Maria Luisa Mauro

The role of auxin in hairy root induction

SummaryWe have investigated the relative role of auxin and of Agrobacterium rhizogenes T-DNA in the induction of hairy roots. By infecting carrot discs with suitably constructed bacterial strains containing different T-DNA complements, we have shown that both auxin and the presence of T-DNA in the carrot cells are required for root growth on the discs. Auxin added alone or in combination with cytokinin is not sufficient to induce rooting on uninfected discs. Also cells transformed by T-DNA containing only auxin synthetic genes very rarely differentiate into roots. On the other hand auxin is necessary for hairy root induction since A. rhizogenes devoid of T-DNA-borne auxin genes is not capable of eliciting symptoms in the absence of hormone. Auxin is not required for either T-DNA transfer or T-DNA expression in the transformed host. Cells infected in the absence of auxin, which do not respond by rooting, do contain T-DNA whose expression is shown by the synthesis of hairy root opines; subsequent addition of auxin to these quiescent transformed cells results in root development. A model for hairy root induction where the action of T-DNA is envisaged as conferring auxin responsiveness to the transformed cells is discussed.

Localization of agropine-synthesizing functions in the TR region of the root-inducing plasmid of Agrobacterium rhizogenes 1855.

The region of the Ri plasmid pRi 1855 that encodes agropine synthesis has been identified through its sequence homology with the equivalent genes of the octopine Ti plasmid pTi ACH5. Interestingly the agropine genes lie outside the so-far identified T-DNA of pRi 1855, and are separated from this latter by a long sequence of non integrated plasmid DNA. The presence of this additional T-DNA (TRight DNA) in hairy roots was demonstrated by Southern blot analysis and by the presence of specific transcripts. The genes for agropine synthesis are arranged in the Ri plasmid in a reversed order as compared to their orientation in the Ti plasmid pTi ACH5.

Identification of the genetic locus responsible for non-polar root induction by Agrobacterium rhizogenes 1855.

SummaryRoot proliferation can be induced by Agrobacterium rhizogenes on carrot discs both on the apical and basal surface (facing the root apex and base, respectively) or on the apical surface only, depending on the bacterial strain. This differential response on the two surfaces is denominated polarity. We correlate the polarity of some strains with the absence of an Ri plasmid genetic locus, present in non polar strains such as A. rhizogenes 1855, which bears sequence homology with the auxin genes of Ti plasmid T-DNA. We demonstrate that this locus is responsible for root induction on the basal surface since insertion of a transposon in this region of pRi1855 induces polarity in this strain.

Sirtinol Treatment Reduces Inflammation in Human Dermal Microvascular Endothelial Cells

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC), a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement.

Bacterial plant oncogenes: the rol genes' saga.

Therol genes are part of the T-DNA which is transferred byAgrobacterium rhizogenes in plant cells, causing neoplastic growth and differentiation. Each of these bacterial oncogenes deeply influences plant development and is finely regulated once transferred into the plant host. Both from the study of the effects and biochemical function of therol genes and from the analysis of their regulation, important insight in plant development can be derived. Some of the most intriguing aspects of past, current and future research on this gene system are highlighted and discussed.

THE ROLD GENE FROM AGROBACTERIUM RHIZOGENES IS DEVELOPMENTALLY REGULATED IN TRANSGENIC TOBACCO

SummaryTherolD gene fromAgrobacterium rhizogenes has recently been shown to induce striking precocity of flowering in transgenic tobacco. A transcriptional fusion between 578 bp of therolD upstream regulating sequence and the GUS reporter gene has been transferred to tobacco plants and its expression analysed throughout the whole life cycle of the plant. A detailed histological analysis revealed thatrolD is strongly expressed in elongating and differentiating tissues of each organ, from the mature embryo to the adult plant. In organs with determinate growth, such as cotyledons, leaves and floral parts,rolD expression follows the age gradient of the tissues. In mature tissues,rolD expression is high, begins to decrease with ageing and is switched off when senescence occurs.RolD is also active in the vascular system, in the procambium, pith- and receptacular-meristems. Our data show that expression ofrolD is under developmental control as it correlates with, and may represent a molecular marker for, the elongation/expansion and maturation phases of the plant tissues.

Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants

Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world. Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b6/f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (Fv/Fm, rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis.

The rolD oncogene promotes axillary bud and adventitious root meristems in Arabidopsis.

The expression of the Agrobacterium rhizogenes rolD oncogene induces precocious floral transition and strong flowering potential in tobacco and tomato. Here, we describe specific developmental effects induced by expression of rolD in Arabidopsis. We show that floral transition, as histologically monitored, occurred in rolD- plants earlier than in wild type, and this was coupled with a premature and enhanced formation of vegetative and reproductive axillary bud meristems. Furthermore, CYP79F1/SUPERSHOOT/BUSHY (SPS), a gene that negatively controls shoot branching in Arabidopsis and involved in glucosinolate metabolism and in cytokinin and auxin homeostasis, was down-regulated in rolD plants. The multiplication of post-embryonic meristems was also observed in the root system, with enhanced adventitious root formation. This result was confirmed by thin cell layer response in vitro, both under hormone-free and standard rooting conditions. However, the formation of lateral root meristems was not affected by rolD expression. Our results show that rolD accelerates and enhances specific post-embryonic meristems in Arabidopsis.

Specific interaction between a Nicotians tabacum nuclear protein and the Agrobacterium rhizogenes rolB promoter

Summary Nuclear protein extracts from Nicotiana tabacum plants and cells culture were utilized in gel retardation assays and in vitro and in vivo footprinting experiments in order to identify nuclear proteins, that would bind to the 5′ non-coding region of the plant oncogene rolB from the Ri plasmid of Agrobacterium rhizogenes . A factor, RBF1 (Rol Binding Factor) was identified that specifically binds to a sequence located between positions −553 and −530 from the translational start codon. This DNA segment is located within a domain controlling the level of expression of rolB and its inducibility in the non meristematic cells in the root apex (in particular protoderm and root cap). The binding sequence for RBF1 has been identified. The factor is present in extracts prepared from leaves and cells culture of untransformed N. tabacum as well as in transgenic plants, expressing rolA, rolB , and rolC (Spena et al., 1987), in apparendy the same amount. Comparing the in vitro footprinting data with an in vivo high-resolution footprinting, obtained by means of a linear amplification procedure utilizing the polymerase chain reaction, we find substantially the same DNA core sequence recognized by RBF1 protein. Our results point to RBF1 as a plant endogenous factor that could be involved in controlling the level and/or tissue specificity expression of the rolB gene.

Expression of Agrobacterium rhizogenes rolB gene fusions in Escherichia coli: production of antibodies against the RolB protein

Expression of the rolB gene of Agrobacterium rhizogenes TL-DNA is sufficient to trigger root differentiation in transformed plant cells. To investigate the role of RolB in differentiation, a large portion of rolB, comprising about 90% of its C-terminal coding sequence, was cloned into vectors pEX34 and pEA305 in frame with the truncated N termini of the pL-MS2 phage DNA polymerase and, respectively, the ptac-c Its phage lambda repressor gene. Hybrid proteins were expressed from both fusions and the one from pMTBEX1 was utilized to raise antibodies. These antibodies specifically recognize the RolB moiety in both pL-MS2-rolB and ptac-cI-rolB fusions. Unfused, complete RolB protein was obtained by in vitro translation in a rabbit reticulocyte system of a transcript obtained by in vitro transcription of rolB. RolB protein is specifically immunoprecipitated by the antibodies raised against the hybrid protein MS2-RolB.