Murine Model for the Study of Influenza D Virus.

A novel genus within the Orthomyxoviridae family was identified in the United States and named influenza D virus (IDV). Bovines have been proposed to be the primary host, and three main viral lineages (D/OK-like, D/660-like, and D/Japan-like) have been described. Experimental infections had previously been performed in swine, ferrets, calves, and guinea pigs in order to study IDV pathogenesis. We developed a murine experimental model to facilitate the study of IDV pathogenesis and the immune response. DBA/2 mice were inoculated with 105 50% tissue culture infective dose (TCID50) of D/bovine/France/5920/2014 (D/OK-like). No clinical signs or weight loss were observed. Viral replication was observed mainly in the upper respiratory tract (nasal turbinates) but also in the lower respiratory tract of infected mice, with a peak at 4 days postinfection. Moreover, the virus was also detected in the intestines. All infected mice seroconverted by 14 days postinfection. Transcriptomic analyses demonstrated that IDV induced the activation of proinflammatory genes, such as gamma interferon (IFN-γ) and CCL2. Inoculation of NF-κB-luciferase and Ifnar1-/- mice demonstrated that IDV induced mild inflammation and that a type I interferon response was not necessary in IDV clearance. Adaptation of IDV by serial passages in mice was not sufficient to induce disease or increased pathogenesis. Taken together, present data and comparisons with the calf model show that our mouse model allows for the study of IDV replication and fitness (before selected viruses may be inoculated on calves) and also of the immune response.IMPORTANCE Influenza D virus (IDV), a new genus of Orthomyxoviridae family, presents a large host range and a worldwide circulation. The pathogenicity of this virus has been studied in the calf model. The mouse model is frequently used to enable a first assessment of a pathogen's fitness, replication, and pathogenesis for influenza A and B viruses. We showed that DBA/2 mice are a relevant in vivo model for the study of IDV replication. This model will allow for rapid IDV fitness and replication evaluation and will enable phenotypic comparisons between isolated viruses. It will also allow for a better understanding of the immune response induced after IDV infection.

Molecular epidemiological survey and phylogenetic analysis of bovine influenza D virus in Japan.

The influenza D virus, a new member of the Orthomyxoviridae family, is predominantly found in cattle. Although viral pathology and clinical disease in cattle appear mild, this virus plays an important role as a trigger of bovine respiratory disease (BRD). BRD is a costly illness worldwide. Thus, epidemiological surveys of the influenza D virus are necessary. Here, we conducted a molecular epidemiological survey for the influenza D virus in healthy and respiratory-diseased cattle in Japan. We found that 2.1% (8/377) of the cattle were infected with influenza D. The cattle with and without respiratory symptoms had approximately equal amounts of the virus. A full-genome sequence analysis revealed that the influenza D virus that was isolated in Japan formed an individual cluster that was distinct from the strains found in other countries. These results suggest that this virus might have evolved uniquely in Japan over a long period of time and that the viral pathology of Japanese strains might be different from the strains found in other countries. Continuous surveillance is required to determine the importance of this virus and to characterize its evolution.

Detection of a novel sapelovirus in central nervous tissue of pigs with polioencephalomyelitis in the USA.

An approximately 3,000 finishing swine operation in the United States experienced an outbreak of an atypical neurologic disease in 11-weeks-old pigs with an overall morbidity of 20% and case fatality rate of 30%. The clinical onset and progression of signs in affected pigs varied but included inappetence, compromised ambulation, ataxia, incoordination, mental dullness, paresis, paralysis and decreased response to environmental stimuli. Tissues from affected pigs were submitted for diagnostic investigation. Histopathologic examination of the cerebrum, cerebellum and spinal cord revealed severe lymphoplasmacytic and necrotizing polioencephalomyelitis with multifocal areas of gliosis and neuron satellitosis, suggestive of a neurotropic viral infection. Bacterial pathogens were not isolated by culture of neurologic tissue from affected pigs. Samples tested by polymerase chain reaction (PCR) were negative for pseudorabies virus and atypical porcine pestivirus. Immunohistochemistry for porcine reproductive and respiratory syndrome virus, porcine circovirus and Listeria was negative. Porcine sapelovirus (PSV) was identified in spinal cord by a nested PCR used to detect porcine enterovirus, porcine teschovirus and PSV. Next-generation sequencing of brainstem and spinal cord samples identified PSV and the absence of other or novel pathogens. In addition, Sapelovirus A mRNA was detected in neurons and nerve roots of the spinal cord by in situ hybridization. The PSV is genetically novel with an overall 94% amino acid identity and 86% nucleotide identity to a recently reported sapelovirus from Korea. This is the first case report in the United States associating sapelovirus with severe polioencephalomyelitis in pigs.

Arbuscular mycorrhizal symbiosis regulates physiology and performance of Digitaria eriantha plants subjected to abiotic stresses by modulating antioxidant and jasmonate levels.

This study evaluates antioxidant responses and jasmonate regulation in Digitaria eriantha cv. Sudafricana plants inoculated (AM) and non-inoculated (non-AM) with Rhizophagus irregularis and subjected to drought, cold, or salinity. Stomatal conductance, photosynthetic efficiency, biomass production, hydrogen peroxide accumulation, lipid peroxidation, antioxidants enzymes activities, and jasmonate levels were determined. Stomatal conductance and photosynthetic efficiency decreased in AM and non-AM plants under all stress conditions. However, AM plants subjected to drought, salinity, or non-stress conditions showed significantly higher stomatal conductance values. AM plants subjected to drought or non-stress conditions increased their shoot/root biomass ratios, whereas salinity and cold caused a decrease in these ratios. Hydrogen peroxide accumulation, which was high in non-AM plant roots under all treatments, increased significantly in non-AM plant shoots under cold stress and in AM plants under non-stress and drought conditions. Lipid peroxidation increased in the roots of all plants under drought conditions. In shoots, although lipid peroxidation decreased in AM plants under non-stress and cold conditions, it increased under drought and salinity. AM plants consistently showed high catalase (CAT) and ascorbate peroxidase (APX) activity under all treatments. By contrast, the glutathione reductase (GR) and superoxide dismutase (SOD) activity of AM roots was lower than that of non-AM plants and increased in shoots. The endogenous levels of cis-12-oxophytodienoc acid (OPDA), jasmonic acid (JA), and 12-OH-JA showed a significant increase in AM plants as compared to non-AM plants. 11-OH-JA content only increased in AM plants subjected to drought. Results show that D. eriantha is sensitive to drought, salinity, and cold stresses and that inoculation with AM fungi regulates its physiology and performance under such conditions, with antioxidants and jasmonates being involved in this process.

Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany.

BACKGROUND: Jasmonates are important regulators in plant responses to biotic and abiotic stresses as well as in development. Synthesized from lipid-constituents, the initially formed jasmonic acid is converted to different metabolites including the conjugate with isoleucine. Important new components of jasmonate signalling including its receptor were identified, providing deeper insight into the role of jasmonate signalling pathways in stress responses and development. SCOPE: The present review is an update of the review on jasmonates published in this journal in 2007. New data of the last five years are described with emphasis on metabolites of jasmonates, on jasmonate perception and signalling, on cross-talk to other plant hormones and on jasmonate signalling in response to herbivores and pathogens, in symbiotic interactions, in flower development, in root growth and in light perception. CONCLUSIONS: The last few years have seen breakthroughs in the identification of JASMONATE ZIM DOMAIN (JAZ) proteins and their interactors such as transcription factors and co-repressors, and the crystallization of the jasmonate receptor as well as of the enzyme conjugating jasmonate to amino acids. Now, the complex nature of networks of jasmonate signalling in stress responses and development including hormone cross-talk can be addressed.

Jasmonate biosynthesis in Arabidopsis thaliana--enzymes, products, regulation.

Among the plant hormones jasmonic acid and related derivatives are known to mediate stress responses and several developmental processes. Biosynthesis, regulation, and metabolism of jasmonic acid in Arabidopsis thaliana are reviewed, including properties of mutants of jasmonate biosynthesis. The individual signalling properties of several jasmonates are described.

Novel mode of hormone induction of tandem tomato invertase genes in floral tissues.

The genomic organization of two extracellular invertase genes from tomato (Lin5 and Lin7), which are linked in a direct tandem repeat, and their tissue-specific and hormone-inducible expression are shown. Transient expression analysis of Lin5 promoter sequences fused to the beta-glucuronidase (GUS) reporter gene (uidA) demonstrates a specific expression of Lin5 during tomato fruit development. A Lin5 promoter fragment was fused to the truncated nos promoter to analyse hormone induction via GUS reporter gene activity in transiently transformed tobacco leaves. A specific up-regulation of GUS activity conferred by this Lin5 promoter fragment in response to gibberellic acid (GA), auxin and abscisic acid (ABA) treatment was observed, indicating a critical role of the regulation of Lin5 by phytohormones in tomato flower and fruit development. In situ hybridization analysis of Lin7 shows a high tissue-specific expression in tapetum and pollen. These results support an important role for Lin5 and Lin7 extracellular invertases in the development of reproductive organs in tomato and contribute to unravel the underlying regulatory mechanisms.

Histochemical analysis of phenylphenalenone-related compounds in Xiphidium caeruleum (Haemodoraceae).

Phenylphenalenones represent a typical group of secondary metabolites of the Haemodoraceae. Some of these phenolic compounds show organ-specific distribution within the plant. However, detailed information on cellular localisation is still lacking. To this end, confocal laser-scanning microscopy, microspectral photometry and high-performance liquid chromatography were used to study the tissue localisation of phenylphenalenone-type compounds in Xiphidium caeruleum Aubl. From the autofluorescence potential of these compounds, specific distribution of allophanylglucosides and non-glucosidic compounds of the phenylphenalenone-type in distinct cells of the roots (apical meristem, cortex, cap, epidermis) and the shoot system was revealed. Fluorescence enhancement using "Naturstoff reagent A" (NA) indicated the occurrence of NA-positive natural products in the vacuoles of leaf epidermal cells. The present results provide new insights into the possible functions of phenylphenalenone-related compounds in the context of their localisation. Additionally, the advantages and limitations of the techniques are discussed.

Purification, characterization, immunolocalization and structural analysis of the abundant cytoplasmic beta-amylase from Calystegia sepium (hedge bindweed) rhizomes.

An abundant catalytically active beta-amylase (EC 3.2.1.2) was isolated from resting rhizomes of hedge bindweed (Calystegia sepium). Biochemical analysis of the purified protein, molecular modeling, and cloning of the corresponding gene indicated that this enzyme resembles previously characterized plant beta-amylases with regard to its amino-acid sequence, molecular structure and catalytic activities. Immunolocalization demonstrated that the beta-amylase is exclusively located in the cytoplasm. It is suggested that the hedge bindweed rhizome beta-amylase is a cytoplasmic vegetative storage protein.

Reorganization of tobacco root plastids during arbuscule development.

In the present paper we analyzed plastid populations labeled by the green fluorescent protein in non-mycorrhizal and mycorrhizal roots of tobacco (Nicotiana tahacum L.). We show by confocal laser scanning microscopy (i) a dramatic increase in these plastids in mycorrhizal roots and (ii) the formation of dense plastid networks covering the symbiotic interface of the arbuscular mycorrhiza, the arbuscule. These cytological observations point to an important role of root cortical cell plastids in the functioning of arbuscular mycorrhizal symbiosis.

Tomato ribonuclease LX with the functional endoplasmic reticulum retention motif HDEF is expressed during programmed cell death processes, including xylem differentiation, germination, and senescence.

We have studied the subcellular localization of the acid S-like ribonuclease (RNase) LX in tomato (Lycopersicon esculentum Mill.) cells using a combination of biochemical and immunological methods. It was found that the enzyme, unexpectedly excluded from highly purified vacuoles, accumulates in the endoplasmic reticulum. The evidence that RNase LX is a resident of the endoplasmic reticulum (ER) is supported by an independent approach showing that the C-terminal peptide HDEF of RNase LX acts as an alternative ER retention signal in plants. For functional testing, the cellular distribution of chimeric protein constructs based on a marker protein, Brazil nut (Bertholletia excelsa) 2S albumin, was analyzed immunochemically in transgenic tobacco (Nicotiana tabacum) plants. Here, we report that the peptide motif is necessary and sufficient to accumulate 2S albumin constructs of both vacuolar and extracellular final destinations in the ER. We have shown immunochemically that RNase LX is specifically expressed during endosperm mobilization and leaf and flower senescence. Using immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. These results support a physiological function of RNase LX in selective cell death processes that are also thought to involve programmed cell death. It is assumed that RNase LX accumulates in an ER-derived compartment and is released by membrane disruption into the cytoplasma of those cells that are intended to undergo autolysis. These processes are accompanied by degradation of cellular components supporting a metabolic recycling function of the intracellular RNase LX.

Iris bulbs express type 1 and type 2 ribosome-inactivating proteins with unusual properties.

Two closely related lectins from bulbs of the Dutch iris (Iris hollandica var. Professor Blaauw) have been isolated and cloned. Both lectins, called Iris agglutinin b and Iris agglutinin r, possess N-glycosidase activity and share a high sequence similarity with previously described type 2 ribosome-inactivating proteins (RIP). However, these lectins show only 57% to 59% sequence identity to a previously characterized type 1 RIP from iris, called IRIP. The identification of the iris lectins as type 2 RIP provides unequivocal evidence for the simultaneous occurrence of type 1 and type 2 RIP in iris bulbs and allowed a detailed comparison of type 1 and type 2 RIP from a single plant, which provides further insight into the molecular evolution of RIP. Binding studies and docking experiments revealed that the lectins exhibit binding activity not only toward Gal/N-acetylgalactosamine, but also toward mannose, demonstrating for the first time that RIP-binding sites can accommodate mannose.

Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles.

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis(+)12-oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato. In stems, young leaves and young flowers, AOC mRNA accumulates to a low level, contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g-1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue-specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue-specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink-source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin-mediated wound response of tomato.

The galactose-binding and mannose-binding jacalin-related lectins are located in different sub-cellular compartments.

A galactose-specific and a mannose-specific lectin of the family of the jacalin-related lectins have been localized by immunofluorescence microscopy. The present localization studies provide for the first time unambiguous evidence for the cytoplasmic location of the mannose-specific jacalin-related lectin from rhizomes of Calystegia sepium, which definitely differs from the vacuolar location of the galactose-specific jacalin from Artocarpus integrifolia. These observations support the hypothesis that the galactose-specific jacalin-related lectins evolved from their mannose-specific homologues through the acquisition of vacuolar targeting sequences.

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies.

A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid.

Molecular cloning of allene oxide cyclase. The enzyme establishing the stereochemistry of octadecanoids and jasmonates.

Allene oxide cyclase (EC ) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5'-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13S enantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.

Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development.

Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full-length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co-purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX-derived 9- as well as 13-hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na-salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up-regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis.

Nuclear gamma-tubulin during acentriolar plant mitosis.

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that gamma-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of gamma-tubulin in nuclei increased during the G(2) phase, when cells are synchronized or sorted for particular phases of the cell cycle. gamma-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear gamma-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of gamma-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

Cloning and expression of a new cDNA from monocotyledonous plants coding for a diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from barley (Hordeum vulgare).

From a cDNA library generated from mRNA of white leaf tissues of the ribosome-deficient mutant 'albostrians' of barley (Hordeum vulgare cv. Haisa) a cDNA was isolated carrying 54.2% identity to a recently published cDNA which codes for the diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) hydrolase of Lupinus angustifolius (Maksel et al. (1998) Biochem. J. 329, 313-319), and 69% identity to four partial peptide sequences of Ap4A hydrolase of tomato. Overexpression in Escherichia coli revealed a protein of about 19 kDa, which exhibited Ap4A hydrolase activity and cross-reactivity with an antibody raised against a purified tomato Ap4A hydrolase (Feussner et al. (1996) Z. Naturforsch. 51c, 477-486). Expression studies showed an mRNA accumulation in all organs of a barley seedling. Possible functions of Ap4A hydrolase in plants will be discussed.