An array of signal-specific MoYpd1 isoforms determines full virulence in the pathogenic fungus Magnaporthe oryzae.

Magnaporthe oryzae is placed first on a list of the world's top ten plant pathogens with the highest scientific and economic importance. The locus MGG_07173 occurs only once in the genome of M. oryzae and encodes the phosphotransfer protein MoYpd1p, which plays an important role in the high osmolarity glycerol (HOG) signaling pathway for osmoregulation. Originating from this locus, at least three MoYPD1 isoforms are produced in a signal-specific manner. The transcript levels of these MoYPD1-isoforms were individually affected by external stress. Salt (KCI) stress raised MoYPD1_T0 abundance, whereas osmotic stress by sorbitol elevates MoYPD1_T1 levels. In line with this, signal-specific nuclear translocation of green fluorescent protein-fused MoYpd1p isoforms in response to stress was observed. Mutant strains that produce only one of the MoYpd1p isoforms are less virulent, suggesting a combination thereof is required to invade the host successfully. In summary, we demonstrate signal-specific production of MoYpd1p isoforms that individually increase signal diversity and orchestrate virulence in M. oryzae.

MIF-like domain containing protein orchestrates cellular differentiation and virulence in the fungal pathogen Magnaporthe oryzae.

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of an MIF protein from the devastating plant pathogen Magnaporthe oryzae (Mo). The fungal genome encodes a single MIF protein (MoMIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that MoMIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results suggest that MoMIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection.

Fully Microfabricated Surface Acoustic Wave Tweezer for Collection of Submicron Particles and Human Blood Cells.

Precise manipulation of (sub)micron particles is key for the preparation, enrichment, and quality control in many biomedical applications. Surface acoustic waves (SAW) hold tremendous promise for manipulation of (bio)particles at the micron to nanoscale ranges. In commonly used SAW tweezers, particle manipulation relies on the direct acoustic radiation effect whose superior performance fades rapidly when progressing from micron to nanoscale particles due to the increasing dominance of a second order mechanism, termed acoustic streaming. Through reproducible and high-precision realization of stiff microchannels to reliably actuate the microchannel cross-section, here we introduce an approach that allows the otherwise competing acoustic streaming to complement the acoustic radiation effect. The synergetic effect of both mechanisms markedly enhances the manipulation of nanoparticles, down to 200 nm particles, even at relatively large wavelength (300 µm). Besides spherical particles ranging from 0.1 to 3 µm, we show collections of cells mixed with different sizes and shapes inherently existing in blood including erythrocytes, leukocytes, and thrombocytes.

Identification of the polyketide synthase gene responsible for the synthesis of tanzawaic acids in Penicillium steckii IBWF104-06.

Microorganisms have been used as biological control agents (BCAs) in agriculture for a long time, but their importance has increased dramatically over the last few years. The Penicillium steckii IBWF104-06 strain has presented strong BCA activity in greenhouse experiments performed against phytopathogenic fungi and oomycetes. P. steckii strains generally produce different antifungal tanzawaic acids; interesting compounds known to be catalyzed by polyketide synthetases in other fungi. Since the decalin structure is characteristic for tanzawaic acids, two polyketide synthase genes (PsPKS1 and PsPKS2) were selected for further analysis, which have similarity in sequence and gene cluster structure with genes that are known to be responsible for the biosynthesis of decalin-containing compounds. Subsequently, gene-inactivation mutants of both PsPKS1 and PsPKS2 have been generated. It was found, that the ΔPspks1 mutant cannot produce tanzawaic acids any more, whereas reintegration of the original PsPKS1 gene into the genome of ΔPspks1 reestablished tanzawaic acid production. The mutant ΔPspks2 is not altered in tanzawaic acids production. Interestingly, both mutants ΔPsPKS1 and ΔPsPKS2 still display strong BCA activity, indicating that the mechanism of action is not related to the production of tanzawaic acids.

Data-Independent Acquisition (DIA) Is Superior for High Precision Phospho-Peptide Quantification in Magnaporthe oryzae.

The dynamic interplay of signaling networks in most major cellular processes is characterized by the orchestration of reversible protein phosphorylation. Consequently, analytic methods such as quantitative phospho-peptidomics have been pushed forward from a highly specialized edge-technique to a powerful and versatile platform for comprehensively analyzing the phosphorylation profile of living organisms. Despite enormous progress in instrumentation and bioinformatics, a high number of missing values caused by the experimental procedure remains a major problem, due to either a random phospho-peptide enrichment selectivity or borderline signal intensities, which both cause the exclusion for fragmentation using the commonly applied data dependent acquisition (DDA) mode. Consequently, an incomplete dataset reduces confidence in the subsequent statistical bioinformatic processing. Here, we successfully applied data independent acquisition (DIA) by using the filamentous fungus Magnaporthe oryzae as a model organism, and could prove that while maintaining data quality (such as phosphosite and peptide sequence confidence), the data completeness increases dramatically. Since the method presented here reduces the LC-MS/MS analysis from 3 h to 1 h and increases the number of phosphosites identified up to 10-fold in contrast to published studies in Magnaporthe oryzae, we provide a refined methodology and a sophisticated resource for investigation of signaling processes in filamentous fungi.

Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.

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 protein secretion and set it under control of the strong EF1α-promoter. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.

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 stress response by M. oryzae. Thus, the method described provides a fast, precise, and comprehensive analysis of stress-dependent metabolic adjustments of carbohydrates not only relevant for M. oryzae but also applicable in other systems.

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 Magnaporthe oryzae as a rapid tool for fluorescence-based identification and visualization of fungicides in vivo with the mode of action in the high osmolarity glycerol (HOG)-signaling pathway. The HOG pathway represents an excellent target for antifungal agents such as the phenylpyrrole fungicides, since almost no relevant resistances have occurred to date, despite 30 years of extensive usage of this fungicide class.

Controllable Bypass Suppression in Magnaporthe oryzae.

Evolutionary adaptation of living organisms is commonly thought to be the result of processes that have taken place over long periods of time. By contrast, we found that the filamentous rice blast fungus Magnaporthe oryzae rapidly suppresses the osmosensitive "loss of function" (lof) phenotype in knockout mutants of the high-osmolarity glycerol (HOG) pathway. That suppression occurs highly reproducibly after 4 weeks of continuous growth upon salt stress. Stable mutants reestablished in osmoregulation arise independently out of individual osmosensitive lof mutants of the HOG pathway. The major compatible solute produced upon salt stress by these suppressor strains was found to be glycerol, whereas it is arabitol in the wildtype strain. We aim to address the molecular or biochemical mechanisms behind this rapid suppression and characterize the associated factors and signaling pathways which enable or prevent suppression. Therefore, we present a protocol to generate these suppressor mutants in M. oryzae easily to study the molecular basis of evolutionary processes or even epigenetic modulation. This protocol may be applicable to many other fungi and will open a door for researchers worldwide since the HOG pathway is worked on intensively in many different model organisms.

The Molecular Mechanism of Fludioxonil Action Is Different to Osmotic Stress Sensing.

The group III two-component hybrid histidine kinase MoHik1p in the filamentous fungus Magnaporthe oryzae is known to be a sensor for external osmotic stress and essential for the fungicidal activity of the phenylpyrrole fludioxonil. The mode of action of fludioxonil has not yet been completely clarified but rather assumed to hyperactivate the high osmolarity glycerol (HOG) signaling pathway. To date, not much is known about the detailed molecular mechanism of how osmotic stress is detected or fungicidal activity is initiated within the HOG pathway. The molecular mechanism of signaling was studied using a mutant strain in which the HisKA signaling domain was modified by an amino acid change of histidine H736 to alanine A736. We found that MoHik1pH736A is as resistant to fludioxonil but not as sensitive to osmotic stress as the null mutant ∆Mohik1. H736 is required for fludioxonil action but is not essential for sensing sorbitol stress. Consequently, this report provides evidence of the difference in the molecular mechanism of fludioxonil action and the perception of osmotic stress. This is an excellent basis to understand the successful phenylpyrrole-fungicides' mode of action better and will give new ideas to decipher cellular signaling mechanisms.

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 blast fungus Magnaporthe oryzae is subjected to an increased structural variation and amplified putative isoforms on a cDNA level. We then generated mutant strains overexpressing these isoforms, purified the products, and present here one previously unknown MoYpd1p isoform on a proteome level. Alternative splicing was found to be a valid molecular mechanism to increase the signal diversity in eukaryotic multistep phosphorelay systems.

A comment on the correct boundary conditions for the Cremer impedance.

Mode merging and the creation of exceptional points can be used to create optimum damping in a lined duct, as pointed out by Cremer [Acustica 3, 249-263 (1953)]. The effect of a mean flow has traditionally been analyzed by assuming the Ingard-Myer boundary condition at the wall. For low frequencies, however, the classical boundary condition is a better alternative. This paper shows that this choice removes two problems with the low-frequency solution: the negative real part of the optimum wall impedance and the non-valid solution for the upstream case. Theoretical derivations are complemented by numerical results to support these conclusions.

Two Novel Dimorphism-Related Virulence Factors of Zymoseptoria tritici Identified Using Agrobacterium-Mediated Insertional Mutagenesis.

Diseases caused by dimorphic phytopathogenic and systemic dimorphic fungi have markedly increased in prevalence in the last decades, and understanding the morphogenic transition to the virulent state might yield novel means of controlling dimorphic fungi. The dimorphic fungus Z. tritici causes significant economic impact on wheat production, and yet the regulation of the dimorphic switch, a key first step in successful plant colonization, is still largely unexplored in this fungus. The fungus is amenable to suppression by fungicides at this switch point, and the identification of the factors controlling the dimorphic switch provides a potential source of novel targets to control Septoria tritici blotch (STB). Inhibition of the dimorphic switch can potentially prevent penetration and avoid any damage to the host plant. The aim of the current work was to unveil genetic determinants of the dimorphic transition in Z. tritici by using a forward genetics strategy. Using this approach, we unveiled two novel factors involved in the switch to the pathogenic state and used reverse genetics and complementation to confirm the role of the novel virulence factors and further gained insight into the role of these genes, using transcriptome analysis via RNA-Seq. The transcriptomes generated potentially contain key determinants of the dimorphic transition.

In vivo Ca2+ dynamics in single pancreatic ß cells.

The dynamics of cytoplasmic free Ca2+ concentration ([Ca2+]i) in pancreatic ß cells is central to our understanding of ß-cell physiology and pathology. In this context, there are numerous in vitro studies available but existing in vivo data are scarce. We now critically evaluate the anterior chamber of the eye as an in vivo, non-invasive, imaging site for measuring [Ca2+]i dynamics longitudinally in three dimensions and at single-cell resolution. By applying a fluorescently labeled glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose in vivo, we followed how glucose almost simultaneously distributes to all cells within the islet volume, resulting in [Ca2+]i changes. We found that almost all ß cells in healthy mice responded to a glucose challenge, while in hyperinsulinemic, hyperglycemic mice about 80% of the ß cells could not be further stimulated from fasting basal conditions. This finding indicates that our imaging modality can resolve functional heterogeneity within the ß-cell population in terms of glucose responsiveness. Importantly, we demonstrate that glucose homeostasis is markedly affected using isoflurane compared to hypnorm/midazolam anesthetics, which has major implications for [Ca2+]i measurements. In summary, this setup offers a powerful tool to further investigate in vivo pancreatic ß-cell [Ca2+]i response patterns at single-cell resolution in health and disease.

Rapid adaptation of signaling networks in the fungal pathogen Magnaporthe oryzae.

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 solute produced by these adapted strains to cope with high osmolarity is glycerol, whereas it is arabitol in the wildtype strain. Genome and transcriptome analysis resulted in candidate genes related to glycerol metabolism, perhaps responsible for an epigenetic induced reestablishment of osmoregulation, since these genes do not show structural variations within the coding or promotor sequences. CONCLUSION: This is the first report of a stable adaptation in eukaryotes by producing different metabolites and opens a door for the scientific community since the HOG pathway is worked on intensively in many eukaryotic model organisms.

Noninvasive intravital high-resolution imaging of pancreatic neuroendocrine tumours.

Preclinical trials of cancer drugs in animal models are important for drug development. The Rip1Tag2 (RT2) transgenic mouse, a model of pancreatic neuroendocrine tumours (PNET), has provided immense knowledge about PNET biology, although tumour progression occurs in a location inaccessible for real-time monitoring. To overcome this hurdle we have developed a novel platform for intravital 3D imaging of RT2 tumours to facilitate real-time studies of cancer progression. Pre-oncogenic islets retrieved from RT2 mice were implanted into the anterior chamber of the eye (ACE) of host mice, where they engrafted on the iris, recruited blood vessels and showed continuous growth. Noninvasive confocal and two-photon laser-scanning microscopy through the transparent cornea facilitated high-resolution imaging of tumour growth and angiogenesis. RT2 tumours in the ACE expanded up to 8-fold in size and shared hallmarks with tumours developing in situ in the pancreas. Genetically encoded fluorescent reporters enabled high-resolution imaging of stromal cells and tumour cell migration. Sunitinib treatment impaired RT2 tumour angiogenesis and growth, while overexpression of the vascular endothelial growth factor (VEGF)-B increased tumour angiogenesis though tumour growth was impaired. In conclusion, we present a novel platform for intravital high-resolution and 3D imaging of PNET biology and cancer drug assessment.

Author Correction: Structural basis for delta cell paracrine regulation in pancreatic islets.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis for delta cell paracrine regulation in pancreatic islets.

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.

Visualizing fungicide action: an in vivo tool 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 the nucleus upon fungicide treatment in the MoHOG1::GFP mutant within seconds, but did not do so in the ΔMohik1/HOG1::GFP mutant. CONCLUSION: Here, we developed a rapid in vivo tool for fluorescent-based validation of fungicides targeting the HOG-signaling pathway. Furthermore, using the fluorescent mutants generated in this study, we are able to visualize that fungicide action is dependent on the histidine kinase MoHik1p but operates in a different mechanism of pathway activation compared to osmotic stress. © 2018 Society of Chemical Industry.

Diet-induced ß-cell insulin resistance results in reversible loss of functional ß-cell mass.

Although convincing in genetic models, the relevance of ß-cell insulin resistance in diet-induced type 2 diabetes (T2DM) remains unclear. Exemplified by diabetes-prone, male, C57B1/6J mice being fed different combinations of Western-style diet, we show that ß-cell insulin resistance occurs early during T2DM progression and is due to a combination of lipotoxicity and increased ß-cell workload. Within 8 wk of being fed a high-fat, high-sucrose diet, mice became obese, developed impaired insulin and glucose tolerances, and displayed noncompensatory insulin release, due, at least in part, to reduced expression of syntaxin-1A. Through reporter islets transplanted to the anterior chamber of the eye, we demonstrated a concomitant loss of functional ß-cell mass. When mice were changed from diabetogenic diet to normal chow diet, the diabetes phenotype was reversed, suggesting a remarkable plasticity of functional ß-cell mass in the early phase of T2DM development. Our data reinforce the relevance of diet composition as an environmental factor determining different routes of diabetes progression in a given genetic background. Employing the in vivo reporter islet-monitoring approach will allow researchers to define key times in the dynamics of reversible loss of functional ß-cell mass and, thus, to investigate the underlying, molecular mechanisms involved in the progression toward T2DM manifestation.-Paschen, M., Moede, T., Valladolid-Acebes, I., Leibiger, B., Moruzzi, N., Jacob, S., García-Prieto, C. F., Brismar, K., Leibiger, I. B., Berggren, P.-O. Diet-induced ß-cell insulin resistance results in reversible loss of functional ß-cell mass.