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.

DNA reassociation kinetics in diploid and phylogenetically tetraploid cyprinidae.

Four diploid and three phylogenetically tetraploid Cyprinidae (Ostariophysi) have been characterized as for nuclear DNA content, modal chromosome number and DNA reassociation kinetics (hydroxyapatite chromatography). Among the diploid species nuclear DNA content (10(-12) g DNA/2C) was 1.62 for Tinca tinca, 1.87 for Scardinius erythrophthalmus, 2.53 for Leuciscus cephalus and 2.75 for Alburnus alburnus, while the phylogenetically tetraploid species Carassius auratus, Barbus barbus and Cyprinus carpio attained 3.40, 3.66 and 3.80 respectively. Modal chromosome number was 2n = 48-50 for diploid individuals and 2n = 100-104 for phylogenetically tetraploid ones. In all the species 5--8% of the genome is represented by highly repetitive and foldback DNA. In DNA reassociation kinetics of phylogenetically tetraploid Cyprinidae a distinct plateau separates an intermediate reassociating sequence fraction (about 22% of the genome; with average repetition frequencies between 1,000 and 1,400) from a slow reassociating one (unique DNA; about 72% of the genome). These two genome fractions are not clearly distinguishable from each other in Cot curves of the diploid Cyprinidae, where a similar plateau is not evident. Since simple ploidy changes are not expected to affect DNA reassociation kinetics we suggest a different evolution in the genome organization of the two ploidy groups. Some possible hypotheses are discussed.

Bacterial plant oncogenes: the rol genes' saga.

The rol genes are part of the T-DNA which is transferred by Agrobacterium 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 the rol 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.

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.

Root 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.

The plant oncogene rolD stimulates flowering in transgenic tobacco plants.

The Agrobacterium rhizogenes T-DNA oncogene rolD under the control of its own 5' regulatory region was transferred to day-neutral tobacco plants. The main trait induced by rolD in transgenic plants is a striking precocity in flower setting and a strong enhancement of the flowering potential. In rolD plants, early flowering is followed by the very rapid growth of numerous lateral inflorescences. The analysis of several morphological and histological parameters suggests that some characteristic morphological abnormalities observed in rolD plants can be accounted for by their early reproductive phase transition and points to the involvement in the transition of a greater portion of the plant body than is the case for untransformed tobacco. The in vitro morphogenic potential of tissues from rolD plants was also tested. Superficial thin cell layer explants from rolD plants show an earlier and much enhanced flower organogenesis, compared to controls, both on flowering and on hormone-free medium.