A combined transcriptomic approach to analyse the dialogue between pseudorabies virus and porcine cells.

The pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for the study of virus-host cell dialog. As PrV has a strong tropism for mucous epithelial cells, we chose to follow in vitro the PrV time course-infection of porcine PK15 cells. The viral and cellular transcriptome modifications were simultaneously analysed using a combined SLA/PrV cDNA microarray, the porcine Qiagen-NRSP8 oligonucleotides microarray and real time quantitative PCR.Ahigh increase in viral gene expression was found from 4 h post-infection (PI), concomitantly to the first viral progeny and most viral genes were differentially expressed 12 h PI. No early global cellular shutoff was observed but many cellular genes were downregulated between 8 and 12 h PI, when UL41 transcripts encoding the virion shutoff protein, were first detected. Several genes involved in the MHC class I mediated antigenic pathway were downregulated including SLA-la, TAP1, TAP2, PSMB8 and PSMB9 genes. These results suggested that PrV prevents the viral antigen presentation by epithelial cells to cytotoxic T lymphocytes by decreasing transcription levels of SLA Ia mediated antigenic pathway genes. Other genes involved in the immune response, the apoptosis pathway, nucleic acid metabolism and cytoskeleton also appeared to be regulated during PrV infection. The combined approach will help to decipher host response evasion strategies developed by PrV and to study early cellular modifications.

Identification of differentially expressed genes in spontaneously regressing melanoma using the MeLiM swine model.

Partial and some few cases of complete spontaneous regression have been observed in cutaneous melanoma patients but little is known about the molecular mechanisms involved. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a suitable animal model to study the phenomenon of spontaneous regression because MeLiM pigs exhibit naturally occurring melanomas which regress completely 6 months after birth. In this study, we used suppression subtractive hybridization (SSH) to identify molecular determinants of melanoma regression within swine melanoma tissues and melanoma cell cultures. Several markers involved in cell-adhesion, -communication, -motility, signal transduction, negative regulation of cell proliferation, transport and immune response were identified that correlated with melanoma regression whereas the main genes involved in melanin synthesis showed a strong downregulation. For the most differentially expressed genes, we validated the results obtained by SSH with qRT-PCR and with immunohistochemistry for some of them (CD9, MITF, RARRES1). Most notable, for the first time in melanoma, we identified the retinoic acid responder 1 gene (RARRES1) as a main actor of the regression process in melanoma. This first gene expression study in swine melanoma regression, may contribute to the finding of new therapeutic targets for human melanoma treatment.

Gene expression patterns during the larval development of European sea bass (dicentrarchus labrax) by microarray analysis.

During the larval period, marine teleosts undergo very fast growth and dramatic changes in morphology, metabolism, and behavior to accomplish their metamorphosis into juvenile fish. Regulation of gene expression is widely thought to be a key mechanism underlying the management of the biological processes required for harmonious development over this phase of life. To provide an overall analysis of gene expression in the whole body during sea bass larval development, we monitored the expression of 6,626 distinct genes at 10 different points in time between 7 and 43 days post-hatching (dph) by using heterologous hybridization of a rainbow trout cDNA microarray. The differentially expressed genes (n = 485) could be grouped into two categories: genes that were generally up-expressed early, between 7 and 23 dph, and genes up-expressed between 25 and 43 dph. Interestingly, among the genes regulated during the larval period, those related to organogenesis, energy pathways, biosynthesis, and digestion were over-represented compared with total set of analyzed genes. We discuss the quantitative regulation of whole-body contents of these specific transcripts with regard to the ontogenesis and maturation of essential functions that take place over larval development. Our study is the first utilization of a transcriptomic approach in sea bass and reveals dynamic changes in gene expression patterns in relation to marine finfish larval development.

Developmental regulated mechanisms affect the ability of a fungal pathogen to infect and colonize tobacco leaves.

During tobacco development, a transition state from susceptibility to resistance to fungal pathogen infection is observed. Leaves acquire resistance to Phytophthora parasistica when the plant becomes committed to flowering. The ability to develop resistance does not imply pathogen-induced defence responses as for the onset of systemic acquired resistance (SAR). Throughout flowering growth, fungal establishment is restrained at two levels. The first level is the control of infection effectiveness. Using the salicylic acid non-accumulating NahG plants, we demonstrate that this control does not require salicylic acid accumulation. The intercellular fluids (IFs) from tobacco leaves committed to flowering exhibit a cytotoxic activity on fungal zoospore cells based on in vitro germination assays. Its accumulation is correlated to the control of infection effectiveness that occurs during flowering growth. The expression of this activity appears to constitute a developmental regulated mechanism that inhibits early steps of fungal pathogen installation. A second level of fungal growth control is the restriction of fungal hyphae expansion. In contrast to infection initiation, fungal hyphae spreading appears to be restricted by similar mechanisms induced during SAR as it is attested by the requirement of salicylic acid accumulation and by the correlating apoplastic accumulation of PR1 proteins. These results provide evidence for the activation of a set of at least two regulatory pathways during flowering growth. This activation leads to the induction of mechanisms which control fungal development by affecting the ability of the fungus to both infect and colonise plant tissues.