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Poster sessionPoster Abstracts Investigations of the genetic background of the Börner (Vitis sp.) Phylloxera resistance by means of Microarray Technology L. Blank Geisenheim Research Center, Section of Botany, Geisenheim, Germany l.blank@fa-gm.de
mbudroni@uniss.it Stuck and slow fermentations represent a chronic problem for the wine industry. The reasons for the premature arrests of fermentation have not been completely elucidated. However, it has been demonstrated that the lack of oxygen, together with the absence of lipid nutrients in the growth medium, highly stresses Saccharomyces cerevisiae cells. The aim of this work was to understand the differences in the transcriptome of two S. cerevisiae wine strains, characterized by a different fermentative behaviour, during stress fermentations. In this study the possible relationships between anaerobic stress and lipid nutrition have been analysed by using an experimental design that allowed us to discard the influence of yeast genetic backgrounds and to consider only the metabolic responses due to the adaptation to the stress conditions. The results obtained showed different regulations of some important pathways such as glucose metabolism and mannoprotein synthesis. Further analyses of those pathways will lead us to figure out the basis of yeast adaptation to musts characterized by scarce amounts of lipid nutrients.
celine.camps@bordeaux.inra.fr The Ascomycete Eutypa lata is a vascular fungus causing eutypiosis, a devastating vineyard disease inducing wood decay. In spite of the economical importance of this disease, there is neither available cure nor method for diagnosis. All grapevine cultivars are susceptible to Eutypa dieback, but the severity of the symptoms may vary. Cabernet sauvignon is considered as one of the most susceptible cultivar as the symptoms are more severe than in other cultivars such as Merlot. In this context, we developed a transcriptomic analysis which aims at comparing gene expression in Cabernet Sauvignon grape plants infected by Eutypa lata and plants which never suffered the disease. We used a 15K (70-mer oligonucleotide) grape microarray slide. Three different conditions in vitro, greenhouse and field were compared, each one presenting advantages and disadvantages: The Eutypa strain NE85-1 previously characterized as virulent, was used for controlled experimental infection of in vitro (plantlet) and greenhouse (well lignified cuttings) plants. In vitro samples were collected 2 weeks or 5 weeks post-infection. Greenhouse infections need one year before symptom appearance. Finally, naturally infected samples have also been collected in the vineyard to compare plants affected by Eutypiosis with plants which never suffered the disease. Re-isolation of the fungus after surface wood sterilization with Na hypochlorite and culture on sterile PDA medium allowed us to check whether the uninoculated control was indeed axenic, and to sort out the inoculated plantlets that were actually infected from those that did not (negative re-isolation) or from those that have been infected by other fungus in the case of field material. A good correlation between symptoms notations and the results of re-isolation was observed. PCR identification of Eutypa lata (Lardner, 2005) was also carried out after fast DNA extraction from re-isolated mycelium. This amplification strategy by indirect PCR allowed us to identify clearly the presence of Eutypa lata infected plants. However routine use in vineyard is not possible because of its destructive character. Microarrays comparison of gene expression between infected and non infected plants in each of this three conditions and subsequent results crossing reveal genes that can be potentially good markers of Eutypa disease, thus leading to possible diagnosis. Microarray analysis of pig muscle in divergent populations for cholesterol and lipid metabolism parameters 1 A. Cánovas, 1 R. Quintanilla, 1 L. Varona, 2 I. Díaz., 1 J. Casellas, 1 R.N. Pena 1Genètica i Millora Animal. IRTA-Lleida. Spain, 2Tecnologia dels Aliments. IRTA-Monells. Spain angela.canovas@irta.es In order to detect and identify genes involved in pig lipid metabolism we have used a microarray approach over muscle samples from an experimental Duroc population of 370 castrated males distributed in five half-sib families. A total of 70 samples of the Gluteus medius were processed. They were selected from animals with the most extreme levels (HIGH and LOW lines; 35 animals per line) for an index composed by cholesterol and lipid metabolism parameters, such as plasma lipoprotein and triglycerides levels, percentage of intramuscular fat and fatty acid composition in muscle. Each sample was individually hybridized using GeneChip Porcine Genome® arrays (Affymetrix). After normalizing data with the Robust Multichip Average (RMA) algorithm, comparison between lines was performed using a Bayesian analysis by means of a mixed model with heterogeneous residual variances. The mixed model analysis resulted in a total of 500 genes differently expressed at a significant level (p-value<10-9), 158 of them showed a ratio between classes greater than 1.5. Taking into account the significance level, the ratio of variation and the function of the genes, we have selected a list of 32 genes to be validated by q-PCR. The function of these genes includes a variety of processes such as: transcription factor, lipid metabolism, intracellular signalling and hormones. Development of a DNA array with multiple levels of phylogenetic specificity to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum. B. Lievens a*, L. Claes a, D.J. Vakalounakis b, A.C.R.C. Vanachter a, and B.P.H.J. Thomma c a Scientia Terrae Research Institute, Sint-Katelijne-Waver, Belgium b National Agricultural Research Foundation (N.A.G.RE.F.), Plant Protection Institute, Heraklion, Crete, Greece c Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands * bli@scientiaterrae.org Fusarium oxysporum is a ubiquitous soilborne fungus that harbours pathogenic as well as non-pathogenic or even beneficial strains. Pathogenic strains are characterized by a high degree of host specificity and strains that infect the same host range are organized in so-called formae speciales. Strains for which no host plant has been identified are believed to be non-pathogenic strains. Therefore, identification below the species level is highly desired. Currently, identification of pathogenic F. oxysporum isolates is mainly based on time-consuming and laborious bioassays. Increasingly, attempts are made to replace these methods by straight-forward, culture-independent molecular identification techniques. Ideally, molecular identification of pathogenic strains is based on the detection of genetic targets that are linked to pathogenicity. However, so far the genetic basis of host specificity and virulence in F. oxysporum is unknown. In this study, a random amplified polymorphic DNA (RAPD) marker-based assay was developed to specifically detect and identify the economically important cucumber pathogens F. oxysporum f. sp. cucumerinum and F. oxysporum f. sp. radicis-cucumerinum. Highly reliable markers were implemented in a DNA macro-array with multiple levels of phylogenetic specificity. Amongst others, the DNA array contained genus-, species- and forma specialis-specific detector oligonucleotides. The array was optimized for specific, sensitive and reliable detection of the target organisms in diverse environmental samples. Results illustrating this work are presented in the poster. Construction and validation of a new flexible Grape microarray platform D. Glissant, A. Ferrarini, M. Pezzotti and M. Delledonne Università degli Studi di Verona, Dipartimento Scientifico e Tecnologico. Strada le Grazie, Verona. Italy glissant@sci.univr.it
The honeybee, Apis mellifera, plays a vital role in agriculture by assisting with the pollination of many different crops. The economic importance of the honeybee cannot be underestimated and numerous pathogens and parasites threaten bee health. Most honey bee viruses are single stranded RNA viruses and are very similar in size and shape, making them difficult to distinguish from each other using physical characteristics. Sequences for eight bee viruses were available from Genbank: Deformed Wing Virus (DWV) , Kashmir Bee Virus (KBV), Acute Paralysis Virus (APV), Varroa Destructor Virus (VDV), Sacbrood Virus (SBV), Chronic Paralysis Virus (CPV), Apis Iridescent Virus (AIV) and Black Queen Cell Virus (BQCV). Initial design of 50-mer oligonucleotides to bee virus sequences was carried out using OligoArray 2.0 (Rouillard et al., 2003), followed by manual selection of promising sequences. Between six and ten oligonucleotides were selected for each virus for inclusion on the microarray. Four oligonucleotides were designed to the 18S ribosomal gene of Apis mellifera as internal controls. Initial testing of the low density microarray has been very successful with 60/61 of the oligonucleotide capture probes hybridising with the correct target DNA (in addition, only one oligonucleotide showed cross-hybridisation). Work is currently being undertaken to sequence other bee viruses and design oligonucleotides for inclusion on this array. Reference: Rouillard, J.M., Zuker, M., and Gulari, E. OligoArray 2.0: Design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Research, 2003, 31(12): 3057-3062.
* aju@scientiaterrae.org Storing sugar extracts as thick juice, a form of sucrose syrup, is common practice in the sugar industry. Even under good storage practices, microbiological problems may occur. Improving control of these microflora-related problems requires greater understanding of the microbial dynamics of thick juice storage. Therefore, the main objective of this study was to map the population dynamics during storage and identify the microflora associated with thick juice degradation in order to define better process controls. Terminal restriction fragment-length polymorphism (T-RFLP) and clone library analysis (CLA) (both targeting 16S-rDNA) as well as classical culture-based methods were used to characterize bacterial communities present in non-degraded and heavily degraded thick juice samples. Based on the results obtained by these approaches, a nylon membrane-based DNA array targeting 16S rDNA sequences was developed for all bacterial species detected. For signal amplification, the target DNA was amplified using universal primers that anneal to conserved sequences flanking diagnostic domains, simultaneously labelled, and subsequently hybridized to the array. Detection was performed using the anti-digoxigenin alkaline phosphatase conjugate and CDP-Star substrate. Both CLA and T-RFLP demonstrated that Tetragenococcus halophilus was the most consistent and predominant taxon present in both degraded and non-degraded samples that were examined. Classical plating on rich medium confirmed the dominance of this halophilic Tetragenococcus species, but revealed also other osmophilic bacteria possibly pernicious during sugar production including Bacillus spp., Staphylococcus spp., Kocuria spp., Aerococcus viridans, Leuconostoc mesenteroides and other Tetragenococcus species. In order to monitor the bacterial population dynamics during thick juice storage, a specific thick juice macro-array was developed covering these bacteria. Using the array, a diverse microbial population was found at the beginning of storage, which evolved to dominance of Tetragenococcus species mostly accompanied by Staphylococci. Based on its high density, we believe that T. halophilus potentially plays a role in thick juice degradation. Results illustrating this work are presented in the poster.
outi.lepparanta@evira.fi Enterohaemorrhagic Escherichia coli (EHEC) is a food and waterborne pathogen with a very low infective dose. EHEC infection poses a significant risk of two serious complications, hemorrhagic colitis and haemolytic uremic syndrome. Production of Shiga toxins (stx) and adherence-mediating factor intimin (eaeA) are considered important in the pathogenesis. Most EHEC outbreaks have been caused by E. coli O157 strains, and their main reservoir is considered to be cattle. Microarrays were used for the comparison of gene content of pathogenic and non-pathogenic E. coli strains. Twenty-one E. coli strains were selected for the studies, 19 of them belonging to serogroup O157. Prior to microarray experiments, these strains were divided into groups: I) six strains associated with human disease, II) six strains with eae and stx genes, III) five strains with eae gene but no stx genes, and IV) four non-EHEC strains. Genomic DNA was fluorescently labelled with Cy3 and Cy5 and the samples were hybridized with the probes of OciChip E. coli O157 Arrays (Ocimum Biosolutions). Each hybridization was replicated using two or more biological samples. Logarithmic ratios were computed between the test and reference sample for each probe on the array. The values were normalized within each array using iterative linear regression method. Microarray profile-based similarity between strains was compared with the existing classification of strains described above. Based on microarray profiles, the difference of group IV from all other groups was explained by the absence of ca. one hundred genes. Differences between groups I, II, and III were smaller and there was variation within each group. A set of genes was selected using statistical analysis which best explained the differences between pathogenic (group I) and possibly pathogenic (group III) strains. Based on this set of genes, the strains belonging to group II were computed against the strains of group I and III, in order to categorize the group II strains as pathogenic or possibly pathogenic. Expression analysis at different reproductive stages in a F2 crossbred. M. Martínez*, R. Pena*, J. Casellas*, A. Tomàs§, A. Fernández†, L. Varona*, J.L. Noguera* *Genètica i Millora Animal. IRTA. Lleida, Spain, §Departament de Ciència Animal i dels Aliments. Facultat de Veterinària, UAB. Bellaterra, Spain , †Departamento de Mejora Genética Animal SGIT-INIA. Madrid, Spain maria.martinez@irta.es
National Nanotechnology Laboratory of CNR-INFM, Unità di Ricerca IIT, Lecce, Italy elisabetta.primiceri@libero.it
Here, we present an electrical detection method based on a completely new type of nanoelectrodes [2] functionalized with ssDNA. Electrical detection [3] would be preferable for the identification of specific DNA sequences due to its predisposition to the miniaturization and its compatibility with the microfabrication techniques. In our approach, we immobilise 5'-SH-DNA probes on the gold electrodes and DNA target sequences are conjugated to gold nanoparticles. After hybridization AuNPs self-assembles onto the nanoelectrodes acting as a bridge between the gap and promotes the current flowing in the nanojunction. As a result, we are able to detect also single hybridization events demonstrating the great sensitivity of our device that make unnecessary the use of PCR to amplify the DNA target or other signal amplification techniques as silver enhancement. Our device joins a great sensitivity with an innovative and cheaper nanofabrication method (exploiting optical lithography of a quantum-well), so it would be a fundamental step towards a new generation of highly sensitive and robust low-cost nanosensors. It offers new possibilities in terms of fabrication of nanosized integated systems and point-of-care analysis and it would allow a potential faster and more efficient sequence analysis.
oger@biomserv.univ-lyon1.fr Erwinia chrysanthemi is a significant bacterial phytopathogen, causing soft rot disease on a wide variety of plant hosts, including many economically important crops (vegetables and ornementals). The pathogenicity of E. chrysanthemi is associated with the production of a large set of pectinases which dissolve the various components of the plant cell walls; these degradations first enable the colonisation of the intercellular spaces of the plant (a latent phase of the disease with no visible symptoms), and then, upon certain critical conditions, they completely disorganise the plant parenchymatous tissues resulting in dramatic symptoms. The production of pectinases has been shown to be controlled by several transcriptional regulators (mostly repressors), including KdgR, PecS, PecT, Fur. The study of mutations on these regulators have revealed for some of them an effect on the duration of the latency period. Particularly, a PecS mutant showed no latency before the apparition of symptoms suggesting that the transition between latency and the symptomatic phase is dependent upon the stimuli received by this regulator after infection and that it plays a pivotal role in the expression of the disease. The genome of Erwinia chrysanthemi 3937 has been recently completely sequenced and annotated by an International Erwinia Consortium (http://www.ahabs.wisc.edu/~pernalab/er-winia/index.html) using ASAP (a systematic annotation package for community analysis of geno-mes: https://asap.ahabs.wisc.edu/asap/ASAP1.htm). In order to determine the set of genes regulated by the transcriptional regulator PecS, the transcriptomes of wild type strain and PecS- mutant A3953 grown in vitro to late exponential phase were compared using pangenomic microarrays produced by maskless technology (4753 CDS each represented by 20 oligonucleotidic probes). Statistical analysis (using the package LIMMA of Bioconductor) revealed 437 genes as differentially expressed between the PecS- mutant and the wild-type strains (FDR <0.05) among which 135 genes had a fold-change > 1.33. Eighteen percent of these 135 genes were already known as targets of PecS (an internal validation of the experiments). Targets identified by this assay fell into the following main categories : secreted proteins (pectate lyases, metallo-proteases, flagellum biosynthesis or regulation …) or proteins implicated in secretion, oxidative stress response (indigoidin biosynthesis, ATPases…), fatty acids biosynthesis. Differentially expressed genes tended to group on specific chromosomal locations, partially reflecting the operonic organization. Some of the potential targets newly identified in this assay are being further investigated by mutant analysis in order to validate their implication with pathogenicity. DNA arrays applied to microorganisms detection. Poltronieri P., D'Urso O.F., Cappello M.S, Morea M. ISPA-CNR, Lecce, Italy palmiro.poltronieri@ispa.cnr.it
The DNA extractions from various matrices were optimized depending on the microorganism to be detected. For example in order to extract DNA from microorganisms colonizing surfaces, such as Listeria species, cells were harvested from samples by repeated washings. Additional enrichment steps based on filtration were performed when necessary. Bacteria were lysed in the presence of lysozyme, also using proteinase K in some cases and DNA was captured on silica or magnetized particles. The DNA array protocol took 24 hours to perform all steps, showing to be a fast multiplex analysis when food samples of unknown microbial composition were tested. DNA arrays set up has the capacity to verify the presence of many species in a single run. This characteristic overcomes its longer time to obtain results when compared to other molecular methods such as real time PCR Ongoing work is aimed at designing new oligonucleotides based on single copy genes such as rpoB, tuf and GroEL/Hsp60 chaperone allowing higher species-specific resolution.
Mary.portillo@uclm.es, cescobar@uam.es Sedentary endoparasitic root-knot nematodes (Meloidogyne spp.) are an important pest of crop plants that causes severe economic losses. The second-stage juveniles, J2 hatch and invade the roots. Their secretions that are injected from the stylet, reprogram specific plant cells. Their development depend on these, 3-7 cells from the xylem parenchyma that are transformed in specialized feeding cells called giant cells (GC). Drastic changes in plant gene expression are induced in GC and surrounding cells that constitute the visible gall. Molecular changes that take place during the establishment of the nematode, involve up-regulation and down-regulation of genes and it has been extensively described (Reviewed in Gheysen and Fenoll., 2002, Bar-Or et al., 2005; Jammes et al., 2005). In this work, the differential gene expression profiles between infected and control tissue has been analyzed with RNA from dissected tomato galls 1, 3, 7 and 14 days after infection (dpi) with Meloidogyne javanica. The analysis of the galls transcriptome was obtained by hybridization of the cDNA TOM1 array which contains 12.899 features derived from the tomato genome (http://bti.cornell.edu/CGEP/CGEP.html). Statistical analysis using a P value cut-off <0.05 revealed 3593 genes displaying significant differential expression during nematode infection. Several transcriptional patterns have been identified and genes clustered. The number of genes differentially expressed during very early gall formation (1-3 dpi, 527-386 genes) differs from that at later stages (7-14 dpi, 1366-1314 genes). In addition, the number of up-regulated and down-regulated genes at the different developmental stages is strikingly different, especially at 1 and 3 dpi, stages when the nematode is starting to become sedentary and GC initiate their development. These results, suggest that specific biological processes are required for gall initiation as compared to the maintenance stages (7- 15 dpi). Furthermore, it suggests a subtle and controlled genetic re-programmation at the beginning of the infection. To explore the biological processes in which the differentially regulated genes are involved, we have classified genes according to functional categories. The highest number of genes was grouped in metabolism, in accordance with the giant cell function as nutrient sinks for the nematode. This result is similar to other studies with root-knot nematodes. (Bar-Or et al., 2005; Jammes et al., 2005; Puthoff et al., 2003). The defence genes are a representative group of up regulated in early stages, due to the plant stress response to the nematode infection. However, most of the defence and stress genes were down-regulated at later infection stages. Our global analysis reflects that genes involved in metabolism, defence, stress, proteins synthesis, transcription factors and transduction signals are the groups with a higher contribution to the global changes in gene expression observed in galls.
Gheysen, G., and Fenoll, C. (2002). Gene expression in nematode feeding sites. Annu. Rev. Phytopathol. 40:191-219. Jammes, F., Lecomte, P., de Almeida-Engler, J., Bitton, F., Martin-Magniette, M.L., Renou, J.P., Abad, P., and Favery, B. (2005). Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsis. Plant J. 44: 447-458. Puthoff, D.P., Nettleton, D., Rodermel, S.R., and Baum, T.J. (2003). Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles. Plant J. 33: 911-921. Comparison of oligonucleotide arrays and SPR-biacore sensors for fungal detection: tests of Aspergillus carbonarius Quarta A, Mita G, D'Urso OF, Zezza F, Pascale, M, Logrieco A., Visconti A. Poltronieri P. ISPA-CNR, Lecce, Italy palmiro.poltronieri@ispa.cnr.it
In the DNA macroarray experiments, oligonucleotides specific for A. carbonarius, A. japonicus and A. aculeatus, were provided with an NH2-terminal modification for covalent binding to epoxy-slides (Nexterion, Schott). These immobilised sequences were used as probe to test their specificity in hybridization experiments with the cyanine 3-labeled amplified products. Surface plasmon resonance (SPR) is an emerging technology that enables the label-free detection of biomolecular interactions. In the SPR sensor experiments, the probes were prepared biotinylated in their 5' end and immobilized on a dextran-streptavidin pre-coated gold chip by means of the well known biotin-streptavidin interaction. The probes were tested for biospecific interactions with label-free amplified products. Conclusions: we tested two different methods to verify the specificity of various probes. Fungal DNA amplified with P1For and P2Rev generic primers was used as target. The advantage o Yüklə 414,14 Kb. Dostları ilə paylaş:
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