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.

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.

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.

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.

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.

Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley (Hordeum vulgare L.) leaves.

Leaves of barley (Hordeum vulgare L. cv. Salome) treated with jasmonic acid (JA), its methyl ester (JM), or its amino acid conjugates exhibit up-regulation of specific genes and down-regulation of house-keeping genes. This transcriptional regulation exhibits several specificities. (i) The (-)-enantiomers are more active, and conjugates are mainly active if they carry an L-amino acid moiety. (ii) The various JA-responsive genes respond differentially to enantiomeric and chiralic forms. (iii) Both JA and its amino acid conjugates exhibiting no or negligible interconversion induce/repress genes.

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.

Compartmentation of uridine 5'-triphosphate occurs during synthesis of cytoplasmic and mitochondrial ribosomal RNAs of cultured tomato cells but not during synthesis of cytoplasmic ribosomal and transfer RNAs.

Compartmentation of uridine 5'-triphosphate (UTP) was studied during the nucleolar synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and the synthesis of cytoplasmic transfer RNA (cyt-tRNA) in the nuclear matrix as well as the synthesis of mitochondrial ribosomal RNA (mt-rRNA) in tomato (Lycopersicon esculentum Mill. cv. Lukullus) cell-suspension culture using the approach of Wiegers et al. (Eur. J. Biochem. 64, 535-540, 1976). Before measurements were made, it was ensured that: (i) there was steady-state labeling of all RNAs studied as well as UTP; (ii) there was stability of cyt-tRNA and cyt-rRNA; (iii) there was no label randomization through degradation of [(3)H]uridine; (iv) there were significant differences in the specific radioactivity of UTP, the final immediate precursor of RNA, after supplying the cells with two different exogenous [(3)H]uridine concentrations.By comparing the steady-state specific radioactivity of UTP with that of cyt-tRNA and cyt-18S rRNA during constant [(3)H]uridine supply, we found that the three molecules had equal specific radioactivities which, however, differed significantly from that of the mt-rRNA. With a 20-fold higher uridine concentration, i.e. a 20-fold lower specific radioactivity of exogenous [(3)H]uridine, the specific radioactivity of cyt-rRNA, cyt-tRNA and UTP decreased proportionally whereas that of mt-RNA increased. These results argue against different UTP pools during synthesis of cyt-rRNA and cyt-tRNA, but indicate compartmentation of UTP during rRNA synthesis in the nucleus and the mitochondria of tomato cells.

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.

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.

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.

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.

Methyl jasmonate induces an O-methyltransferase in barley.

We have previously described a truncated cDNA clone for a barley (Hordeum vulgare L. cv. Salome) jasmonate regulated gene, JRG5, which shows homology to caffeic acid O-methyltransferase (COMT). A cDNA encompassing the coding region was amplified by PCR and cloned for overexpression in E. coli. Western blot analyses indicate that the recombinant protein crossreacts with the antibodies directed against the tobacco class II OMT and only weakly with the antibodies for the tobacco class I OMT. An immunoreactive band in the protein extract of jasmonate-treated leaf segments suggests that JRG5 transcripts that accumulate after jasmonate treatment are also translated. Specific methylating activities on caffeic acid and catechol were obtained from the recombinant protein through renaturation of protein extracted from inclusion bodies or from bacteria grown and induced at low temperature. On Northern blots, the JRG5 transcripts were detected in the leaf sheath but not the leaf lamina; stem, root or inflorescence and accumulated in leaf segments after jasmonate application. Several hormone or stress treatments did not induce JRG5 mRNA accumulation. This includes sorbitol stress which is known to lead to enhanced endogenous jasmonate levels and the implications for jasmonate signaling are discussed. Based on quantitative measurements and fluorescence microscopy, jasmonate-induced accumulation of ferulic acid and phenolic polymers in the cell wall were detected and the possibility of cell wall strengthening mediated through phenolic crosslinks is discussed.

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.

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.

Developmental and tissue-specific expression of JIP-23, a jasmonate-inducible protein of barley.

Developmental expression of a 23 kDa jasmonate-induced protein (JIP-23) of barley leaves (Hordeum vulgare cv. Salome) was studied by measuring the time-dependent accumulation of transcript and protein during germination. Tissue-specific expression of JIP-23 was analyzed immunocytochemically and by in situ hybridizations, respectively. During seed germination JIP-23 mRNA was found to accumulate transiently with a maximum at 32 h, whereas the protein was steadily detectable after the onset of expression. The occurrence of new isoforms of JIP-23 during germination in comparison to jasmonate-treated leaves suggests, that the JIP-23 gene family of barley is able to express different subsets of isoforms dependent on the developmental stage. JIP-23 and its transcript were found mainly in the scutellum, the scutellar nodule and in lower parts of the primary leaf of 6 days old seedings. All these tissues exhibited high levels of endogenous jasmonates. In situ hybridization revealed specific accumulation of JIP-23 mRNA in companion cells of the phloem in the nodule plate of the scutellum. In accordance with that, JIP-23 was detected immunocytochemically in phloem cells of the root as well as of the scutellar nodule and in parenchymatic cells of the scutellum. The cell type-specific occurrence of JIP-23 was restricted to cells, which are known to be highly stressed osmotically by active solute transport. This observation suggests, that the expression of this protein might be a response to osmotic stress during development.