Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, 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 preemptive wheat breeding for blast resistance.

Rgs1 is a regulator of effector gene expression during plant infection by the rice blast fungus Magnaporthe oryzae.

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.

The transcriptional landscape of plant infection by the rice blast fungus Magnaporthe oryzae reveals distinct families of temporally co-regulated and structurally conserved effectors.

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.

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.

Applications of a novel radiotelemetry method for the measurement of intrathoracic pressures and physiological rhythms in freely behaving mice.

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.

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus.

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 genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). 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. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. 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.

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 genes involved in dimorphic switch, a plate-based screening system was established. With this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing high amount of mycelium under different incubation conditions. Using genome walking approach previously established, the T-DNA integration sites were recovered and the disrupted genomic loci of corresponding mutants were identified and validated within reverse genetics approach. As prove of concept, two of the random mutants obtained were selected for further investigation using targeted gene inactivation. Both genes deduced were found to encode known factors, previously characterized in other fungi: Ssk1p being constituent of HOG pathway and Ade5,7p involved in de novo purine biosynthesis. The targeted mutant strains defective in these genes exhibit a drastically impaired virulence within infection assays on whole wheat plants. Moreover exploiting further physiological assays the predicted function for both gene products could be confirmed in concordance with conserved biological role of homologous proteins previously described in other fungal organisms.

Central-acting therapeutics alleviate respiratory weakness caused by heart failure-induced ventilatory overdrive.

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 ß-adrenergic (ß-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 ß-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.

The role of glycerol in the pathogenic lifestyle of the rice blast fungus Magnaporthe oryzae.

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.

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 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.

What Patients Really Want: Optimizing the Military Preoperative Evaluation Clinic.

The idea of the preoperative anesthesia clinic as a means of examining and treating the patient so that he will arrive in the operating theater as strong and healthy as possible is well established in practice and literature.However, problems in clinic design and execution often result in high patient waiting times, decreased patient and staff satisfaction, decreased patient capacity, and high clinic costs. Although the details of clinic design, outcomes, and satisfaction have been extensively evaluated at civilian hospitals, we have not found corresponding literature addressing these issues specifically within military preoperative evaluation clinics. We find that changing to an appointment-based (versus walk-in) system and eliminating data collection step redundancies will likely result in lower wait times, higher satisfaction, lower per patient costs, and a more streamlined and resource-efficient structure.

A modular and compact portable mini-endstation for high-precision, high-speed fixed target serial crystallography at FEL and synchrotron sources.

The design and implementation of a compact and portable sample alignment system suitable for use at both synchrotron and free-electron laser (FEL) sources and its performance are described. The system provides the ability to quickly and reliably deliver large numbers of samples using the minimum amount of sample possible, through positioning of fixed target arrays into the X-ray beam. The combination of high-precision stages, high-quality sample viewing, a fast controller and a software layer overcome many of the challenges associated with sample alignment. A straightforward interface that minimizes setup and sample changeover time as well as simplifying communication with the stages during the experiment is also described, together with an intuitive naming convention for defining, tracking and locating sample positions. The setup allows the precise delivery of samples in predefined locations to a specific position in space and time, reliably and simply.

Respiratory muscle weakness in the Zucker diabetic fatty rat.

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.

High osmolarity glycerol (HOG) signalling in Magnaporthe oryzae: Identification of MoYPD1 and its role in osmoregulation, fungicide action, and pathogenicity.

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.

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 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.

Prostaglandins induce vasodilatation of the microvasculature during muscle contraction and induce vasodilatation independent of adenosine.

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.

The role of the Tra1p transcription factor of Magnaporthe oryzae in spore adhesion and pathogenic development.

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.

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

Identifying reaction intermediates and catalytic active sites through in situ characterization techniques.

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.