bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of ITS and IGS1 regions for strain typing of clinical and non-clinical isolates of Pichia anomala
Authors: Sutar Rajeshwari , David Joseph K. , Ganesan K. , Ghosh Anup K. , Singhi Sunit , Chakrabarti Arunaloke , Bachhawat Anand K.
Published: January 2004

Pichia anomalais an emerging nosocomial pathogen and there is a need for methods that distinguish between differentP. anomalastrains. In the typing of several clinical as well as non-clinicalP. anomalastrains, the sequence variation of the internal transcribed spacer (ITS) was found to be inadequate for typing purposes. The intergenic spacer 1 (IGS1) region of the rDNA of severalP. anomalastrains was therefore investigated in detail. The IGS1 region (which varied from 1213 to 1231 bp in length) was interspersed with repeats and had more variation than the ITS regions. Comparative analysis in cases where analysis by the ITS was ambiguous clearly revealed the IGS1 region to be a more discriminatory tool in the typing ofP. anomalastrains.
Clonal similarity of salivary and nasopharyngeal Fusobacterium nucleatum in infants with acute otitis media experience
Authors: Haraldsson Gunnsteinn , Holbrook W. Peter , Könönen Eija
Published: January 2004

The environment of an infant's nasopharynx during acute otitis media (AOM) favours the growth of anaerobic bacteria, which can be recovered frequently during infection, but hardly at all if the infant is healthy. The aim of this investigation was to identify the potential source and inoculation route of anaerobes that were present in the nasopharynx. Eleven Fusobacterium nucleatum isolates that were collected through the nasal cavity from the nasopharynx of eight infants with a history of AOM, and 161 F. nucleatum isolates from the saliva of the same infants, were typed to the clonal level by using arbitrarily primed PCR (AP-PCR). In five of the eight infants examined, identical AP-PCR types were found among nasopharyngeal and salivary isolates. As anaerobes seem to be present only transiently in the nasopharynx and salivary contamination of the nasopharyngeal samples can be excluded, this observation indicates that the source of nasopharyngeal anaerobes is the oral cavity and that saliva is their transmission vehicle.
Long-term predominance of two pan-European clones among multi-resistant Acinetobacter baumannii strains in the Czech Republic
Authors: Nemec Alexandr , Dijkshoorn Lenie , van der Reijden Tanny J.K.
Published: January 2004

In a recent study, a large proportion of multi-drug-resistant (MDR) Acinetobacter baumannii strains that were isolated from hospitalized patients in the Czech Republic was found to belong to two major groups (A and B). These groups appeared to be similar to epidemic clones I and II, respectively, which were identified previously among outbreak strains from north-western European hospitals. The aim of the present study was to assess in detail the genetic relatedness of Czech A. baumannii strains and those of epidemic clones I and II by using ribotyping with HindIII and HincII and by AFLP fingerprinting. The study collection included 70 MDR strains that were isolated in 30 Czech hospitals in 1991–2001, 15 susceptible Czech strains from 1991 to 1996 and 13 reference strains of clones I and II from 1982 to 1990. One major HindIII/HincIII ribotype (R1-1) was observed in 38 MDR Czech strains and eight reference strains of clone I, whereas another major ribotype (R2-2) was observed in 11 MDR Czech strains and in three reference strains of clone II. A selection of 59 Czech strains (representative of all ribotypes) and the 13 reference strains were investigated by AFLP fingerprinting. At a clustering level of 83 %, two large clusters could be distinguished: cluster 1 included all reference strains of clone I and 25 MDR Czech strains, whilst cluster 2 contained all reference strains of clone II and 11 MDR Czech strains. There was a clear correlation between the groupings by AFLP analysis and by ribotyping, as all strains with ribotype R1-1 and four strains with slightly different ribotypes were found in AFLP cluster 1, whereas all strains with ribotype R2-2 and seven strains with similar ribotypes were in AFLP cluster 2. Thus, 41 and 21 MDR Czech strains could be classified as belonging to clones I and II, respectively. The remaining eight MDR and 15 susceptible strains were highly heterogeneous and were distinct from clones I and II by both AFLP fingerprinting and ribotyping. These results indicate that the two predominant groups observed among MDR Czech A. baumannii strains from the 1990s are genetically congruent with the north-western European epidemic clones that were found in the 1980s. Recognition of these clinically relevant, widespread clones is important in infection prevention and control; they are also interesting subjects to study genetic mechanisms that give rise to their antibiotic resistance and epidemic behaviour.
Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing: genotype spectrum of patients from the west German area
Authors: Speich Norbert , Schmitt Christoph , Bollmann Reinhard , Bollmann Magdolna
Published: January 2004

Human papillomaviruses (HPVs) are aetiological agents for cervical cancer. More than 70 different HPV types that infect genital mucosa have been found. In order to develop a sensitive and specific detection and typing assay, a PCR/direct sequencing approach was used. Two pairs of consensus primers were used for amplification of HPV DNA and the PCR products obtained were analysed by automated sequencing. Sequences were compared with those in GenBank by using the blast program. In this study, 2916 cytological samples were screened for HPV, as well as for triage. Nine hundred and forty-eight (32.5 %) samples were positive for HPV, of which 134 harboured more than one HPV type. Of the 948 PCR-positive samples, 648 were typed. Thirty-nine different HPV types were identified by sequencing. The two most frequently found HPV types, 16 and 31, together accounted for 36.3 % of the sequences (26.2 and 10.1 %, respectively). This group was followed by HPV types 6 (5.7 %), 18 (5.3 %), 58 (4.5 %), 61 (4.5 %), 53 (4.4 %), 42 (4.3 %) and 51 (4.0 %). All other types were detected at frequencies <4 % and eight types were detected only once. PCR/direct sequencing is a reliable method for routine detection of HPV in cytological samples. The data presented here suggest a complex distribution of HPV types in the population tested. The results accentuate the importance of PCR-based techniques in HPV diagnosis, as hybridization-based methods can only detect a limited number of infections. This method can also be applied easily to the analysis of tissue samples and it therefore also allows type-specific follow-up of women who have been treated for cervical intraepithelial neoplasia.
Mycobacterium tuberculosis isolates belonging to katG gyrA group 2 are associated with clustered cases of tuberculosis in Italian patients
Authors: Dolzani L. , Rosato M. , Sartori B. , Banfi E. , Lagatolla C. , Predominato M. , Fabris C. , Tonin E. , Gombac F. , Monti-Bragadin C.
Published: January 2004

Fifty-one consecutive isolates of Mycobacterium tuberculosis, collected during a 2-year period in the north-east of Italy, were subjected to IS6110-RFLP analysis to detect the presence of clusters and assigned to one of the three genotypic groups delineated by single nucleotide polymorphisms in the genes katG and gyrA. All the isolates collected from the local population belonged to group 2 or 3, while group 1 isolates were found only in specimens collected from African immigrants. Clustered cases of tuberculosis, which are likely to be related to recently transmitted infection, were found to be significantly associated with katG gyrA group 2. In the local situation, strains belonging to this group may therefore present a higher risk of transmission.
Chlorate: a reversible inhibitor of proteoglycan sulphation in Chlamydia trachomatis-infected cells
Authors: Fadel Sanaa , Eley Adrian
Published: January 2004

Sulphated glycosaminoglycans, such as heparan sulphate, have been shown to be essential for the infectivity of many organisms. The aims of this study were to verify the role of sulphated glycosaminoglycans in chlamydial infection and to investigate whether they are present on chlamydia or chlamydial host cells. The effect of undersulphation of host cells and chlamydial elementary bodies was examined using sodium chlorate. Also studied was whether any inhibitory effect was reversible. The results strongly suggest that Chlamydia trachomatis does not produce heparan sulphate and that heparan sulphate of the host cell is necessary and sufficient to mediate chlamydial infection. The essential role played by the sulphate constituents of the host-cell glycosaminoglycan in the infectivity of LGV serovars, and to a lesser extent of serovar E, was also confirmed.
Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) regulates triglyceride metabolism by activation of the nuclear receptor FXR
Authors: Zhang Yanqiao , Castellani Lawrence W. , Sinal Christopher J. , Gonzalez Frank J. , Edwards Peter A.
Published: January 2004

Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) has been shown to regulate adaptive thermogenesis and glucose metabolism. Here we show that PGC-1α regulates triglyceride metabolism through both farnesoid X receptor (FXR)-dependent and -independent pathways. PGC-1α increases FXR activity through two pathways: (1) it increases FXR mRNA levels by coactivation of PPARγ and HNF4α to enhance FXR gene transcription; and (2) it interacts with the DNA-binding domain of FXR to enhance the transcription of FXR target genes. Ectopic expression of PGC-1α in murine primary hepatocytes reduces triglyceride secretion by a process that is dependent on the presence of FXR. Consistent with these in vitro studies, we demonstrate that fasting induces hepatic expression of PGC-1α and FXR and results in decreased plasma triglyceride levels in wild-type but not in FXR-null mice. Our data suggest that PGC-1α plays an important physiological role in maintaining energy homeostasis during fasting by decreasing triglyceride production/secretion while it increases fatty acid β-oxidation to meet energy needs.
p63 is the molecular switch for initiation of an epithelial stratification program
Authors: Koster Maranke I. , Kim Soeun , Mills Alea A. , DeMayo Francesco J. , Roop Dennis R.
Published: January 2004

Development of stratified epithelia, such as the epidermis, requires p63 expression. The p63 gene encodes isoforms that contain (TA) or lack (ΔN) a transactivation domain. We demonstrate that TAp63 isoforms are the first to be expressed during embryogenesis and are required for initiation of epithelial stratification. In addition, TAp63 isoforms inhibit terminal differentiation, suggesting that TAp63 isoforms must be counterbalanced by ΔNp63 isoforms to allow cells to respond to signals required for maturation of embryonic epidermis. Our data demonstrate that p63 plays a dual role: initiating epithelial stratification during development and maintaining proliferative potential of basal keratinocytes in mature epidermis.
The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3′UTR
Authors: Vella Monica C. , Choi Eun-Young , Lin Shin-Yi , Reinert Kristy , Slack Frank J.
Published: January 2004

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3′UTR of the mRNA of its target gene, lin-41, to down-regulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3′UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for down-regulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3′UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo.
A Glutamate Receptor–Interacting Protein homolog organizes muscle guidance in Drosophila
Authors: Swan Laura E. , Wichmann Carolin , Prange Ulrike , Schmid Andreas , Schmidt Manuela , Schwarz Tobias , Ponimaskin Evgeni , Madeo Frank , Vorbrüggen Gerd , Sigrist Stephan J.
Published: January 2004

During Drosophila embryogenesis, developing muscles extend growth-cone–like structures to navigate toward specific epidermal attachment sites. Here, we show that the homolog of Glutamate Receptor–Interacting Proteins (DGrip) acts as a key component of proper muscle guidance. Mutations in dgrip impair patterning of ventral longitudinal muscles (VLMs), whereas lateral transverse muscles (LTMs) that attach to intrasegmental attachment sites develop normally. Myoblast fusion, stabilization of muscle contacts, and general muscle function are not impaired in the absence of DGrip. Instead, the proper formation of cellular extensions during guidance fails in dgrip mutant VLMs. DGrip protein concentrates at the ends of VLMs while these muscles guide toward segment border attachment sites. Conversely, LTMs overexpressing DGrip form ectopic cellular extensions that can cause attachment of these muscles to other muscles at segment borders. Our data suggest that DGrip participates in the reception of an attractive signal that emanates from the epidermal attachment sites to direct the motility of developing muscles. This dgrip phenotype should be valuable to study mechanistic principles of Grip function.
Induction of Salt and Osmotic Stress Tolerance by Overexpression of an Intracellular Vesicle Trafficking Protein AtRab7 (AtRabG3e)
Authors: Mazel Alexander , Leshem Yehoram , Tiwari Budhi Sagar , Levine Alex
Published: January 2004

Adaptation to stress requires removal of existing molecules from various cellular compartments and replacing them with new ones. The transport of materials to and from the specific compartments involved in the recycling and deposition of macromolecules is carried out by an intracellular vesicle trafficking system. Here, we report the isolation of a vesicle trafficking-regulating gene, AtRabG3e (formerly AtRab7), from Arabidopsis. The gene was induced during programmed cell death after treatment of intact leaves with superoxide and salicylic acid or infection with necrogenic pathogens. Transgenic plants that expressed the AtRabG3e gene under the constitutive 35S promoter from cauliflower mosaic virus exhibited accelerated endocytosis in roots, leaves, and protoplasts. The transgenic plants accumulated sodium in the vacuoles and had higher amounts of sodium in the shoots. The transgenic plants also showed increased tolerance to salt and osmotic stresses and reduced accumulation of reactive oxygen species during salt stress. These results imply that vesicle trafficking plays an important role in plant adaptation to stress, beyond the housekeeping function in intracellular vesicle trafficking.
Binding of Sulfonylurea by AtMRP5, an Arabidopsis Multidrug Resistance-Related Protein That Functions in Salt Tolerance
Authors: Lee Eun Kyung , Kwon Minjae , Ko Jae-Heung , Yi Hochul , Hwang Moo Gak , Chang Soochul , Cho Myeon Haeng
Published: January 2004

Recently, a new member of the ABC transporter superfamily of Arabidopsis, AtMRP5, was identified and characterized. In the present work, we found that AtMRP5 can bind specifically to sulfonurea when it is expressed in HEK293 cells. We also present evidence for a new role of AtMRP5 in the salt stress response of Arabidopsis. We used reverse genetics to identify an Arabidopsis mutant (atmrp5-2) in which the AtMRP5 gene was disrupted by transferred DNA insertion. In root-bending assays using Murashige and Skoog medium supplemented with 100 mm NaCl, root growth of atmrp5-2 was substantially inhibited in contrast to the almost normal growth of wild-type seedlings. This hypersensitive response of the atmrp5-2 mutant was not observed during mannitol treatment. The root growth of the wild-type plant grown in Murashige and Skoog medium supplemented with the MRP inhibitor glibenclamide and NaCl was inhibited to a very similar extent as the root growth of atmrp5-2 grown in NaCl alone. The Na+-dependent reduction of root growth of the wild-type plant in the presence of glibenclamide was partially restored by diazoxide, a known K+ channel opener that reverses the inhibitory effects of sulfonylureas in animal cells. Moreover, the atmrp5-2 mutant was defective in 86Rb+ uptake. When seedlings were treated with 100 mm NaCl, atmrp5-2 seedlings accumulated less K+ and more Na+ than those of the wild type. These observations suggest that AtMRP5 is a putative sulfonylurea receptor that is involved in K+ homeostasis and, thus, also participates in the NaCl stress response.
Differential Expression of Sucrose Transporter and Polyol Transporter Genes during Maturation of Common Plantain Companion Cells
Authors: Ramsperger-Gleixner Martina , Geiger Dietmar , Hedrich Rainer , Sauer Norbert
Published: January 2004

The cDNAs of two sorbitol transporters, common plantain (Plantago major) polyol transporter (PLT) 1 and 2 (PmPLT1 and PmPLT2), were isolated from a vascular bundle-specific cDNA library from common plantain, a dicot plant transporting Suc plus sorbitol in its phloem. Here, we describe the kinetic characterization of these sorbitol transporters by functional expression in Brewer's yeast (Saccharomyces cerevisiae) and in Xenopus sp. oocytes and for the first time the localization of plant PLTs in specific cell types of the vascular tissue. In the yeast system, both proteins were shown to be uncoupler sensitive and could be characterized as low-affinity and low-specificity polyol symporters. The K  m value for the physiological substrate sorbitol is 12 mm for PmPLT1 and even higher for PmPLT2, which showed an almost linear increase in sorbitol transport rates up to 20 mm. These data were confirmed in the Xenopus sp. system, where PmPLT1 was analyzed in detail and characterized as a H+ symporter. Using peptide-specific polyclonal antisera against PmPLT1 or PmPLT2 and simultaneous labeling with the monoclonal antiserum 1A2 raised against the companion cell-specific PmSUC2 Suc transporter, both PLTs were localized to companion cells of the phloem in common plantain source leaves. These analyses revealed two different types of companion cells in the common plantain phloem: younger cells expressing PmSUC2 at higher levels and older cells expressing lower levels of PmSUC2 plus both PLT genes. The putative role of these low-affinity transporters in phloem loading is discussed.
Gene Trapping with Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes
Authors: Alvarado Martha C. , Zsigmond Laura M. , Kovács Izabella , Cséplö Ágnes , Koncz Csaba , Szabados László M.
Published: January 2004

To monitor the expression of T-DNA-tagged plant genes in vivo, a collection of 20,261 transgenic lines of Arabidopsis (Columbia-0) were generated with the promoter trap vector pTluc, which carries a promoterless firefly luc (luciferase) reporter gene linked to the right T-DNA border. By detection of bioluminescence in 3-week-old seedlings, 753 lines were identified showing constitutive, organ-specific, and stress-responsive luciferase expression patterns. To facilitate the identification of well-defined luciferase expression patterns, a pooled seed stock was established. Several lines showed sugar, salt, and abscisic acid (ABA)-inducible luciferase activity. Segregation analysis of 215 promoter trap lines indicated that about 50% of plants contained single insertions, whereas 40% carried two and 10% carried three or more T-DNA tags. Sequencing the T-DNA insert junctions isolated from 17 luciferase-expressing lines identified T-DNA tags in 5′- and 3′-transcribed domains and translational gene fusions generated by T-DNA insertions in exons and introns of Arabidopsis genes. Tissue specific expression of eight wild-type Arabidopsis genes was confirmed to be similar to the luminescence patterns observed in the corresponding luciferase-tagged lines. Here, we describe the characterization of a transcriptional luc reporter gene fusion with the WBC-type ABC transporter gene At1g17840. Expression of wild-type and luciferase-tagged At1g17840 alleles revealed similar induction by salt, glucose, and ABA treatments and gibberellin-mediated down-regulation of ABA-induced expression. These results illustrate that luciferase gene traps are well suited for monitoring the expression of stress-responsive Arabidopsis genes in vivo.
An Invertase Inhibitor from Maize Localizes to the Embryo Surrounding Region during Early Kernel Development
Authors: Bate Nicholas J. , Niu Xiping , Wang Yuwen , Reimann Kellie S. , Helentjaris Timothy G.
Published: January 2004

Invertase activity is thought to play a regulatory role during early kernel development by converting sucrose originating from source leaves into hexoses to support cell division in the endosperm and embryo. Invertases are regulated at the posttranslational level by small protein inhibitors, INVINHs. We found that in maize (Zea mays), an invertase inhibitor homolog (ZM-INVINH1) is expressed early in kernel development, between 4 and 7 d after pollination. Invertase activity is reduced in vitro in the presence of recombinant ZM-INVINH1, and inhibition is attenuated by pre-incubation with sucrose. The presence of a putative signal peptide, fractionation experiments, and ZM-INVINH1::green fluorescent protein fusion experiments indicate that the protein is exported to the apoplast. Moreover, association of ZM-INVINH1 with the glycoprotein fraction by concanavalin A chromatogaphy suggests that ZM-INVINH1 interacts with an apoplastic invertase during early kernel development. ZM-INVINH1 was localized to the embryo surrounding region by in situ analysis, suggesting that this region forms a boundary, compartmentalizing apoplast invertase activity to allow different embryo and endosperm developmental rates.
Tobacco Nectarin V Is a Flavin-Containing Berberine Bridge Enzyme-Like Protein with Glucose Oxidase Activity
Authors: Carter Clay J. , Thornburg Robert W.
Published: January 2004

Ornamental tobacco (Nicotiana langsdorffii X N. sanderae) secretes a limited array of proteins (nectarins) into its floral nectar. Careful sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of tobacco nectar revealed that a broad protein band from 61 to 65 kD actually consists of five discrete protein bands. N-terminal sequencing and tryptic peptide mass spectrometry fingerprint analysis demonstrated that the upper three bands are isoforms of the same protein, NEC5 (Nectarin V), whereas the lower two bands, NEC4 (Nectarin IV), are related to each other but not to NEC5. Reverse transcription-polymerase chain reaction (RT-PCR) based upon N-terminal sequence of NEC5 generated a short cDNA that encoded the N terminus of the NEC5 protein. Two rounds of inverse-PCR using genomic DNA permitted the isolation of approximately one-half of the coding region of the nec5 gene along with 787 nucleotides of the 5′-flanking region. This DNA fragment was used as a probe to isolate a near full-length nec5 clone from a nectary-derived cDNA library. BLAST analysis identified the nec5 cDNA as a berberine bridge enzyme-like protein. Approximately 40% of the cDNA sequence corresponded to peptides that were identified by tryptic peptide mass spectrometry fingerprint analysis of the NEC5 protein, thereby confirming that this cDNA encoded the NEC5 protein. In-gel assays also demonstrated that NEC5 contains a covalently linked flavin, and it possesses glucose oxidase activity. RT-PCR-based expression analyses showed that nec5 expression is limited exclusively to the nectary gland during late stages of floral development.
The Galactose Residues of Xyloglucan Are Essential to Maintain Mechanical Strength of the Primary Cell Walls in Arabidopsis during Growth
Authors: Peña María J. , Ryden Peter , Madson Michael , Smith Andrew C. , Carpita Nicholas C.
Published: January 2004

In land plants, xyloglucans (XyGs) tether cellulose microfibrils into a strong but extensible cell wall. The MUR2 and MUR3 genes of Arabidopsis encode XyG-specific fucosyl and galactosyl transferases, respectively. Mutations of these genes give precisely altered XyG structures missing one or both of these subtending sugar residues. Tensile strength measurements of etiolated hypocotyls revealed that galactosylation rather than fucosylation of the side chains is essential for maintenance of wall strength. Symptomatic of this loss of tensile strength is an abnormal swelling of the cells at the base of fully grown hypocotyls as well as bulging and marked increase in the diameter of the epidermal and underlying cortical cells. The presence of subtending galactosyl residues markedly enhance the activities of XyG endotransglucosylases and the accessibility of XyG to their action, indicating a role for this enzyme activity in XyG cleavage and religation in the wall during growth for maintenance of tensile strength. Although a shortening of XyGs that normally accompanies cell elongation appears to be slightly reduced, galactosylation of the XyGs is not strictly required for cell elongation, for lengthening the polymers that occurs in the wall upon secretion, or for binding of the XyGs to cellulose.
Histochemical Analysis Reveals Organ-Specific Quantitative Trait Loci for Enzyme Activities in Arabidopsis
Authors: Sergeeva Lidiya I. , Vonk Jenneke , Keurentjes Joost J.B. , van der Plas Linus H.W. , Koornneef Maarten , Vreugdenhil Dick
Published: January 2004

To identify genetic loci involved in the regulation of organ-specific enzyme activities, a specific histochemical staining protocol was used in combination with quantitative trait locus (QTL) analysis. Using phosphoglucomutase (PGM) as an example, it is shown that enzyme activity can specifically, and with high resolution, be visualized in non-sectioned seedlings of Arabidopsis. The intensities of staining were converted to quantitative data and used as trait for QTL analysis using Landsberg erecta × Cape Verde Islands recombinant inbred lines. Independently, PGM activities were quantified in whole-seedling extracts, and these data were also used for QTL analysis. On the basis of extract data, six significant (P < 0.05) loci affecting PGM activity were found. From the histochemical data, one or more specific QTLs were found for each organ analyzed (cotyledons, shoot apex, hypocotyl, root, root neck, root tip, and root hairs). Loci detected for PGM activity in extracts colocated with loci for histochemical staining. QTLs were found coinciding with positions of (putative) PGM genes but also at other positions, the latter ones supposedly pointing toward regulatory genes. Some of this type of loci were also organ specific. It is concluded that QTL analysis based on histochemical data is feasible and may reveal organ-specific loci involved in the regulation of metabolic pathways.
Pseudomonas aeruginosa-Plant Root Interactions. Pathogenicity, Biofilm Formation, and Root Exudation
Authors: Walker Travis S. , Bais Harsh Pal , Déziel Eric , Schweizer Herbert P. , Rahme Laurence G. , Fall Ray , Vivanco Jorge M.
Published: January 2004

Pseudomonas aeruginosa is an opportunistic human pathogen capable of forming a biofilm under physiological conditions that contributes to its persistence despite long-term treatment with antibiotics. Here, we report that pathogenic P. aeruginosa strains PAO1 and PA14 are capable of infecting the roots of Arabidopsis and sweet basil (Ocimum basilicum), in vitro and in the soil, and are capable of causing plant mortality 7 d postinoculation. Before plant mortality, PAO1 and PA14 colonize the roots of Arabidopsis and sweet basil and form a biofilm as observed by scanning electron microscopy, phase contrast microscopy, and confocal scanning laser microscopy. Upon P. aeruginosa infection, sweet basil roots secrete rosmarinic acid (RA), a multifunctional caffeic acid ester that exhibits in vitro antibacterial activity against planktonic cells of both P. aeruginosa strains with a minimum inhibitory concentration of 3 μg mL-1. However, in our studies RA did not attain minimum inhibitory concentration levels in sweet basil's root exudates before P. aeruginosa formed a biofilm that resisted the microbicidal effects of RA and ultimately caused plant mortality. We further demonstrated that P. aeruginosa biofilms were resistant to RA treatment under in vivo and in vitro conditions. In contrast, induction of RA secretion by sweet basil roots and exogenous supplementation of Arabidopsis root exudates with RA before infection conferred resistance to P. aeruginosa. Under the latter conditions, confocal scanning laser microscopy revealed large clusters of dead P. aeruginosa on the root surface of Arabidopsis and sweet basil, and biofilm formation was not observed. Studies with quorum-sensing mutants PAO210 (ΔrhlI), PAO214 (ΔlasI), and PAO216 (ΔlasI ΔrhlI) demonstrated that all of the strains were pathogenic to Arabidopsis, which does not naturally secrete RA as a root exudate. However, PAO214 was the only pathogenic strain toward sweet basil, and PAO214 biofilm appeared comparable with biofilms formed by wild-type strains of P. aeruginosa. Our results collectively suggest that upon root colonization, P. aeruginosa forms a biofilm that confers resistance against root-secreted antibiotics.
Ethylene-Dependent and -Independent Processes Associated with Floral Organ Abscission in Arabidopsis
Authors: Patterson Sara E. , Bleecker Anthony B.
Published: January 2004

Abscission is an important developmental process in the life cycle of the plant, regulating the detachment of organs from the main body of the plant. This mechanism can be initiated in response to environmental cues such as disease or pathogen, or it can be a programmed shedding of organs that no longer provide essential functions to the plant. We have identified five novel dab (delayed floral organ abscission) mutants (dab1-1, dab2-1, dab3-1, dab3-2, and dab3-3) in Arabidopsis. These mutants each display unique anatomical and physiological characteristics and are governed by three independent loci. Scanning electron microscopy shows delayed development of the flattened fracture plane in some mutants and irregular elongation in the cells of the fracture plane in other mutants. The anatomical observations are also supported by breakstrength measurements that show high breakstrength associated with broken cells, moderate levels for the flattened fracture plane, and low levels associated with the initial rounding of cells. In addition, observations on the expression patterns in the abscission zone of cell wall hydrolytic enzymes, chitinase and cellulose, show altered patterns in the mutants. Last, we have compared these mutants with the ethylene-insensitive mutants etr1-1 and ein2-1 to determine if ethylene is an essential component of the abscission process and find that although ethylene can accelerate abscission under many conditions, the perception of ethylene is not essential. The role of the dab genes and the ethylene response genes during the abscission process is discussed.
Transcriptional Regulation of Sorghum Defense Determinants against a Phloem-Feeding Aphid
Authors: Zhu-Salzman Keyan , Salzman Ron A. , Ahn Ji-Eun , Koiwa Hisashi
Published: January 2004

When attacked by a phloem-feeding greenbug aphid (Schizaphis graminum), sorghum (Sorghum bicolor) activates jasmonic acid (JA)- and salicylic acid (SA)-regulated genes, as well as genes outside known wounding and SA signaling pathways. A collection of 672 cDNAs was obtained by differential subtraction with cDNAs prepared from sorghum seedlings infested by greenbug aphids and those from uninfested seedlings. Subsequent expression profiling using DNA microarray and northern-blot analyses identified 82 transcript types from this collection responsive to greenbug feeding, methyl jasmonate (MeJA), or SA application. DNA sequencing analyses indicated that these encoded proteins functioning in direct defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell maintenance, and photosynthesis, as well as proteins of unknown function. In response to insect feeding, sorghum increased transcript abundance of numerous defense genes, with some SA-dependent pathogenesis-related genes responding to greenbug more strongly than to SA. In contrast, only weak induction of MeJA-regulated defense genes was observed after greenbug treatment. However, infestation tests confirmed that JA-regulated pathways were effective in plant defense against greenbugs. Activation of certain transcripts exclusively by greenbug infestation was observed, and may represent unique signal transduction events independent of JA- and SA-regulated pathways. Results indicate that plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, but also suggest that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery, possibly through their particular mode of feeding.
Xylem Wall Collapse in Water-Stressed Pine Needles
Authors: Cochard Hervé , Froux Fabienne , Mayr Stefan , Coutand Catherine
Published: January 2004

Wall reinforcement in xylem conduits is thought to prevent wall implosion by negative pressures, but direct observations of xylem geometry during water stress are still largely lacking. In this study, we have analyzed the changes in xylem geometry during water stress in needles of four pine species (Pinus spp.). Dehydrated needles were frozen with liquid nitrogen, and xylem cross sections were observed, still frozen, with a cryo-scanning electron microscope and an epifluorescent microscope. Decrease in xylem pressure during drought provoked a progressive collapse of tracheids below a specific threshold pressure (P  collapse) that correlates with the onset of cavitation in the stems. P  collapse was more negative for species with smaller tracheid diameter and thicker walls, suggesting a tradeoff between xylem efficiency, xylem vulnerability to collapse, and the cost of wall stiffening. Upon severe dehydration, tracheid walls were completely collapsed, but lumens still appeared filled with sap. When dehydration proceeded further, tracheids embolized and walls relaxed. Wall collapse in dehydrated needles was rapidly reversed upon rehydration. We discuss the implications of this novel hydraulic trait on the xylem function and on the understanding of pine water relations.
Biocontrol ofBacillus subtilisagainst Infection of Arabidopsis Roots byPseudomonas syringaeIs Facilitated by Biofilm Formation and Surfactin Production
Authors: Bais Harsh Pal , Fall Ray , Vivanco Jorge M.
Published: January 2004

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 μg mL-1), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.
Environmentally Induced Plasticity of Root Hair Development in Arabidopsis
Authors: Müller Margarete , Schmidt Wolfgang
Published: January 2004

Postembryonic development of plants is dependent on both intrinsic genetic programs and environmental factors. The plasticity of root hair patterning in response to environmental signals was investigated in the Columbia-0 wild type and 19 Arabidopsis mutants carrying lesions in various parts of the root hair developmental pathway by withholding phosphate or iron (Fe) from the nutrient medium. In the aging primary root and in laterals of the wild type, the number of root hairs increased in response to phosphate and Fe deficiency in a manner typical of each growth type. Although an increase in root hair density in -phosphorus plants was mainly achieved by the formation of extra hairs over both tangential and radial wall of underlying cortical cells, roots of -Fe plants were characterized by a high percentage of extra hairs with two tips. Root hair patterning and hair length was differentially affected by the presence or absence of phosphate and Fe among the genotypes under investigation, pointing to separate cascades of gene activation under all three growth conditions. Divergence in root hair patterning was most pronounced among mutants with defects in genes that affect the first stages of differentiation, suggesting that nutritional signals are perceived at an early stage of epidermal cell development. During elongation of the root hairs, no differences in the requirement of gene products between the growth types were obvious. The role of genes involved in root hair development in the aging primary root of Arabidopsis under the various growth conditions is discussed.
AmSUT1, a Sucrose Transporter in Collection and Transport Phloem of the Putative Symplastic Phloem LoaderAlonsoa meridionalis
Authors: Knop Christian , Stadler Ruth , Sauer Norbert , Lohaus Gertrud
Published: January 2004

A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80–91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and K  m value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.
Biosynthesis of Camptothecin. In Silico and in Vivo Tracer Study from [1-13C]Glucose
Authors: Yamazaki Yasuyo , Kitajima Mariko , Arita Masanori , Takayama Hiromitsu , Sudo Hiroshi , Yamazaki Mami , Aimi Norio , Saito Kazuki
Published: January 2004

Camptothecin derivatives are clinically used antitumor alkaloids that belong to monoterpenoid indole alkaloids. In this study, we investigated the biosynthetic pathway of camptothecin from [1-13C]glucose (Glc) by in silico and in vivo studies. The in silico study measured the incorporation of Glc into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-13C]Glc. The in vivo study followed incorporation of [1-13C]Glc into camptothecin with hairy roots of Ophiorrhiza pumila by 13C nuclear magnetic resonance spectroscopy. The 13C-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-d-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-d-erythritol 4-phosphate pathway and the shikimate pathway. This study provides the innovative example for how a computer-aided comprehensive metabolic analysis will refine the experimental design to obtain more precise biological information.
Modeling Nitrogen Uptake in Oilseed Rape cv Capitol during a Growth Cycle Using Influx Kinetics of Root Nitrate Transport Systems and Field Experimental Data
Authors: Malagoli Philippe , Lainé Philippe , Le Deunff Erwan , Rossato Laurence , Ney Bertrand , Ourry Alain
Published: January 2004

The use of kinetic equations of NO3  - transport systems in oilseed rape (Brassica napus), determined by 15NO3  - labeling under controlled conditions, combined with experimental field data from the INRA-Châlons rape database were used to model NO3  - uptake during the plant growth cycle. The quantitative effects of different factors such as day/night cycle, ontogenetic stages, root temperature, photosynthetically active radiation, and soil nitrate availability on different components of the constitutive high-affinity transport systems, constitutive low-affinity transport systems, inducible low-affinity transport systems, and inducible high-affinity transport systems of nitrate were then determined to improve the model's predictions. Simulated uptake correlated well with measured values of nitrogen (N) uptake under field conditions for all N fertilization rates tested. Model outputs showed that the high-affinity transport system accounted for about 89% of total NO3  - uptake (18% and 71% for constitutive high-affinity transport systems and inducible high-affinity transport systems, respectively) when no fertilizer was applied. The low-affinity transport system accounted for a minor proportion of total N uptake, and its activity was restricted to the early phase of the growth cycle. However, N fertilization in spring increased the duration of its contribution to total N uptake. Overall, data show that this mechanistic and environmentally regulated approach is a powerful means to simulate total N uptake in the field with the advantage of taking both physiologically regulated processes at the overall plant level and specific nitrate transport system characteristics into account.
A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family
Authors: Mudgil Yashwanti , Shiu Shin-Han , Stone Sophia L. , Salt Jennifer N. , Goring Daphne R.
Published: January 2004

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.
Chlamydomonas reinhardtii Secretes Compounds That Mimic Bacterial Signals and Interfere with Quorum Sensing Regulation in Bacteria
Authors: Teplitski Max , Chen Hancai , Rajamani Sathish , Gao Mengsheng , Merighi Massimo , Sayre Richard T. , Robinson Jayne B. , Rolfe Barry G. , Bauer Wolfgang D.
Published: January 2004

The unicellular soil-freshwater alga Chlamydomonas reinhardtii was found to secrete substances that mimic the activity of the N-acyl-l-homoserine lactone (AHL) signal molecules used by many bacteria for quorum sensing regulation of gene expression. More than a dozen chemically separable but unidentified substances capable of specifically stimulating the LasR or CepR but not the LuxR, AhyR, or CviR AHL bacterial quorum sensing reporter strains were detected in ethyl acetate extracts of C. reinhardtii culture filtrates. Colonies of C. reinhardtii and Chlorella spp. stimulated quorum sensing-dependent luminescence in Vibrio harveyi, indicating that these algae may produce compounds that affect the AI-2 furanosyl borate diester-mediated quorum sensing system of Vibrio spp. Treatment of the soil bacterium Sinorhizobium meliloti with a partially purified LasR mimic from C. reinhardtii affected the accumulation of 16 of the 25 proteins that were altered in response to the bacterium's own AHL signals, providing evidence that the algal mimic affected quorum sensing-regulated functions in this wild-type bacterium. Peptide mass fingerprinting identified 32 proteins affected by the bacterium's AHLs or the purified algal mimic, including GroEL chaperonins, the nitrogen regulatory protein PII, and a GTP-binding protein. The algal mimic was able to cancel the stimulatory effects of bacterial AHLs on the accumulation of seven of these proteins, providing evidence that the secretion of AHL mimics by the alga could be effective in disruption of quorum sensing in naturally encountered bacteria.