Cost-meetings Eden Roc, esp may 2007

B. Session 2a

Session 2a: Current Questions and Applications
Chair: Marta Mendes (COST DC BMBS)

Rapporteur: François Hatey (COST 861),

Keynote Speaker: Christian Bendixen

Transcript profiling in farm animals, novel tools and strategies
C. Bendixen

University of Aarhus, Tjele, Denmark

[e-mail]
Selected examples of the development and application of tools for expression analysis in pigs and cattle will be described. In particular, the development of porcine cDNA- and oligonucleotide-arrays and the use of these resources in different experimental designs will be presented. Data will include descriptive profiling of gene-expression across a wide range of tissues as well as data from experiments exploring transcript profiles related to quantitative traits. New transcript profiling strategies based upon massively parallel sequencing technologies will be discussed and different studies comparing alternative approaches will be shown. Particular focus will be on the additional information obtainable from novel technologies and their potential power relative to array technology.
On the Outside of Fleshy Fruit: A Study of Gene Expression and Metabolic Pathways in Peel of Normal and Mutant Tomato
T. Mandel, S. Mintz, A. Adato, I. Venger, I. Rogachev and A. Aharoni

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel

[e-mail]
The fruit skin provides an interface between the plant tissue and its environment, acting as a protective barrier, as well as a medium for the exchange of gases, water and nutrients. In fleshy fruit, the skin is normally composed of hypodermal cells and a continuous layer of epidermal cells that are covered by a thick, waxy cuticle. The building blocks for construction of the cuticle are generated in the epidermis. In this study we conducted extensive gene expression profiling of tomato fruit peel in order to understand the genetic regulation of the various biosynthetic pathways involved in cuticle formation. Peel and flesh tissues derived from tomato fruit at 5 developmental stages (from green to red fruit) were used for hybridization to the Affymetrix GeneChips representing 9,200 tomato transcripts. Approximately 400 genes showed preferential expression in the peel compared to the flesh tissue. Genes encoding enzymes involved in cuticle metabolism such as wax, cutin and flavonoid biosynthesis were highly represented and clustered together in the course of fruit development. Comparison of the tomato peel associated genes to those preferentially expressed in epidermis tissue of Arabidopsis stems revealed similar genes expressed in both tissues. We also examined gene expression in a natural tomato mutant termed "y" showing a colourless peel phenotype compared to the yellow-coloured normal peel. Peel and flesh tissues derived from three developmental stages of the "y" mutant were profiled. Data interpretation by means of PCA showed that most changes in the "y" mutant occur in the orange/red stages of development. In the "y" mutant, major changes in gene expression were detected in the flavonoid pathway leading to the formation of naringenin chalcone, the yellow pigment accumulating in tomato fruit cuticle. We are currently conducting non-targeted metabolite analysis on the same sample set and results from the combined transcriptome-metabolome data will be presented.
Comparative gene expression analysis between the chilean native strawberry, Fragaria chiloensis, and its world-wide cultivated relative, Fragaria x ananassa.
F. Carbone^a, G. Saud^b,c,*, P. DS Caligari^a,b and G. Perrotta^a
a Ente per le Nuove Tecnologie, l'Energia e l'Ambiente (ENEA), Italy, ^b Universidad de Talca, Chile, ^c Centro de Investigación en Biotecnología Silvoagrícola (CIBS), Chile

[e-mail]

The cultivated strawberry, Fragaria x ananassa, is a tasty and highly nutritive fruit well known all over the world. It has its origins in an inter-specific hybridization between wild species native to the Americas. One of the parentals of this hybrid was the white fruited chilean native strawberry, Fragaria chiloensis, nowadays a species with high potential to become an exotic crop it self. In order to investigate the transcriptome of these species and to perform an inter-specific gene expression analysis, cDNA microarray experiments were carried out at different stages of fruit development in both species. As it was expected, results evidenced the activation or up-regulation of an important number of coding sequences putatively encoding ripening related proteins in both species. Notably, differential transcriptional profiles were detected in putative genes important for fruit development and quality between both species. Also, an important number of clones with no similarity to any other proteins deposited in the databases were observed to be regulated in the microarray experiments, representing an interesting opportunity for data-mining. The information provided in this report, together with phenotypic evidence obtained in other available and future researches, could be of great utility to further characterize the ripening syndrome in strawberries.
Utilization of a peach microarray for gene expression profiling in strawberry.
L. Trainotti, A.Tadiello, G. Casadoro.

Department of Biology, University of Padua, Padova, Italy.

[e-mail]
During ripening, fleshy fruits undergo a number of physical, chemical and physiological changes affecting pigmentation, texture, flavour, aroma, that make the fruit more attractive and palatable. All these changes are the results of a co-ordinated modulation of gene expression, regulated by complex and interrelated mechanisms which are affected by internal and external factors. Monitoring transcriptome changes is widely used to unravel complex biological features, such as fruit development and ripening, and microarrays represent one of the techniques employed to achieve it. This technique, albeit being very expensive, has been used by plant biologists since its appearance and now is largely diffused. Since the development of a microarray platform is expensive and time-consuming, initially it has been pursued mainly for those species where the investment was thought to be profitable, that is for the main crops (e.g. corn, rice, maize, tomato) or for the model species Arabidopsis. Anyway, being the technology based on DNA sequence hybridization, a given microarray might theoretically be used also with species that are taxonomically close to the one for which the platform has been developed, given that sufficient sequence conservation among orthologous genes exists.

We have developed µPEACH, an oligo-based microarray with more that 4800 unique probes that is being used to investigate peach fruit development and ripening. Yet, since both peach and strawberry belong to the Rosaceae family, we have tested µPEACH with strawberry RNA. In particular, we have compared transcriptome changes occurring during the transition from the pre-climacteric to the climacteric stage in peach, with the ones occurring during the transition from white to red ripe strawberry. This analysis has allowed us to identify 60 and 57 genes, respectively, that are either up-regulated or down-regulated during ripening of both fruits. Among the up-regulated genes there are five gene of unknown function but also a leucoanthocyanidin dioxygenase and other genes involved in flavonoid biosynthesis, an AP2 and an ERE-BP transcription factors and several genes involved in sugar metabolism. Among the down-regulated genes, the most abundant class is still the one of unknown function (7 genes), several genes involved in the formation of the ubiquitin complex and some in photosynthesis. Cell wall genes have been found in both classes, as it was expected in ripening fruits.

The use of a single platform has the significant advantage to reduce the development costs (i.e. probe design and synthesis, but also database managing). Moreover, it simplifies the data handling and analyses, besides allowing a direct comparison of the expression data collected among the different fruit species. In the future it could be a useful tool also for other small fruits for which the development of a dedicated array could be too expensive.
Application of microarray technology to examine the local and systemic plant responses to root-knot nematode interaction.
S. Sanz-Alférez

Universidad Autónoma de Madrid. Department of Biology. Madrid - Spain.

[e-mail]
Sedentary endoparasitic nematodes are plant parasites that interact with their host to establish a permanent feeding site. Root-knot nematodes, Meloidogyne sp., establish a compatible interaction with plants by reprogramming root cells to feed, develop and reproduce. The selected root cells are modified to become enlarged and multinucleated, called giant cells. These cells seem to be metabolically active, with a thick cell wall that increases the solute uptake from the vascular system. In parallel to the giant cells formation, the surrounding cortical cells suffer a hypertrophy, then the infected root present the typical gall or root-knot shape. The molecular signals inducing the feeding cells remain unclear, but nematode secretions and from surface molecules are good candidates to play a role in the parasitism event. Furthermore, the infected plant has to induce responses against the pathogen, both locally and systemically, using a complex network of signal transduction pathways. In general, the nematode infection elicits important changes in plant gene expression. Previous studies, bases on differential gene expression, analyses of known genes by promoter GUS fusion or in situ hybridization, and promoter traps strategies have identified around fifty plant genes upregulated, and ten that are downregulated in giant cells. The majority of changes seem to be induced through the establishment of the feeding site. Induced genes are mainly related to metabolic pathways, cell-cycle progression, cell-wall expansion and water transport. However, the biological processes underlying the plant-nematode interaction remain unclear.

Microarray technology makes it possible to generate large-scale information about patterns of gene expression during plant-nematode interactions. Chips containing a large number of tomato cDNAs have allowed to perform microarrays for an important number of genes keep in the Lycopersicon esculentum genome. We have obtained a large-scale host gene-expression profile of a compatible interaction between Lycopersicon esculentum and Meloidogyne javanica carried out by a microarray strategy. Both systemically and local changes have been studied, using leaf and root tissues of infected and non-infected tomato plants. Quantitative PCR analysis and promoter GUS fusion examination are carrying out to confirm the changes in mRNA levels observed in the microarray experiment. The data will be presented and the role of changes in gene expression will be discussed.

Monitoring plant gene expression after fungal infection.
F. Förster, A. Felk, F. Maier, W. Schäfer

University of Hamburg, Biocenter Klein Flottbek, Hamburg, Germany

[e-mail]
The fungal pathogen F. graminearum is the most common causal agent of Fusarium head blight (FHB) of small grain cereals and of cob rot of maize. The threat posed by this fungus is due to yield decreases and mycotoxin contamination. The cereal-Fusarium pathosystem is characterized by non-race and non-hostspecific relationships. The outcome of infection varies between total destruction of the host and minor damage. Up until now, no F. graminearum resistant plant cultivars exist in the field.

We want to identify plant genes involved in defence against fungal infection. We generated fungal mutants differing in their ability to infect barley and compared the plant gene expression between infection of these mutants and wild type infection.

Therefore, we infected barley with: Firstly, Gpmk1 MAP kinase disruption mutants of F. graminearum which are apathogenic and cannot infect the inoculated spikelet. Secondly, a fungal mutant deficient in a secreted fungal lipase able to infect the point of inoculation, i.e. the spikelet, but unable to colonize the spike. Thirdly, wild type F. graminearum, readily infecting the entire spike. To monitor the infection process we transformed all strains to constitutively express the green fluorescent protein. We harvested the inoculated spikelet and part of the adjacent plant rachis five days post infection and generated cDNA libraries. Using these probes we hybridised Affymetrix microarrays covering the transcriptom of barley. Using this experimental set up we now can monitor plant gene expression in response to infection of near isogenic F. graminearum strains differing dramatically in their ability to colonize barley.

3. Rapporteurs Remarks 2

The keynote lecture by C Bendixen described the developments of new tools for expression analyses in pigs and cattle. Among them were subtraction libraries to identify genes coregulated with important diseases like those induced by Pleuropneumonia, Mycoplasma-like pathogens. Among the new technologies applied are also 454 sequencing strategies and QTL-searching strategies to identify genome parts correlated with resistance to Pleuropneumonia where 5 QTLs have been identified. Experiments are underway to convert these to mendelian traits allowing a more extensive study using array approaches.

The subsequent lecture by A. Aharoni described intensive research around the main interface between fleshy fruits like tomato and the environment, the skin. Using Affymetrix chips with 9200 tomato traits the transcriptome and metabolome date were sampled along the development of fruit to ripeness. Questions to be asked covered the key genes involved in skin formation and assembly.

Microarrays based on 1811 ESTs on a DNA chip were described by F. Carbone, as tools to compare the red strawberry F. x ananassa versus the wild species F. chiloensis with respect to transcritomics during the ripening stage. The results were substantiated by qPCR. This will help to understand the physiology of fruit ripening and aid to breed new strawberry varieties.

In order to reduce chip development costs, a peach microarray was tested for its suitability to study also other crop plants belonging to the same taxonomic family, i.e. strawberries. The analyses showed that the peach chip could also be applied to detect differences in the transcriptome during the transition from white to red strawberry fruits. These results led L. Trainotti to suggest that this peach array could be successfully applied also for other small fruit crops from the same family to save developmental costs.

S. Sanz-Alférez described in her talk the microarray studies undertaken to more exactly understand the interaction taking place between the sedentary nematode Meloidogyne spec. and its plant host tomato. When establishing a successful inoculation the female nematodes elicit the formation of giant cells as feeding sites and in addition induce systemic signals for the plant host. This leads to several up- and downregulated genes in the plant host, among them mostly genes involved in metabolic pathways, cellcycle progression, cell wall expansion and water transport. The array results have been confirmed by other techniques like qPCR and promotor- GUS-fusion experiments in different kinds of tomato tissue.

Finally F. Förster together with W. Schäfer described their results from comparative inoculation experiments of barley with wild type Fusarium graminearum and two different pathogenicity defective mutants produced by knocking out the Gpmk 1 MAP kinase and a secreted lipase, respectively. The plant tissue where the block of fungal spread was observed was used to construct cDNAs which were hybridised on Affymetrix barley-chips. With this experimental approach the plant genes for a successful infection by the fungus could be identified. They could be substantiated by qPCR experiments.