Cyclic AMP is a well known second messenger which regulates a wide variety of cellular responses in living organisms such as bacteria, fungi, and animals. On the contrary, its presence and its role in plants have been debated for decades. The skepticism was finally overcome with the use of mass spectrometry that provided unequivocal evidence of its presence in higher plants. The information on the biological function of cAMP in plants remains very limited, mainly because its content in plant cells is significantly lower than in other organisms. To date, the cAMP involvement in several processes of higher plants, including cell cycle regulation, growth and reorientation of the pollen tube, seed germination and defense processes has been reported. However, in plants, the mechanisms involved in the cAMP-dependent signal transduction are yet unknown, especially for the failure to identify a kinase that responds specifically to cellular changes in cAMP concentration. Understanding both the biological events specifically attributable to cAMP, and the mechanisms by which these processes are regulated, through the combination of quantitative data with mathematical models, is a challenge for the study of plant signal transduction. In order to obtain more information on the role of cAMP in plants we generated tobacco Bright yellow-2 (TBY-2) lines that constitutively express a non-invasive tool, the “cAMP-sponge”, able to selectively perturb the cAMP concentration (Lefkimmiatis et al, 2009). The cAMP-sponge is composed of two high-affinity cAMP binding domains of the regulatory subunits I beta of human protein kinase A (PKARIbeta). These domains have been engineered to be unable to interact with the catalytic subunit of PKA itself or to homodimerize. This construct binds with high affinity cAMP but not cGMP. The cAMP-sponge in frame with the reporter gene mCherry has been inserted in the binary vector pGreenII (kan) under the control of the strong constitutive promoter CaMV 35S.The construct has been transferred into Agrobacterium tumefaciens GV3101 strain through electroporation and mobilized into TBY-2 cells via A. tumefaciens -mediated transformation. Transgenic TBY-2 lines have been selected in the presence of kanamycin, and several independent transgenic lines obtained. Transformed lines have been analyzed through PCR, RT-PCR and immunoblotting to assess trans-gene integration and mRNA and protein expression. Finally the effect of the lower levels of cAMP on the growth of TBY-2 cells has been analyzed.

Cyclic AMP is an important second messenger in all living organisms; however its existence and its role in plants has been debated for long time. Nowadays it is clear that also in plants, cAMP is involved in several physiological processes, including cell cycle regulation, seed germination, growth and reorientation of the pollen tube. It has also been reported that cAMP is involved in the activation of defence mechanisms in response to biotic and abiotic stresses. In order to verify the involvement of cAMP in plant stress response the effect of moderate heat stress (35°C) in wild type and transgenic tobacco BY-2 cells, with reduced intracellular level of cAMP (cAS cells), has been studied. The obtained results show that heat stress negatively affects cell growth inhibiting cell cycle progression and cell expansions and increasing cell death. cAMP depletion makes tobacco BY-2 cells more susceptible to heat stress determining an increase in cell death. The involvement of heat shock proteins and antioxidants in determining the high susceptibility of cAS cells to heat stress has been also studied and discussed

Pathogens exploit different infection st rategies to infect plants and stomat a represent a direct pathway by which microbes can enter the plant. To counter this, guard cells have evolved the ability to detect conserved microbial molecules and close stomata. In our attempts to understa nd the full nature of the interactions that occur between a potential pathogen and its host, we are focussing on stomatal immunity. The plasma membrane receptor FLS2 confers plant immunity through perceptio n of bacterial flagellin (flg22), which tr iggers stomatal closure. Despite the importance of stomatal immunity, the pathways underlying stomatal behavior and their interaction with immunity control remain largely unknown. To addre ss this, we determined the genetic fr amework of stomatal immunity. I will present our high-throughput imaging pipeline of detecting stomatal apertures, and will discuss some of our recent findings of stomatal immunity control. This includes some recent works on ESCRT-I components that are required for plant immunity at the level of stomata closure. Taken to gether, advanced bioimaging allows us to tackle the role of stomatal closure involved in the in teraction between plants and microbes.

Mitochondria are considered to be the powerhouse of the cell. In mitochondria the degradation of the carbohydrates is coupled with the synthesis of high-energy molecules such as ATP, which powers up the vast majority of chemical reactions of the cell. The biogenesis and function of mitochondria is the result of coordination between the nuclear genome, where there is more than 95% of mitochondrial genes, and the mitochondrial genome. This coordination is particularly rigorous also because the protein complexes both of respiratory chain (OXPHOS) and mitochondrial ribosomes are assembled according to wellestablished stoichiometric relationships. In higher eukaryotes has been reported that this type of coordination is achieved mainly at the transcriptional level. Various experimental observations suggest the presence of a fine-tuned communication between mitochondrial and nuclear genomes that results in the interdependent expression of OXPHOS genes and ribosomal genes encoded by mtDNA and nuclear genome. Having previously identified, by using a series of bioinformatics analysis, a group of five DNA motifs that, for the distribution and frequency in putative regulatory regions of OXPHOS, TCA, and ribosomal genes, could be regarded as excellent regulatory sequences, we decided to validate this analysis by means of the One-Hybrid Assay. The One-hybrid assay was performed using two different DNA motifs as bait, the Site II motif (ref) and the Ac/tTGT motif. This analysis showed that some proteins are able to interact "in vivo" with those motifs. We have identified a transcription factor belonging of the family AP2/ERF/B3 (AtERF#115) that binds Site II motif. The transcription factor is part of the regulative cascade of ethylene, this finding could help explain the mechanisms by which ethylene is able to influence the respiration (e.g. climacteric). We have also identified two transcription factors belonging to the family of bZIP (AtbZIP18 and AtbZIP52), when we used the Ac/tTGT motif as a DNA-bait. The transcription factors are involved in the process of growth and development of the plant. The family of bZIP transcription factors is required in the process of growth and development of the plant and therefore it can be possible that AtbZIP18 and AtbZIP52 may be involved in response to those processes.

The widespread occurrence of epigenetic alterations as a consequence of polyploidization in plants indicates that DNA methylation systems may be perturbed by polyploidy changes. DNA methylation genes are involved in many vital developmental and physiological processes of plants. In this work, we seek novel information on epigenetic consequences of autopolyploidization in alfalfa (Medicago sativa). In particular, genes responsible for the DNA methylation status are studied in 2x, 3x and 4x genotypes progenies obtained by crossing two 2x plants that produce both n and 2n eggs and pollen, respectively. To identify DNA methyltransferase genes and their expression patterns we examined some of the elements of three major DNA methyltransferase families MET1, CMT and DRM. Methylation at CG nucleotides is maintained in plants by the the enzymes of the MET1 family typical of higher eukariotes. The second family, called Domain Rearranged Methyltransferase (DRM) has characteristic rearrangement of conserved motifs in the catalytic domain and probably catalyzes methylation of native DNA. The chromomethylases (CMT) are unique of higher plants. These enzymes maintain methylation of CHG trinucleotides. In silico searches have lead to the identification of M. sativa methyltransferase genes homologous to known plant methyltransferase genes. Oligonucleotides have been designed from these sequences in order to analize the expression pattern of these genes. Gene expression changes induced by polyploidization are being investigated using qRT-PCR and expression data will be validated and jointly analyzed to identify ploidy-affected genes.

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5' UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its e V ects on the activity of the enhancer in transiently transfected Drosophila S2R+ cell line. Moreover, we show that the new insulator is able to a V ect signi W cantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.

Cyclic AMP is a well known second messenger involved in different cellular responses in all living organisms. In higher plants, its role as second messenger has been widely debated, due to its low content and to the difficulty of measuring it. However, its natural occurrence and the existence of adenylyl-cyclases and cAMP phosphodiesterases, that constitute the on-off switches needed for its action as second messenger, have been demonstrated. Data accumulated in the last three decades show the involvement of cAMP in several processes of higher plants, including cell cycle regulation, growth and reorientation of the pollen tube, seed germination and defense responses. However little is known on the mechanisms involved in the cAMP-dependent signal transduction in plants. To shed light on cAMP role in plant signaling pathways, Arabidopsis plants, whose genome has been completely sequenced, and tobacco Bright Yellow-2 (BY-2) cells, that are highly synchronizable, have been chosen as model systems. For this purpose, both Arabidopsis plants and tobacco BY-2 cells were transformed with the "cAMP-sponge", a non invasive tool able to selectively reduce cAMP concentration (Lefkimmiatis et al, 2009). The cAMP-sponge is composed of two high-affinity cAMP binding domains of the regulatory subunits I beta of human protein kinase A (PKARIbeta) that specifically bind cAMP and not cGMP. The construct containing the cAMP-sponge in frame with the reporter gene mCherry were mobilized into TBY-2 cells and Arabidopsis via A. tumefaciens -mediated transformation. Transgenic TBY-2 calli and Arabidopsis plants were selected in the presence of appropriate antibiotics, and several independent transgenic lines were obtained. Trans-gene integration and its expression in Arabidospsis and tobacco transformed lines were verified by PCR, RT-PCR and immunoblotting analyses. The low levels of cAMP negatively affected the growth of TBY-2 cells whereas no distinctive phenotype was observed in Arabidobsis plants. However, a stress condition was evidenced for both tobacco BY-2 cells and Arabidopsis plants, as shown by the alteration of their cellular redox state, analysed by ascorbate and glutathione measurements

A set of 67 novel LTR-retrotransposon has been identified by in silico analyses of the Culex quinquefasciatus genome using the LTR_STRUC program. The phylogenetic analysis shows that 29 novel and putatively functional LTR-retrotransposons detected belong to the Ty3/gypsy group. Our results demonstrate that, by considering only families containing potentially autonomous LTR-retrotransposons, they account for about 1% of the genome of C. quinquefasciatus. In previous studies it has been estimated that 29% of the genome of C. quinquefasciatus is occupied by mobile genetic elements. The potential role of retrotransposon insertions strictly associated with host genes is described and discussed along with the possible origin of a retrotransposon with peculiar Primer Binding Site region. Finally, we report the presence of a group of 38 retrotransposons, carrying tandem repeated sequences but lacking coding potential, and apparently lacking ‘‘master copy’’ elements from which they could have originated. The features of the repetitive sequences found in these non- autonomous LTR retrotransposons are described, and their possible role discussed. These results integrate the existing data on the genomics of an important virus-borne disease vector.

Background: Herpes Simplex virus, type 1 (HSV-1) commonly produces lytic mucosal lesions. It invariably initiates latent infection in sensory ganglia enabling persistent, lifelong infection. Acute HSV-1 encephalitis is rare and definitive evidence of latent infection in the brain is lacking. However, exposure untraceable to encephalitis has been repeatedly associated with impaired working memory and executive functions, particularly among schizophrenia patients. Method: Patterns of HSV-1 infection and gene expression changes were examined in human induced pluripotent stem cell (iPSC)-derived neurons. Separately, differences in blood oxygenation level dependent (BOLD) responses to working memory challenges using letter nback tests were investigated using functional MRI (fMRI) among schizophrenia cases/controls. Results: HSV-1 induced lytic changes in iPSC-derived glutamatergic neurons and neuroprogenitor cells. In neurons, HSV-1 also entered a quiescent state following co-incubation with anti-viral drugs, with distinctive changes in gene expression related to functions such as glutamatergic signaling. In the fMRI studies, main effects of SZ (p=0.001) and HSV-1 exposure(1-back, p=1.76x10-4; 2-back, p=1.39x10-5) on BOLD responses were observed. We also noted increased BOLD responses in the fronto-parietal, thalamus and midbrain regions among HSV-1 exposed schizophrenia cases and controls, compared with unexposed persons. Conclusions: The lytic/quiescent cycles in iPSC-derived neurons indicate that persistent neuronal infection can occur, altering cellular function. The fMRI studies affirm the associations between non-encephalitic HSV-1 infection and functional brain changes linked with working memory impairment. The fMRI and iPSC studies together provide putative mechanisms for the cognitive impairments linked to HSV-1 exposure.