Genome-Wide Transcriptional Changes and Lipid Profile Modifications Induced by Medicago truncatula N5 Overexpression at an Early Stage of the Symbiotic Interaction with Sinorhizobium meliloti.

Plant lipid-transfer proteins (LTPs) are small basic secreted proteins, which are characterized by lipid-binding capacity and are putatively involved in lipid trafficking. LTPs play a role in several biological processes, including the root nodule symbiosis. The Medicago truncatula nodulin 5 (MtN5) LTP has been proved to positively regulate the nodulation capacity, controlling rhizobial infection and nodule primordia invasion. To better define the lipid transfer protein MtN5 function during the symbiosis, we produced MtN5-downregulated and -overexpressing plants, and we analysed the transcriptomic changes occurring in the roots at an early stage of Sinorhizobium meliloti infection. We also carried out the lipid profile analysis of wild type (WT) and MtN5-overexpressing roots after rhizobia infection. The downregulation of MtN5 increased the root hair curling, an early event of rhizobia infection, and concomitantly induced changes in the expression of defence-related genes. On the other hand, MtN5 overexpression favoured the invasion of the nodules by rhizobia and determined in the roots the modulation of genes that are involved in lipid transport and metabolism as well as an increased content of lipids, especially galactolipids that characterize the symbiosome membranes. Our findings suggest the potential participation of LTPs in the synthesis and rearrangement of membranes occurring during the formation of the infection threads and the symbiosome membrane

Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis.

The success of in vitro plant regeneration and the competence of genetic transformation greatly depends on the genotype of the species of interest. In previous work, we developed a method for the efficient Agrobacterium-mediated genetic transformation via organogenesis of V. vinifera cultivar Thompson Seedless, by using meristematic bulk (MB) as starting tissue. In this study, we applied this method for the regeneration and transformation of MBs obtained from the Italian cultivar Ciliegiolo and two of the commonly used Vitis rootstocks, 110 Richter and Kober 5BB, in comparison with Thompson Seedless. The A. tumefaciens strain EHA105, harbouring pK7WG2 binary vector, was used for the transformation trials, which allowed selection through the enhanced-green fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII) gene. Putative transformed tissues and/or shoots were identified by either a screening based on the eGFP expression alone or its use in combination with kanamycin in the medium. MBs obtained from Thompson Seedless showed the highest regeneration and transformation cell competence, which subsequently allowed the recovery of stably transformed plants. Ciliegiolo, 110 Richter, and Kober 5BB, produced actively growing transgenic calli showing eGFP fluorescence, more consistently on selective media, but had no regenerative competence

Double-Stranded RNA Targeting Dicer-Like Genes Compromises the Pathogenicity of Plasmopara viticola on Grapevine

Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine, attacking all green parts of the plant. The damage is severe when the infection at flowering stage is left uncontrolled. P. viticola management consumes a significant amount of classical pesticides applied in vineyards, requiring efficient and environmentally safe disease management options. Spray-induced gene silencing (SIGS), through the application of exogenous double-stranded RNA (dsRNA), has shown promising results for the management of diseases in crops. Here, we developed and tested the potential of dsRNA targeting P. viticola Dicer-like (DCL) genes for SIGS-based crop protection strategy. The exogenous application of PvDCL1/2 dsRNA, a chimera of PvDCL1 and PvDCL2, highly affected the virulence of P. viticola. The reduced expression level of PvDCL1 and PvDCL2 transcripts in infected leaves, treated with PvDCL1/2 dsRNA, was an indication of an active RNA interference mechanism inside the pathogen to compromise its virulence. Besides the protective property, the PvDCL1/2 dsRNA also exhibited a curative role by reducing the disease progress rate of already established infection. Our data provide a promising future for PvDCL1/2 dsRNA as a new generation of RNA-based resistant plants or RNA-based agrochemical for the management of downy mildew disease in grapevine

po 126 survival probability of human breast carcinoma cells to radiation treatment role of cell fusion and of a syncytin1 homologous protein

Introduction The success of radiotherapy depends on the ability to inhibit tumour growth, and relapse after therapy is determined by cells that retain their clonogenic potential. The radiation sensitivity of isolated tumour cell clones in vitro is routinely determined with clonogenic assays. In solid tumours, however, clonogenic cells are not isolated and we carried out experiments to measure the influences of cell-cell contact on their proliferative potential. To this end we developed a new experimental approach to measure the effects of radiation on tumour cell populations. The observations can be understood with the help of a novel stochastic model with a well-defined biological basis. Material and methods T47D cells (human breast carcinoma) were grown at various concentrations in F(flat)-bottom and V-bottom wells of 96-well culture plates. The spheroid outgrowth method was also used to obtain densely-packed tissue cell cultures. A Gammacell40 irradiator equipped with a 137Cs source was used to treat cell cultures. Cell fusion was assessed by confocal microscopy. Syncytin 1 expression was assessed by RT-PCR and by flow cytometry using an anti-HERV antibody (clone ab7115, Abcam). Results and discussions The probability of cell survival after 8 Gy radiation treatment increased ~4.7 times when the cells were grown in V-bottom wells as compared to cells grown in F-bottom wells (p(survival)=0.0113 and 0.0024, respectively). Microscopic inspections of tissue-like cultures showed that after treatment cell populations were mostly composed of giant cells with multiple nuclei. Cytoplasmic bridges joining different cells were clearly visible. Giant cells and cytoplasmic bridges disappeared at later times (>600 hours) when the cells displayed normal morphology and started to proliferate again. Sequence analysis of cloned RT-PCR products showed that cells expressed a Syncytin 1 homologous protein (Sp). Flow cytometry assays confirmed cytoplasmic expression of Sp and revealed that Sp translocated to the cell surface of irradiated cells committed to death. The fraction of cells surviving 8 Gy treatment was significantly reduced in cultures treated with anti-Sp antibodies. Conclusion Our experimental findings indicate that recovery of breast tumours from radiation is very likely to involve complex pathways that act at the cell population level and that include events of cell fusion mediated by a protein homologous to Syncytin 1

Is biotechnology (more) acceptable when it enables a reduction in phytosanitary treatments? A European comparison of the acceptability of transgenesis and cisgenesis

Reduced pesticide use is one of the reasons given by Europeans for accepting new genetic engineering techniques. According to the advocates of these techniques, consumers are likely to embrace the application of cisgenesis to apple trees. In order to verify the acceptability of these techniques, we estimate a Bayesian multilevel structural equation model, which takes into account the multidimensional nature of acceptability and individual, national, and European effects, using data from the Eurobarometer 2010 73.1 on science. The results underline the persistence of clear differences between European countries and whilst showing considerable defiance, a relatively wider acceptability of vertical gene transfer as a means of reducing phytosanitary treatments, compared to horizontal transfer

Can the two carboxypeptidase inhibitors of tomato act as signalling peptides during fruit development?

The tomato TCMP-1 and TCMP-2 genes code for metallocarboxypeptidase inhibitors, a subclass of the cystine-knot peptides family. The two transcripts display a sequential expression during flower/fruit development, with TCMP-1 highly expressed in flower buds before anthesis and TCMP-2 in ripe fruits. The alteration of their endogenous levels by expressing the TCMP-1 coding sequence under the control of the TCMP-2 promoter revealed that their relative levels are crucial for the fruit set timing (Molesini et al., 2018). The two mature TCMPs are 32% identical and highly similar in structure to the potato carboxypeptidase inhibitor (PCI). Both TCMPs and PCI share structural homology with some mammalian growth factors such as the epidermal growth factor (EGF) and vascular epidermal growth factor (VEGF), and are bioactive in mammalian cells. PCI competes with EGF for the binding to EGF receptor, inhibiting its activation (Blanco-Aparicio et al., 1998); TCMPs inhibit angiogenesis both in vitro in human umbilical vascular cells and in vivo in zebrafish by affecting the VEGF receptor activation (Cavallini et al., 2011; Treggiari et al., 2015). Thus, TCMPs can interfere with growth factor signalling pathways in animal cells. We can speculate that TCMPs could exert a similar activity also in plant cells. By sequence comparison we searched for plant receptors containing extracellular EGF-like domains that could represent good candidates for TCMPs recognition. EGF repeats in plants are found in receptor-like kinases of the wall-associated kinases-type (WAKs-type) and S-locus receptor kinases-type (SRKs-type). WAK and SRK members are shown to be involved in stress responses but also in growth and development

Study of the role of a putative acetylornithine deacetylase on fruit development in Arabidopsis thaliana

The aim of this work is to investigate the role of N-acetylornithine deacetylase (NAOD) in fruit development. To study its biological role, we have generated silenced lines by transforming Arabidopsis plants with a hairpin construct and searched for T-DNA null mutants. We obtained two silenced lines that showed approximately 90%reduction in NAOD transcript level in comparison with wild type. The phenotypical analysis of these lines revealed a reduced growth and a lower number of siliques in comparison with control plants. A T-DNA insertional line was obtained from the SALK collection. The expression of the gene was almost completely abolished in the homozygous mutant line

Study of Mtn5 transcriptional control and of its involvement in Medicago truncatula nodulation pathway

The symbiosis between legumes and rhizobia starts with an exchange of molecular signals between the two partners. In response to the plant-derived flavonoids, bacteria synthesize Nod Factors (NFs), which are able to induce a series of events, such as ion fluxes, root hair deformation and the expression of the early nodulin genes, that eventually lead to the formation of root nodules. We previously demonstrated that MtN5 is an early nodulin, required for the establishment of the symbiosis and also present in mature nodules. In order to investigate the role of MtN5 in root nodules induction pathway, its expression profile during the early stages of infection was studied. MtN5promoter::GUS fusion showed that the promoter was active in epidermis and root hairs a few hours after inoculation, whilst in mature nodules, GUS was observed in the distal zone.In a time course nodulation experiment, MtN5 showed to be co-expressed with early markers of rhizobia infection, such as RIP1, NIN and ENOD11, and resulted to be more precocious than ENOD20 and MtN6. In transgenic adventitious root silenced for MtN5 expression (MtN5hp roots), we observed that upon rhizobia infection the nodulin MtNIN was not induced, whilst ENOD11 was strongly upregulated with respect to control roots. Furthermore, in MtN5hp roots the expression of FLOT4, a nodulin gene known to be involved in the infection thread growth, was unaffected by the inoculation with symbiotic bacteria, in contrast with what observed in control roots

Zu lieben und zu sterben

Traduzione in lingua tedesca del romanzo storico di Andrea Molesini "Non tutti i bastardi sono di Vienna" (Palermo, Sellerio,2010