A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.     

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.     

The movement protein of cowpea mosaic virus binds GTP and single-stranded nucleic acid in vitro

The movement protein (MP) of Cowpea mosaic virus forms tubules in plasmodesmata to enable the transport of mature virions. Here it is shown that the MP is capable of specifically binding riboguanosine triphosphate and that mutational analysis suggests that GTP binding plays a role in the targeted transport of the MP. Furthermore, the MP is capable of binding both single-stranded RNA and single-stranded DNA in a non-sequence-specific manner, and the GTP- and RNA-binding sites do not overlap.     

Turnover of nitrogenase and leghemoglobin in root nodules of Pisum sativum

Turnover rates of the two nitrogenase components and leghemoglobin in root nodules of pea plants nodulated with Rhizobium leguminosarum were determined with three different methods: 1, Kinetics of 35S incorporation into protein; 2, pulse-chase experiments; 3, chloramphenicol inhibition of bacteroid protein synthesis. Methods 1 and 3 revealed that the turnover rates of the two nitrogenase components and leghemoglobin are identical to the average rate of bacteroid and plant nodule protein turnover. The t1/2 times of component I and II and leghemoglobin were about 2 days. Pulse-chase experiments with 35SO(2-)4 appeared to be rather unsuitable for determination of turnover rates in pea root nodules.     

ag13 is expressed in Alnus glutinosa nodules in infected cells during endosymbiont degradation and in the nodule pericycle

We isolated and characterized an Alnus glutinosa cDNA clone, pAg13, which corresponds to a gene expressed at higher levels in nodules induced by Frankia than in roots. The deduced polypeptide sequence is rich in glutamic acid and proline and contains a putative signal peptide indicating an extracellular location of Ag13. In situ hybridization showed that ag13 is expressed in the pericycle of the nodule vascular bundle and in infected cells that exhibited degradation of the endosymbiont.     

Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur.

Three novel nodulation (Nod) factors were synthesized from chitotetraose and three structurally different fluorescent BODIPY-tagged fatty acids. With fluorescence spectroscopic and microscopic techniques, the following aspects were studied: whether these amphiphilic molecules insert in membranes, whether they transfer between different membranes, and whether they are able to transfer from a membrane to a legume root hair. Fluorescence correlation spectroscopy showed that fluorescent Nod factors are present as monomers in PBS buffer at a concentration of 10 nM, but that when either Triton X-100 micelles or dioleoylphosphatidylcholine (DOPC) vesicles are present, the Nod factors are associated with these particles. With time-correlated single-photon counting fluorescence spectroscopy, it was shown that upon Nod factor insertion in the membrane, the rotation of the fluorescent acyl chain was markedly reduced. A fluorescence resonance energy transfer assay was used to study the transfer of Nod factors from one membrane to the other, or from vesicles to root hairs. Nod factors transfer rapidly between membranes or from vesicles to root hair cell walls. However, they do not flip-flop between membrane leaflets. The results provide novel insights for the mode of secretion and transfer of Nod factors during the early steps of the Rhizobium-legume interaction.     

Non-specific interactions are sufficient to explain the position of heterochromatic chromocenters and nucleoli in interphase nuclei

The organization of the eukaryote nucleus into functional compartments arises by self-organization both through specific protein–protein and protein–DNA interactions and non-specific interactions that lead to entropic effects, such as e.g. depletion attraction. While many specific interactions have so far been demonstrated, the contributions of non-specific interactions are still unclear. We used coarse-grained molecular dynamics simulations of previously published models for Arabidopsis thaliana chromatin organization to show that non-specific interactions can explain the in vivo localization of nucleoli and chromocenters. Also, we quantitatively demonstrate that chromatin looping contributes to the formation of chromosome territories. Our results are consistent with the previously published Rosette model for Arabidopsis chromatin organization and suggest that chromocenter-associated loops play a role in suppressing chromocenter clustering.