Search

The rice blast fungus Magnaporthe oryzae causes a devastating disease that threatens global rice (Oryza sativa) production. Despite intense study, the biology of plant tissue invasion during blast disease remains poorly understood. Here we report a high-resolution transcriptional profiling study of the entire plant-associated development of the blast fungus. Our analysis revealed major temporal changes in fungal gene expression during plant infection. Pathogen gene expression could be classified into 10 modules of temporally co-expressed genes, providing evidence for the induction of pronounced shifts in primary and secondary metabolism, cell signaling, and transcriptional regulation. A set of 863 genes encoding secreted proteins are differentially expressed at specific stages of infection, and 546 genes named MEP (Magnaportheeffector protein) genes were predicted to encode effectors. Computational prediction of structurally related MEPs, including the MAX effector family, revealed their temporal co-regulation in the same co-expression modules. We characterized 32 MEP genes and demonstrate that Mep effectors are predominantly targeted to the cytoplasm of rice cells via the biotrophic interfacial complex and use a common unconventional secretory pathway. Taken together, our study reveals major changes in gene expression associated with blast disease and identifies a diverse repertoire of effectors critical for successful infection.

To cause rice blast disease, the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on the selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signaling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls the expression of at least 60 temporally coregulated effector genes, preventing their transcription during the prepenetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for the orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.

To cause rice blast disease the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signalling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls expression of at least 60 temporally co-regulated effector genes, preventing their transcription during the pre-penetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.

The rice blast fungus Magnaporthe oryzae causes a devastating disease which threatens global rice production. In spite of intense study, the biology of plant tissue invasion during blast disease remains poorly understood. Here we report a high resolution, transcriptional profiling study of the entire plant-associated development of the blast fungus. Our analysis revealed major temporal changes in fungal gene expression during plant infection. Pathogen gene expression could be classified into 10 modules of temporally co-expressed genes, providing evidence of induction of pronounced shifts in primary and secondary metabolism, cell signalling and transcriptional regulation. A set of 863 genes encoding secreted proteins are differentially expressed at specific stages of infection, and 546 were predicted to be effectors and named MEP (Magnaportheeffector protein) genes. Computational prediction of structurally-related MEPs, including the MAX effector family, revealed their temporal co-regulation in the same co-expression modules. We functionally characterised 32 MEP genes and demonstrate that Mep effectors are predominantly targeted to the cytoplasm of rice cells via the biotrophic interfacial complex (BIC), and use a common unconventional secretory pathway. Taken together, our study reveals major changes in gene expression associated with blast disease and identifies a diverse repertoire of effectors critical to successful infection.

Techniques to comprehensively evaluate pulmonary function carry a variety of limitations, including the ability to continuously record intrathoracic pressures (ITP), acutely and chronically, in a natural state of freely behaving animals. Measurement of ITP can be used to derive other respiratory parameters, which provide insight to lung health. Our aim was to develop a surgical approach for the placement of a telemetry pressure sensor to measure ITP, providing the ability to chronically measure peak pressure, breath frequency, and timing of the respiratory cycle to facilitate circadian analyses related to breathing patterns. Applications of this technique are shown using a moderate hypoxic challenge. Male C57Bl/6 mice were implanted with radiotelemetry devices to record heart rate, temperature, activity, and ITP during 24-h normoxia, 24-h hypoxia ([Formula: see text] = 0.15), and return to 48-h normoxia. Radiotelemetry of ITP permitted the detection of hypoxia-induced increases in “the ITP equivalent” of ventilation, which were driven by increases in breathing frequency and ITP on a short-term time scale. Respiratory frequency, derived from pressure waveforms, was increased by a decrease in expiratory time without changes in inspiratory time. Chronically, telemetric recording allowed for circadian analyses of respiratory drive, as assessed by inspiratory pressure divided by inspiratory time, which was increased by hypoxia and remained elevated for 48 h of recovery. Furthermore, respiratory frequency demonstrated a circadian rhythm, which was disrupted through the recovery period. In conclusion, radiotelemetry of ITP is a viable, long-term, chronic methodology that extends traditional methods to evaluate respiratory function in mice. NEW & NOTEWORTHY We have demonstrated for the first time in mice that radiotelemetry is an effective tool for the continuous and chronic recording of intrathoracic pressure (ITP) to facilitate circadian rhythm analyses. We show that continuous 24-h hypoxic stress alters the circadian rhythms of heart rate, body temperature, activity, and respiratory parameters, acutely and perpetually, through normoxic recovery. Radiotelemetry of ITP can complement traditional methods for evaluating respiratory function and better our understanding of respiratory pathophysiology.

Wheat, the most important food crop, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America, and to guide pre-emptive wheat breeding for blast resistance.

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.

We report the compilation of MagnaGenes , a database which summarises all the available studies reporting phenotypic data about gene function in the blast fungus Magnaporthe oryzae. MagnaGenes includes information for 1637 genes and allows them to be sorted by putative function. For example, those with a particular role, such as ‘conidiation’ can be defined and grouped together. Magnagenes also contains clickable hyperlinks to the associated primary literature and to gene information held in the Ensembl and Uniprot databases. Magnagenes highlights some of the strengths and weaknesses in the Magnaporthe research community’s effort to understand the genetic basis of the ability of M. oryzae to cause blast disease. It can serve as a guide to the understudied aspects of the blast fungus biology. We provide Magnagenes to the community as part of the OpenRiceBlast and Open WheatBlast initiatives. We aim to release regular updates to Magnagenes and welcome additions or corrections from the blast research community to expand the database. &nbsp

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 close to MoPKS19, resulted in a mutant exclusively producing dihydropyriculol and dihydropyriculariol. By contrast, overexpression of MoC19OXR1 resulted in a mutant strain only producing pyriculol. The MoPKS19 cluster, furthermore, comprises two transcription factors MoC19TRF1 and MoC19TRF2, which were both found individually to act as negative regulators repressing gene expression of MoPKS19. Additionally, extracts of ΔMopks19 and ΔMoC19oxr1 made from axenic cultures failed to induce lesions on rice leaves compared to extracts of the wild-type strain. Consequently, pyriculol and its isomer pyriculariol appear to be the only lesion-inducing secondary metabolites produced by M. oryzae wild-type (MoWT) under these culture conditions. Interestingly, the mutants unable to produce pyriculol and pyriculariol were as pathogenic as MoWT, demonstrating that pyriculol is not required for infection.

The Magnaporthe oryzae genes FLB3 and FLB4, orthologues of the Aspergillus nidulans regulators of conidiation FlbC and FlbD, were inactivated. These genes encode C2H2 zinc finger and Myb-like transcription factors, respectively, in A. nidulans. Analysis of the resultant mutants demonstrated that FLB4 is essential for spore formation and that strains lacking this gene are fluffy in their colony morphology due to an inability to complete conidiophore formation. Meanwhile, FLB3 is required for normal levels of aerial mycelium formation. We identified genes dependent on both transcription factors using microarray analysis. This analysis revealed that the transcription of several genes encoding proteins implicated in sporulation in Magnaporthe oryzae and other filamentous fungi are affected by FLB3 or FLB4 inactivation. Furthermore, the microarray analysis indicates that Flb3p may effectively reprogramme the cell metabolically by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. Additionally, qRT-PCR was employed and showed that FLB3 and FLB4 transcripts are enriched in synchronously sporulating cultures, as were the transcripts of other genes that are necessary for normal conidiation, consistent with a role for their gene products in this process.

The rice blast fungus Magnaporthe oryzae elaborates a specialized cell called an appressorium, which is used to breach the tough outer cuticle of a rice leaf, enabling the fungus entry to host plant cells. The appressorium generates enormous turgor by accumulating glycerol to very high concentrations within the cell. Glycerol accumulation and melanization of the appressorium cell wall collectively drive turgor-mediated penetration of the rice leaf. In this review, we discuss the potential metabolic sources of glycerol in the rice blast fungus and how appressorium turgor is focused as physical force at the base of the infection cell, leading to the formation of a rigid penetration peg. We review recent studies of M. oryzae and other relevant appressorium-forming fungi which shed light on how glycerol is synthesized and how appressorium turgor is regulated. Finally, we provide some questions to guide avenues of future research that will be important in fully understanding the role of glycerol in rice blast disease.

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific gene editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based gene editing method. It has previously been reported in M. oryzae that transformation with plasmids expressing Cas9 can generate specific mutations using sgRNAs, directing the endonuclease to specific genes. We show, however, that expression of Cas9 is highly toxic to M. oryzae, rendering this approach impractical. We demonstrate that using purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs), provides an alternative and very effective gene editing procedure. When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.

Dyspnea and reduced exercise capacity, caused, in part, by respiratory muscle dysfunction, are common symptoms in patients with heart failure (HF). However, the etiology of diaphragmatic dysfunction has not been identified. To investigate the effects of HF on diaphragmatic function, models of HF were surgically induced in CD-1 mice by transverse aortic constriction (TAC) and acute myocardial infarction (AMI), respectively. Assessment of myocardial function, isolated diaphragmatic strip function, myofilament force-pCa relationship, and phosphorylation status of myofilament proteins was performed at either 2 or 18 wk postsurgery. Echocardiography and invasive hemodynamics revealed development of HF by 18 wk postsurgery in both models. In vitro diaphragmatic force production was preserved in all groups while morphometric analysis revealed diaphragmatic atrophy and fibrosis in 18 wk TAC and AMI groups. Isometric force-pCa measurements of myofilament preparations revealed reduced Ca2+sensitivity of force generation and force generation at half-maximum and maximum Ca2+activation in 18 wk TAC. The rate of force redevelopment ( ktr) was reduced in all HF groups at high levels of Ca2+activation. Finally, there were significant changes in the myofilament phosphorylation status of the 18 wk TAC group. This includes a decrease in the phosphorylation of troponin T, desmin, myosin light chain (MLC) 1, and MLC 2 as well as a shift in myosin isoforms. These results indicate that there are multiple changes in diaphragmatic myofilament function, which are specific to the type and stage of HF and occur before overt impairment of in vitro force production.

This 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 stress and fludioxonil application resulted in the phosphorylation of the MoHog1p in a MoYpd1p-dependent manner. We therefore consider MoYpd1p to be essential for the regulation capability of the HOG pathway and the fungicide action of fludioxonil, but dispensible for viability. The results indicate that MoYpd1p functions as signal transfer protein between MoSln1p, MoHik1p, and MoSsk1p. Manipulations of the HOG signalling pathway affects the infection-related morphogenesis in M. oryzae, since the mutant strain ΔMoypd1 has a white and fluffy phenotype on complete media, is not able to form spores in various conditions and fails to colonize rice plants.

Herein, we describe the development of a novel dual air-bearing fixed-χ diffractometer for beamline I19 at Diamond Light Source. The diffractometer is designed to facilitate the rapid data collections possible with a Dectris Pilatus 2M pixel-array photon-counting detector, while allowing remote operation in conjunction with a robotic sample changer. The sphere-of-confusion is made as small as practicably possible, through the use of air-bearings for both the ω and φ axes. The design and construction of the new instrument is described in detail and an accompanying paper by Johnson et al. (also in this issue) will provide a user perspective of its operation.

Diaphragmatic weakness is a feature of heart failure (HF) associated with dyspnea and exertional fatigue. Most studies have focused on advanced stages of HF, leaving the cause unresolved. The long-standing theory is that pulmonary edema imposes a mechanical stress, resulting in diaphragmatic remodeling, but stable HF patients rarely exhibit pulmonary edema. We investigated how diaphragmatic weakness develops in two mouse models of pressure overload-induced HF. As in HF patients, both models had increased eupneic respiratory pressures and ventilatory drive. Despite the absence of pulmonary edema, diaphragmatic strength progressively declined during pressure overload; this decline correlated with a reduction in diaphragm cross-sectional area and preceded evidence of muscle weakness. We uncovered a functional codependence between angiotensin II and β-adrenergic (β-ADR) signaling, which increased ventilatory drive. Chronic overdrive was associated with increased PERK (double-stranded RNA-activated protein kinase R-like ER kinase) expression and phosphorylation of EIF2α (eukaryotic translation initiation factor 2α), which inhibits protein synthesis. Inhibition of β-ADR signaling after application of pressure overload normalized diaphragm strength, Perk expression, EIF2α phosphorylation, and diaphragmatic cross-sectional area. Only drugs that were able to penetrate the blood-brain barrier were effective in treating ventilatory overdrive and preventing diaphragmatic atrophy. These data provide insight into why similar drugs have different benefits on mortality and symptomatology, despite comparable cardiovascular effects.

Key points The role of prostaglandins in the changes in blood flow and microvascular vasodilation associated with exercise and muscle contraction is controversial. Whether prostaglandins are produced independently during muscle contraction or whether their production is dependent on the production of adenosine is not well understood. We show that prostaglandins are an important component of the microvascular vasodilation associated with muscle contraction but only under specific contractile conditions. Further, we show that microvascular vasodilation in response to adenosine is not dependent on prostaglandins. Therefore, we conclude that there are specific contractile conditions under which prostaglandins are an important component of the vasodilation induced by muscle contraction and we propose that prostaglandin-induced microvascular vasodilation during exercise is independent of adenosine. Abstract Blood flow data from contracting muscle in humans indicates that adenosine (ADO) stimulates the production of nitric oxide (NO) and vasodilating prostaglandins (PG) to produce arteriolar vasodilatation in a redundant fashion such that when one is inhibited the other can compensate. We sought to determine whether these redundant mechanisms are employed at the microvascular level. First, we determined whether PGs were involved in active hyperaemia at the microvascular level. We stimulated four to five skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of 2A arterioles (maximum diameter 40 μm, third arteriolar level up from the capillaries) at a site of overlap with the stimulated muscle fibres before and after 2 min of contraction [stimulus frequencies: 4, 20 and 60 Hz at 15 contractions per minute (CPM) or contraction frequencies of 6, 15 or 60 CPM at 20 Hz; 250 ms train duration]. Muscle fibres were stimulated in the absence and presence of the phospholipase A2 inhibitor quinacrine. Further, we applied a range of concentrations of ADO (10−7–10−5 m) extraluminally, (to mimic muscle contraction) in the absence and presence of l-NAME (NO synthase inhibitor), indomethacin (INDO, cyclooxygenase inhibitor) and l-NAME + INDO and observed the response of 2A arterioles. We repeated the latter experiment on a different level of the cremaster microvasculature (1A arterioles) and on the microvasculature of a different skeletal muscle (gluteus maximus, 2A arterioles). We observed that quinacrine inhibited vasodilatation during muscle contraction at intermediate and high contraction frequencies (15 and 60 CPM). l-NAME, INDO and l-NAME + INDO were not effective at inhibiting vasodilatation induced by any concentration of ADO tested in 2A and 1A arterioles in the cremaster muscle or 2A arterioles in the gluteus maximus muscle. Our data show that PGs are involved in the vasodilatation of the microvasculature in response to muscle contraction but did not obtain evidence that extraluminal ADO causes vasodilatation through NO or PG or both. Thus, we propose that PG-induced microvascular vasodilation during exercise is independent of ADO.

This article presents results from experimental studies and techno-economic analysis of a catalytic process for the conversion of whole biomass into drop-in aviation fuels with maximal carbon yields. The combined research areas highlighted include biomass pretreatment, carbohydrate hydrolysis and dehydration, and catalytic upgrading of platform chemicals. The technology centers on first producing furfural and levulinic acid from five- and six-carbon sugars present in hardwoods and subsequently upgrading these two platforms into a mixture of branched, linear, and cyclic alkanes of molecular weight ranges appropriate for use in the aviation sector. Maximum selectivities observed in laboratory studies suggest that, with efficient interstage separations and product recovery, hemicellulose sugars can be incorporated into aviation fuels at roughly 80% carbon yield, while carbon yields to aviation fuels from cellulose-based sugars are on the order of 50%. The use of lignocellulose-derived feedstocks rather than commercially sourced model compounds in process integration provided important insights into the effects of impurity carryover and additionally highlights the need for stable catalytic materials for aqueous phase processing, efficient interstage separations, and intensified processing strategies. In its current state, the proposed technology is expected to deliver jet fuel-range liquid hydrocarbons for a minimum selling price of $4.75 per gallon assuming nth commercial plant that produces 38 million gallons liquid fuels per year with a net present value of the 20 year biorefinery set to zero. Future improvements in this technology, including replacing precious metal catalysts by base metal catalysts and improving the recyclability of water streams, can reduce this cost to $2.88 per gallon.

Transcription factors play a critical regulatory role in development by binding DNA and initiating alterations in gene transcription. The transcript of the putative Magnaporthe oryzae transcription factor-encoding gene TRA1 accumulates during germination and this accumulation was previously found to depend on the transcription factor Con7p. In the current work tra1 − mutants were generated and these strains were found to exhibit a reduced attachment, germination, appressorium formation and virulence. Adhesion to artificial and plant surfaces was affected, and FITC-labelled concanavalin A, a lectin which inhibits attachment of Magnaporthe spores, showed a reduced affinity for mutant spore tip where it normally preferentially binds. We used microarray analysis to identify Tra1p-dependent genes from two different sources: aerial structures and conidia. Mutation of 11 Tra1p-dependent genes showed that the predicted transcription factor encoding gene TDG2 is required for normal adhesion and virulence, that the genes TDG7 and TDG4 are required for normal sporulation and that TDG6 is required for wild-type levels of spore adhesion.

The conversion of glucose, furan and maple wood has been investigated over different types of ZSM-5 catalyst in semi-batch and fixed-bed reactors. The aromatic yield from glucose conversion goes through a maximum as a function of the framework silica-to-alumina ratio (SAR) of ZSM-5 with an optimum at SAR = 30. This suggests that the concentration of acid sites inside the zeolite is critical for maximizing aromatic yield. Creating hierarchical mesopores within the zeolite slightly increased of coke formation and decreased the formation of the monocyclic aromatics. Mesoporous ZSM-5 was also observed to favor the production of larger alkylated monoaromatics. The selective removal of external acid sites from the ZSM-5 catalysts only slightly increased the catalyst activity but also decreased the selectivity to the desired aromatic products.

In a screening for inhibitors of infection-related morphogenesis in the rice blast fungus Magnaporthe oryzae, a series of 10 coumarin derivatives were isolated from Pedilanthus tithymaloides (Euphorbiaceae). Seven of these compounds turned out to be known while three represent previously unreported natural products. Their structures were established on the basis of spectroscopic data and X-ray crystallography. Nine out of 10 coumarin derivatives were found to inhibit conidial germination in the phytopathogenic fungus at low concentrations.

The hydrodeoxygenation (HDO) of m-cresol is investigated as a model for the HDO of phenolic compounds from lignin pyrolysis. Pt catalysts supported on γ-Al2O3 and SiO2 are effective for the conversion of m-cresol to toluene and methylcyclohexane at 533 K and 0.5 atm H2. Experiments using Pt/γ-Al2O3 show that the reaction proceeds by a combination of Pt-catalyzed hydrogenation and acid-catalyzed dehydration reactions. Dehydration of a partially hydrogenated oxygenate intermediate is most likely the dominant reaction pathway to toluene. The acidity of the γ-Al2O3 support was modified by base (K2CO3) and acid (NH4F) treatments, and increasing the number and strength of acid sites was found to increase the rate of HDO. Pt/SiO2 was more active for m-cresol HDO than Pt/Al2O3. The reaction rate on Pt/Al2O3 and Pt/SiO2 decreased after 5 h on stream, but Pt/Al2O3 regained initial reactivity after reductive treatment in H2.

Zeolite beta has been prepared in dilute (H2O/SiO2 = 80) and concentrated (H2O/SiO2 = 20) conditions. The rates of zeolite beta nucleation and growth in dilute colloidal solutions have been characterized using dynamic light scattering and small angle X-ray scattering. By blending TEA+ and Na+ in dilute solutions at low Al concentration (Si/Al = 50) it has been determined that the presence of Na+ decreases the nucleation rate and increases the growth rate (i.e., decreases the colloidal stability of the precursor nanoparticles) because Na+ competes with TEA+ for surface adsorption sites and occlusion into the precursor particles. Using more concentrated synthesis solutions the role of ion pairs (e.g., [[ SiO]− TEA+], [[AlO2]− TEA+], and [[AlO2]− Na+]) in the precursor particles and the zeolite beta product was investigated in detail. Thermal gravimetric analyses have helped determine relation between synthesis conditions and the ion pairs formed in the zeolite beta product. On the basis of the energies associated with these ion pairs a state diagram of zeolite beta including surface structure and phase selectivity has been developed. The relative rates of zeolite beta nucleation and growth have also been investigated at several ratios of TEA+ to Na+ in concentrated solutions. The synergy between these cations and anionic surface adsorption sites has led to synthesis of nearly monodisperse zeolite beta crystal particles having sizes from about 100 to 500 nm.

We investigate the influence of zeolite pore size and shape selectivity on the conversion of glucose to aromatics. Zeolites having a variety of pore size and shape (small pore ZK-5, SAPO-34, medium pore Ferrierite, ZSM-23, MCM-22, SSZ-20, ZSM-11, ZSM-5, IM-5, TNU-9, and large pore SSZ-55, Beta zeolite, Y zeolite) were synthesized, characterized, and tested in a pyroprobe reactor coupled with GC–MS for the conversion of glucose to aromatics. The aromatic yield was a function of the pore size of the zeolite catalyst. Small pore zeolites did not produce any aromatics with oxygenated products (from pyrolysis of glucose), CO, CO2 and coke as the major products. Aromatic yields were highest in the medium pore zeolites with pore sizes in the range of 5.2–5.9 A. High coke yield, low aromatic yields, and low oxygenate yields were observed with large pore zeolites, suggesting that the large pores facilitate the formation of coke. In addition to pore window size, internal pore space and steric hindrance play a major role for aromatic production. Medium pore zeolites with moderate internal pore space and steric hindrance (ZSM-5 and ZSM-11) have the highest aromatic yield and the least amount of coke. The kinetic diameters of the products and reactants were estimated to determine whether the reactions occur inside the pores or at external surface sites for the different zeolite catalysts. This analysis showed that the majority of the aromatic products and the reactants can fit inside the zeolite pores of most of the medium and large pore zeolites. However, in some of the smaller pore zeolites the polycyclic aromatics may form by secondary reactions on the catalyst surface, either directly or via reaction of the smaller aromatics.

Summary A strain harbouring an insertion within the promoter of the CON7 gene of Magnaporthe grisea was isolated. This gene was previously shown to be essential for appressorium formation and growth in planta and is predicted to encode a transcription factor. Microarray-based gene expression analysis was used to identify several genes whose transcription during germination depends on Con7p. These include the pathogenicity factor-encoding gene PTH11 and several other genes which like PTH11 are predicted to encode G protein-coupled receptors. Microarray analysis also revealed several Con7p-dependent genes which may encode factors determining cell wall structure or function, either through the synthesis/degradation of cell wall components or by association with the cell exterior. One Con7p-dependent gene predicted to encode a class VII chitin synthase was deleted, leading to dramatic consequences on the pathogenic development of the resultant strain. Within the con7– mutant, a 29% reduction in chitin content of germinated spores was found and the mutant was hypersensitive to the chitin synthase inhibitor nikkomycin Z. A green fluorescent protein-tagged Con7p was found to have nuclear localization within spores. Taken together, these observations suggest that Con7p encodes a transcription factor required for the transcription of several genes which participate in disease-related morphogenesis in M. grisea.

SUMMARY Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host–pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea, the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1, an NRPS gene, and a gene encoding an l-ornithine N5-monooxygenase (OMO1) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N5 hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea.

The relationship of trehalose metabolism to fungal virulence was explored in the rice blast fungus Magnaporthe grisea. To determine the role of trehalose synthesis in pathogenesis, we identified and deleted TPS1, encoding trehalose-6-phosphate synthase. A Deltatps1 mutant failed to synthesize trehalose, sporulated poorly and was greatly attenuated in pathogenicity. Appressoria produced by Deltatps1 did not develop full turgor or elaborate penetration hyphae efficiently. To determine the role of subsequent trehalose breakdown, we deleted NTH1, which encodes a neutral trehalase. Nth1 mutants infected plants normally, but showed attenuated pathogenicity due to a decreased ability to colonize plant tissue. A second trehalase was also identified, required both for growth on trehalose and mobilization of intracellular trehalose during infection-related development. TRE1 encodes a cell wall-localized enzyme with characteristics of both neutral and acidic trehalases, but is dispensable for pathogenicity. Our results indicate that trehalose synthesis, but not its subsequent breakdown, is required for primary plant infection by M.grisea, while trehalose degradation is important for efficient development of the fungus in plant tissue following initial infection.

The obesity epidemic is considered one of the most serious public health problems of the modern world. Physical therapy is the most accessible form of treatment; however, compliance is a major obstacle due to exercise intolerance and dyspnea. Respiratory muscle atrophy is a cause of dyspnea, yet little is known of obesity-induced respiratory muscle dysfunction. Our objective was to investigate whether obesity-induced skeletal muscle wasting occurs in the diaphragm, the main skeletal muscle involved in inspiration, using the Zucker diabetic fatty (ZDF) rat. After 14 wk, ZDF rats developed obesity, hyperglycemia, and insulin resistance, compared with lean controls. Hemodynamic analysis revealed ZDF rats have impaired cardiac relaxation ( P = 0.001) with elevated end-diastolic pressure ( P = 0.006), indicative of diastolic dysfunction. Assessment of diaphragm function revealed weakness ( P = 0.0296) in the absence of intrinsic muscle impairment in ZDF rats. Diaphragm morphology revealed increased fibrosis ( P < 0.0001), atrophy ( P < 0.0001), and reduced myosin heavy-chain content ( P < 0.001), compared with lean controls. These changes are accompanied by activation of the myostatin signaling pathway with increased serum myostatin ( P = 0.017), increased gene expression ( P = 0.030) in the diaphragm and retroperitoneal adipose ( P = 0.033), and increased SMAD2 phosphorylation in the diaphragm ( P = 0.048). Here, we have confirmed the presence of respiratory muscle atrophy and weakness in an obese, diabetic model. We have also identified a pathological role for myostatin signaling in obesity, with systemic contributions from the adipose tissue, a nonskeletal muscle source. These findings have significant implications for future treatment strategies of exercise intolerance in an obese, diabetic population.

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 affected by osmotic or sugar stress. In contrast to yeast, the HOG signaling cascade in phytopathogenic fungi apparently comprises more elements. Furthermore, vegetative growth of the mutants ΔMohik5 and ΔMohik9 was found to be sensitive to hypoxia-inducing NaNO2 -treatment. Additionally, it was monitored that NaNO2 -treatment resulted in MoHog1p phosphorylation. As a consequence we assume a first simplified model for hypoxia signaling in M. oryzae including the HOG pathway and the HIKs MoHik5p and MoHik9p.

Impairments in O2 delivery are a common feature of many chronic diseases (e.g., COPD, diabetes, and anemia). It reduces quality of life and increases morbidity, mortality and hospitalizations in patients. Circadian rhythms in mammals, including humans, are essential to health whereas disruptions exacerbate disease pathogenesis. While severe hypoxia can abolish circadian rhythm, moderate hypoxia is more clinically relevant but its effects on circadian rhythm are unknown. Our objective was to investigate the effects of moderate hypoxia on circadian rhythm. Methods: Mice were implanted with telemetry devices to measure heart rate (HR) and body temperature (Tb). Baseline measurements were obtained and animals subjected to 24 hours of moderate hypoxia (O2 = 15%) followed by 24 hours of normoxic recovery. Results: Moderate hypoxia disrupted, but did not abolish, the circadian rhythm of HR and Tb. Amplitude of circadian rhythm for HR was altered during both sleep and active states whereas Tb amplitude was altere...

Exercise intolerance, characterized by dyspnea and early onset of exertional fatigue, is a chief complaint among patients with heart failure (HF). Limb muscle dysfunction is a known contributor to ...

The fission yeast cdc23 gene is required for correct DNA replication: cdc23 mutants show reduced rates of DNA synthesis and become elongated after cell-cycle arrest. We have cloned the Schizosaccharomyces pombe cdc23 gene by complementation of the temperature-sensitive phenotype of cdc23-M36 and confirmed the identity of the gene by integrative mapping. Analysis of the DNA sequence reveals that cdc23 can encode a protein of 593 amino acids (Mr=67 kDa) with 22% overall identity and many structural homologies with the product of the Saccharomyces cerevisiae DNA43 (MCM10) gene which is required for correct initiation of DNA synthesis at chromosomal origins of replication. Construction of a cdc23 null allele has established that the cdc23 gene is essential for viability, with cdc23 deletion mutant spores germinating but undergoing arrest with undivided nuclei in the first or second cell cycle. The S. pombe cdc23 gene on an expression plasmid is able to complement the S. cerevisiae dna43-1 mutant. These structural and functional homologies between two distantly related species suggest that cdc23 and DNA43 may represent genes for a conserved essential eukaryotic DNA replication function.

This tutorial review centers on recent advances and applications of experimental techniques that help characterize surface species and catalyst structures under in situ conditions. We start by reviewing recent applications of IR spectroscopy of working catalysis, emphasizing newer approaches such as Sum Frequency Generation and Polarization Modulation-infrared reflection absorption spectroscopy. This is followed by a section on solid-state NMR spectroscopy for the detection of surface species and reaction intermediates. These two techniques provide information mainly about the concentration and identity of the prevalent surface species. The following sections center on methods that provide structural and chemical information about the catalyst surface. The increasingly important role of high-pressure X-ray photoelectron spectroscopy in catalyst characterization is evident from the new and interesting information obtained on supported catalysts as presented in recent reports. X-Ray absorption spectroscopy (XANES and EXAFS) is used increasingly under reaction conditions to great advantage, although is inherently limited to systems where the bulk of the species in the sample are surface species. However, the ability of X-rays to penetrate the sample has been used cleverly by a number of groups to understand how changing reaction conditions change the structure and composition of surface atoms on supported catalyst.

This tutorial review centers on recent advances and applications of experimental techniques that help characterize surface species and catalyst structures under in situ conditions. We start by reviewing recent applications of IR spectroscopy of working catalysis, emphasizing newer approaches such as Sum Frequency Generation and Polarization Modulation-infrared reflection absorption spectroscopy. This is followed by a section on solid-state NMR spectroscopy for the detection of surface species and reaction intermediates. These two techniques provide information mainly about the concentration and identity of the prevalent surface species. The following sections center on methods that provide structural and chemical information about the catalyst surface. The increasingly important role of high-pressure X-ray photoelectron spectroscopy in catalyst characterization is evident from the new and interesting information obtained on supported catalysts as presented in recent reports. X-Ray absorption spectroscopy (XANES and EXAFS) is used increasingly under reaction conditions to great advantage, although is inherently limited to systems where the bulk of the species in the sample are surface species. However, the ability of X-rays to penetrate the sample has been used cleverly by a number of groups to understand how changing reaction conditions change the structure and composition of surface atoms on supported catalyst.

Mycosphaerella graminicola is a dimorphic fungus which causes Septoria tritici leaf blotch. This report describes the examination of the role of several components of the Pmk1p/Fus3p mitogen-activated protein kinase (MAPK) signalling pathway in the development of this species. The genes encoding the MAPK kinase kinase MgSte11p and the MAPK kinase MgSte7p were found to be indispensible for pathogenicity while the deletion of the gene encoding the proposed scaffold protein MgSte50p led to a reduction in virulence. These phenotypes were attributed to a reduced ability to form filaments on the plant surface which prevented penetration. A delayed disease progression was observed on deletion of the gene MGSTE12. The MGSTE7, MGSTE50 and MGSTE12 genes were able to complement mutants of Magnaporthe grisea lacking the orthologous genes. Interactions between the My. graminicola signalling components were also investigated. Furthermore genes whose MgSte12p/Mst12p dependence is conserved between My. graminicola and Ma. grisea were identified.

The rapid emergence of fungicide resistance has brought a strong demand for plant protectants with a new mode of action. One of the challenges for modern plant pathology research is the identification of genes and gene products essential for the establishment of pathogenic interactions between host and pathogen. Genome-wide technologies, such as transcriptomics, metabolomics, and proteomics represent a new opportunity to identify such targets in phytopathogenic fungi. This chapter reviews genome-wide approaches that have emerged in the post-genomics era, e.g. comparative genomics and gene expression profiling experiments such as microarray-based transcriptional profiling, serial analysis of gene expression, and massively parallel signature sequencing. It also discusses prospects for exploiting these modern technologies for the development of plant protectants and system biology.

The plant pathogenic fungus Magnaporthe grisea is able to enter its host via appressorium‐mediated penetration. Earlier investigations have shown that these infection structures are rich in the cell wall polysaccharide chitin. Previously, we have described how the transcription of a class VII chitin synthase‐encoding gene CHS7 is completely dependent on the putative transcription factor Con7p during the germination of conidia, and how con7 (−) mutants are unable to form appressoria under any conditions tested. Because of the pleiotropic effects of the con7 (−) mutation, we examined the consequences of the targeted deletion of CHS7. The chs7 (−) mutants generated were unable to form appressoria on artificial surfaces, except following the application of the exogenous inducers 1,16‐hexadecanediol and cyclic adenosine monophosphate. The appressoria formed had a reduced chitin content and were often found to be smaller and misshapen compared with the wild‐type. chs7 (−) mutants were significantly reduced in their ability to enter rice plants, but growth in planta was not affected. Reverse transcriptase‐polymerase chain reaction analysis demonstrated that CHS7 transcription was strongly induced on germination of spores, and a green fluorescent protein‐tagged Chs7p protein was found to be produced abundantly during infection‐related morphogenesis. Together, these data suggest that the class VII chitin synthase Chs7p of M. grisea is required for normal appressorium formation and function.

The plant pathogenic fungus Magnaporthe grisea excretes siderophores of the coprogen-type for iron acquisition and uses ferricrocin for intracellular iron storage. In the present report we characterize mutants with defects in extracellular siderophore biosynthesis. Deletion of the M. grisea SSM2 gene, which encodes a non-ribosomal peptide synthetase, resulted in a loss of the production of all coprogens. The mutant strains had a reduced growth rate, produced fewer conidia and were more sensitive to oxidative stress. Ferricrocin production was not affected. Upon deletion of M. grisea OMO1, a gene predicted to encode an L-ornithine-N(5)-monooxygenase, no siderophores of any type were detected, the strain was aconidial, growth rate was reduced and sensitivity to oxidative stress was increased. Abundance of several proteins was affected in the mutants. The Deltassm2 and Deltaomo1 mutant phenotypes were complemented by supplementation of the medium with siderophores or reintroduction of the respective genes.

The kingdom fungi comprises a highly diverse array of species. Where species diversification has been studied in detail, fungal species can be 6 times as numerous as those of flowering plants. On this basis, since approximately 270,000 flowering plants are known today, it is estimated that more than 1.5 million fungal species may exist (Carlile and Watkinson 1994; Hawksworth 2001)). Fungi are able to colonize a broad range of substrata; they may live as saprophytes, associated with plants as mycorrhiza and as pathogens of plants, animals and microorganisms (Dix and Webster 1995). As saprophytes, fungi contribute to the ecological balance of the environment by degrading organic materials, such as decaying plant material, dung and organic pollutants. In mycorrhiza, fungi provide phosphate for the associated plant while they gain carbohydrates in exchange (Smith and Read 1997). However, many plant-associated fungi do not provide any mutual benefit for the colonized hosts. Whereas biotrophic fungi rely on the living host for nutrient provision, others (necrotrophs) often kill and exploit the surrounding tissue. The high biodiversity of fungal species is also reflected by exceedingly rich diversity of metabolism and the corresponding metabolites that affect the attacked plant. A large group of pathogens known as necrotrophs secrete phytotoxic secondary metabolites to kill the host tissue and to avoid initiation of defense responses. Secondary metabolites have been defined as metabolic products not essential for growth and without obvious function for the producing cell during its life cycle (Aharonowitz and Demain 1980). Secondary metabolite production depends both on the genotype of the organism and the environmental conditions under which growth takes place, for example the composition of the medium used to culture the organism (Weinberg 1974; Bu’Lock 1975). So far, several thousand sec