Use of a transgenic early flowering approach in apple (Malus?×?domestica Borkh.) to introgress fire blight resistance from cultivar Evereste

The long juvenile phase of Malus spp. has always been a major drawback for the rapid introgression of agronomically relevant traits (e.g. disease resistances) from wild apples into domestic apple cultivars (M.?×?domestica Borkh.). Several agro-technical approaches have been investigated but none was able to reduce the juvenile phase to less than 18?months. Recently, an early flowering transgenic line named T1190 was obtained by over-expressing the BpMADS4 gene from silver birch (Betula pendula Roth.) in the apple cultivar Pinova. In this study, we report on the acceleration of the first two introgression cycles (F1 and BC??1) of the highly efficacious fire blight resistance locus Fb_E from the ornamental apple cultivar Evereste, using the BpMADS4-transgenic line T1190. A background selection based on simple sequence repeats (SSR) markers regularly distributed over the apple genome was applied to the 24 BC??1 seedlings carrying the BpMADS4 transgene and the Fb_E locus. Two early flowering BC??1 seedlings estimated to carry less than 15% of the genome of Evereste were identified. They are currently (July 2011) being used in reciprocal crosses with the apple cultivar Royal Gala to continue the introgression of the Fb_E locus. Additionally, the strong phenotypic effect of the Fb_E locus from Evereste was confirmed by artificially inoculating a T1190?×?Evereste F1 progeny with the causal agent of fire blight, Erwinia amylovora. Possible ways of enhancing the fast introgression of disease resistance genes in domestic apple using the transgenic line T1190 are discussed.     

Development of a transgenic early flowering pear (Pyrus communis L.) genotype by RNAi silencing of PcTFL1-1 and PcTFL1-2

Trees require a long maturation period, known as juvenile phase, before they can reproduce, complicating their genetic improvement as compared to annual plants. 'Spadona', one of the most important European pear (Pyrus communis L.) cultivars grown in Israel, has a very long juvenile period, up to 14?years, making breeding programs extremely slow. Progress in understanding the molecular basis of the transition to flowering has revealed genes that accelerate reproductive development when ectopically expressed in transgenic plants. A transgenic line of 'Spadona', named Early Flowering-Spadona (EF-Spa), was produced using a MdTFL1 RNAi cassette targeting the native pear genes PcTFL1-1 and PcTFL1-2. The transgenic line had three T-DNA insertions, one assigned to chromosome 2 and two to chromosome 14 PcTFL1-1 and PcTFL1-2 were completely silenced, and EF-Spa displayed an early flowering phenotype: flowers developed already in tissue culture and on most rooted plants 1-8?months after transfer to the greenhouse. EF-Spa developed solitary flowers from apical or lateral buds, reducing vegetative growth vigor. Pollination of EF-Spa trees generated normal-shaped fruits with viable F1 seeds. The greenhouse-grown transgenic F1 seedlings formed shoots and produced flowers 1-33?months after germination. Sequence analyses, of the non-transgenic F1 seedlings, demonstrated that this approach can be used to recover seedlings that have no trace of the T-DNA. Thus, the early flowering transgenic line EF-Spa obtained by PcTFL1 silencing provides an interesting tool to accelerate pear breeding.     

The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.     

Synthesis and aminoacyl-tRNA synthetase inhibitory activity of aspartyl adenylate analogs

Three nonhydrolyzable aspartyl adenylate analogs have been prepared and tested as inhibitors of E. coli aspartyl-tRNA synthetase. 5'-O-[N-(L-Aspartyl)sulfamoyl]adenosine is a potent competitive inhibitor (K(i) = 15 nM) whereas L-aspartol adenylate is a weaker inhibitor (K(i) = 45 microM) with respect to aspartic acid. The corresponding ketomethylphosphonate (a novel isosteric replacement) is also a strong inhibitor (K(i) = 123 nM).     

Serum increases CYP1A1 induction by 3-methylcholanthrene

Mice with a targeted null mutation of the serotonin 5-HT(2C) receptor gene exhibit hyperphagia that leads to a late-onset obesity. Here we show that oxygen consumption was decreased in fed and fasted obese mutants. No phenotypic differences were observed in uncoupling protein-1 (UCP-1) mRNA levels in brown adipose tissues and UCP-3 mRNA in skeletal muscle. UCP-2 mRNA levels were significantly increased in white adipose tissue (4-fold) and skeletal muscle (47%) in older obese mutant mice, whereas UCP-2 mRNA in liver are significantly increased in both young lean (54% increase) and older obese (52% increase) mutant mice. In contrast, 5-HT(2C) receptor mutants displayed age-dependent decreases in beta 3-adrenergic receptor (beta 3-AR) mRNA levels in white adipose tissue, however, no such changes were observed in brown adipose tissue. These results indicate that a mutation of 5-HT(2C) receptor gene leads to a secondary decrease in beta 3-AR gene expression that is related to enhanced adiposity.     

Laminin-induced capping and receptor expression at cell surface in a rat rhabdomyosarcoma cell line: Involvement in cell adhesion and migration on laminin substrates

The present study reveals the dynamic distribution of membrane laminin receptors induced by laminin binding in a rat rhabdomyosarcoma cell line RMS S4. The treatment of the cells with soluble laminin did not modify cell adhesion to laminin-coated substrates in in vitro attachment assays. Fluorescent labeling of membrane-bound laminin revealed that occupied receptors were induced to cluster and cap. New free membrane binding sites were made evident after capping of bound laminin by a double labeling technique. Cytochalasin D (CD) treatment prevented the capping process. The adhesion of CD-treated cells to laminin-coated substrates was inhibited by cell preincubation with soluble laminin. Cycloheximide treatment had no effect on the ability of RMS S4 cells to adhere to adsorbed laminin after preincubation in the presence of soluble laminin. These results taken as a whole suggest that free receptors may arise from an intracellular pool that could be maintained by membrane receptor recycling. Since capping and motility seem related events, migration of RMS S4 cells on laminin was studied in the agarose drop assay. Immobilized laminin stimulated basic cell motility by more than 200%. E8 laminin fragment retained partially the motility stimulating property of laminin while P1 pepsinic fragment had no effect. The presence of constantly available receptors at the cell surface could be determinant in the ability of cells to migrate on laminin substrates.