOG) signaling pathway in the filamentous fungus Magnaporthe oryzae are viable, but impaired in osmoregulation. Results After long-term cultivation upon high osmolarity, stable individuals with reestablished osmoregulation capacity arise independently from each of the mutants with inactivated HOG pathway. This phenomenon is extremely reproducible and occurs only in osmosensitive mutants related to the HOG pathway – not in other osmosensitive Magnaporthe mutants. The major compatible so…
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Additional file 7: Figure S6. qPCR results of selected genes. qRT-PCR analysis of putative genes in MoWT, the “lof” mutants ΔMohog1 and ΔMohog1(adapted). The M. oryzae cultures were grown for 96 h in CM at 26 °C and 100 rpm. Each of the cultures was separated into two samples, one mixed with 0.5 M KCl and one untreated control further grown in CM at 26 °C and 100 rpm). Samples were taken after 25 min. The RNA was isolated from the mycelium samples and the results of transcript abundance given relative to quantification in the MoWT untreated control. Three biological replicates were used of each.
Visualizing fungicide action: anin vivotool for rapid validation of fungicides with target location HOG pathway
BACKGROUND The mitogen-activated protein kinase MoHog1p was fused with a green fluorescent protein (GFP) in the filamentous fungus Magnaporthe oryzae. The MoHOG1::GFP mutant was found to be an excellent tool visualizing in vivo fungicide-dependent translocation of MoHog1p into the nucleus. Validation of pathway specificity was achieved by generating fluorescence-labelled MoHog1p in the ΔMohik1 'loss of function' mutant strain. RESULTS GFP-labelled MoHog1p expressed in the wildtype and in ΔMohik1 demonstrates that fludioxonil is acting on the HOG pathway and even more precisely that fungicide action is dependent on the group III histidine kinase MoHik1p. GFP-tagged MoHog1p translocated into …
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Additional file 3: Figure S2. Investigation of the â adaptation-frequencyâ in Magnaporthe oryzae mutants with inactivated components of the HOG signaling cascade.
Unravelling the biosynthesis of pyriculol in the rice blast fungus Magnaporthe oryzae
Pyriculol was isolated from the rice blast fungus Magnaporthe oryzae and found to induce lesion formation on rice leaves. These findings suggest that it could be involved in virulence. The gene MoPKS19 was identified to encode a polyketide synthase essential for the production of the polyketide pyriculol in the rice blast fungus M. oryzae. The transcript abundance of MoPKS19 correlates with the biosynthesis rate of pyriculol in a time-dependent manner. Furthermore, gene inactivation of MoPKS19 resulted in a mutant unable to produce pyriculol, pyriculariol and their dihydro derivatives. Inactivation of a putative oxidase-encoding gene MoC19OXR1, which was found to be located in the genome cl…
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Additional file 4: Figure S3. Mycelium dry weight of the Magnaporthe oryzae wildtype strain, mutants with inactivated components of the HOG signaling cascade and the “adapted” strains after growth in liquid culture upon sorbitol-stress. The fungal colonies were grown in 250 ml complete medium inclusive 1,5 M sorbitol for 6 d at 26 °C and 120 rpm. Error bars represent the standard deviation of three biological replicates of each strain.
Fungicide resistance towards fludioxonil conferred by overexpression of the phosphatase gene Mo PTP 2 in Magnaporthe oryzae
The fungicide fludioxonil causes hyperactivation of the Hog1p MAPK within the high-osmolarity glycerol signaling pathway essential for osmoregulation in pathogenic fungi. The molecular regulation of MoHog1p phosphorylation is not completely understood in pathogenic fungi. Thus, we identified and characterized the putative MoHog1p-interacting phosphatase gene MoPTP2 in the filamentous rice pathogen Magnaporthe oryzae. We found overexpression of MoPTP2 conferred fludioxonil resistance in M. oryzae, whereas the 'loss of function' mutant ΔMoptp2 was more susceptible toward the fungicide. Additionally, quantitative phosphoproteome profiling of MoHog1p phosphorylation revealed lower phosphorylati…
ChemInform Abstract: Total Synthesis of (-)-Hymenosetin.
Hymenosetin (I) and its N-methyl analogue are prepared in 11 and 8 steps, respectively, from citronellal employing an intramolecular Diels-Alder reaction as the key step.
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Additional file 6: Figure S5. VENN diagram of putative structural variations in promotor [A] and in coding sequences (CDS) [B] within the genome of ΔMohog1, ΔMohog1(adapted) and ΔMopbs2(adapted). Numbers in the intersection regions represent overlapping SNPs among the strains. Numbers in parentheses show the corresponding relative percentage of genes harbouring the SNPs.
Total Synthesis of (-)-Hymenosetin.
The 3-decalinoyltetramic acid (−)-hymenosetin and its N-methyl analogue were prepared in 11 and 8 steps, respectively, from (+)-citronellal using an intramolecular Diels–Alder reaction as the key step. This method represents the first example for the synthesis of a 3-decalinoyltetramic acid with a free NH moiety. The stereochemistry of the title compound, an unnatural diastereomer, and of a decalin building block was studied in detail using circular dichroism spectroscopy in the IR and UV/VIS freqeuncy range. This allowed to determine the absolute configuration of the natural product and to plan the synthetic route.
A Target-Based In Vivo Test System to Identify Novel Fungicides with Mode of Action in the HOG Pathway
Resistance management plays a key role in modern plant protection. There is a growing need to identify new fungicide targets and new modes of action. In this context, it is also mandatory to find new compounds acting on successful target locations. For the latter, so-called target-site-specific test systems emerged to search for inhibitors. Most of them are based on in vitro assays, in which interaction between a compound and a purified target protein is demonstrated. Consequently, getting essential information about potentially toxic effects in the living cell or in the whole organism is not possible. Thus, we present a fluorescent-labelled mutant strain of the rice blast fungus Magnaporth…
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Additional file 5: Figure S4. Pathogenicity assay of the MoWT, the lof mutants and the â adaptedâ strains. The plant infection assays were carried out as described in experimental procedures. The error bars represent the standard deviation of three experiments with three replicates each.
The exceptionality of stress response in Magnaporthe oryzae: a set of “salt stress-induced” genes unique to the rice blast fungus
The ability of pathogens to signal perception and adaptation to environmental changes is an important prerequisite for successful colonization of the host organism. Filamentous phytopathogenic fungi, for example, have to cope with rapid changes in the environment during invasive growth in planta. Consequently, they have evolved a range of specific factors contributing to environmental adaptation facilitating host invasion. In addition to conserved pathways, including genes participating in stress response, unique/individual genes within the pathogens might represent determinants of pathogenicity. Therefore, identification of unique genes could provide a set of excellent candidates for novel…
The Determination of Carbohydrates by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD)
Chromatography techniques are widely used to separate, identify, and quantify molecules depending on their physicochemical properties. Standard methods range from simple size exclusion to separation based on affinity or ion exchange. Here, we present a method for the direct analysis of carbohydrates in Magnaporthe oryzae using high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). The combination of HPAEC with PAD provides the highest selectivity and sensitivity with minimal sample preparation and cleanup time. Utilizing our HPAEC-PAD approach, we obtain reliable and highly reproducible determination of carbohydrates produced as osmotic stre…
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Additional file 2: Figure S1. Schematic presentation and verification of the MoWT, the lof-mutants and the adapted strains by southern hybridization within the Magnaporthe oryzae genome. Southern blot analysis of gene deletion/disruption mutants in M. oryzae with gene specific probes. Genomic DNA of M. oryzae strain 70â 15 and the mutants was isolated and restricted with restriction enzymes. The probes which we used for hybridization with the genomic DNA of the wildtype strain and the corresponding mutant strains were always identical.
High osmolarity glycerol (HOG) signalling in Magnaporthe oryzae: Identification of MoYPD1 and its role in osmoregulation, fungicide action, and pathogenicity
AbstractThis study comprises a first functional analysis of an YPD1-homologue in filamentous phytopathogenic fungi and its role in the HOG signalling pathway. We generated a gene deletion mutant of the gene MoYPD1 in Magnaporthe oryzae and characterized the resulting mutant strain. We have shown that MoYpd1p is a component of the phosphorelay system acting in the HOG pathway due to its Y2H protein interaction with the HKs MoHik1p and MoSln1p as well as with the response regulator MoSsk1p. Fungicidal activity of fludioxonil was reported to be based on the inhibition of MoHik1p resulting in hyperactivation of the HOG signalling pathway and lethality. Western analysis proved that both, osmotic…
Hog1p activation by marasmic acid through inhibition of the histidine kinase Sln1p
BACKGROUND The histidine kinase (HK) MoHik1p within the high-osmolarity glycerol (HOG) pathway is known to be the target of the fungicide fludioxonil. Treatment of the fungus with fludioxonil causes an uncontrolled hyperactivation of the pathway and cell death. In this study, we used a target-based in vivo test system with mutant strains of the rice blast fungus Magnaporthe oryzae to search for new fungicidal compounds having various target locations within the HOG pathway. Mutants with inactivated HOG signalling are resistant to fungicides having the target located in the HOG pathway. RESULTS The HK MoSln1p was identified as being involved in the new antifungal mode of action of marasmic a…
Reactive Layer Assembly Sustains an Interlocked Structure in Green Processed and Scalable High-Performance Layered Wood
Wood can be processed into structural materials by partial removal of lignin and subsequent hot compression. The fundamental challenge of this process is achieving a cost-effective, green, and scal...
Evidence of a New MoYpd1p Phosphotransferase Isoform in the Multistep Phosphorelay System of Magnaporthe oryzae
Different external stimuli are perceived by multiple sensor histidine kinases and transmitted by phosphorylation via the phosphotransfer protein Ypd1p in the multistep phosphorelay system of the high osmolarity glycerol signaling pathway of filamentous fungi. How the signal propagation takes place is still not known in detail since multiple sensor histidine kinase genes in most filamentous fungi are coded in the genome, whereas only one gene for Ypd1p exists. That raises the hypothesis that various Ypd1p isoforms are produced from a single gene sequence, perhaps by alternative splicing, facilitating a higher variability in signal transduction. We found that the mRNA of MoYPD1 in the rice bl…
Identification of factors involved in dimorphism and pathogenicity of Zymoseptoria tritici
A forward genetics approach was applied in order to investigate the molecular basis of morphological transition in the wheat pathogenic fungus Zymoseptoria tritici. Z. tritici is a dimorphic plant pathogen displaying environmentally regulated morphogenetic transition between yeast-like and hyphal growth. Considering the infection mode of Z. tritici, the switching to hyphal growth is essential for pathogenicity allowing the fungus the host invasion through natural openings like stomata. We exploited a previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate a mutant library by insertional mutagenesis including more than 10,000 random mutants. To identify gene…
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Additional file 1: SVs summary.