Interchromatid and interhomolog recombination in Arabidopsis thaliana

Intermolecular recombination events were monitored in Arabidopsis thaliana lines using specially designed recombination traps consisting of tandem disrupted beta-glucuronidase or luciferase reporter genes in direct repeat orientation. Recombination frequencies (RFs) varied between the different lines, indicating possible position effects influencing intermolecular recombination processes. The RFs between sister chromatids and between homologous chromosomes were measured in plants either hemizygous or homozygous for a transgene locus. The RFs in homozygous plants exceeded those of hemizygous plants by a factor of >2, implying that in somatic plant cells both sister chromatid recombination and recombination between homologous chromosomes exist for recombinational DNA repair. In addition, different DNA-damaging agents stimulated recombination in homozygous and hemizygous plants to different extents in a manner dependent on the type of DNA damage and on the genomic region. The genetic and molecular analysis of recombination events showed that most of the somatic recombination events result from gene conversion, although a pop-out event has also been characterized.     

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.     

Single-stranded DNA Plant Pathogens in Eilat

An international conference on ``Inter- and Intracellular Dynamics of ssDNA Plant Pathogens: Implications for Improving Resistance' was sponsored by the United States-Israel Binational Agricultural Research and Deveoplment Fund (BARD) and organized in Eilat, Israel in November 2005. The topic of this meeting was single-stranded plant pathogens, their inter- as well as intra-cellular dynamics and their implications for improving resistance. Most of the talks concentrated on new and very new findings on principles of virus and bacterium-host interactions, studies that no doubt will lead eventually to the establishment of plants resistant to viral and bacterial infections.     

Cross-species functionality of pararetroviral elements driving ribosome shunting

Background

Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5′-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.

Methodology/Principal Findings

Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.

Conclusions/Significance

Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism.We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany.     

The CaMV transactivator/viroplasmin interferes with RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis

Several RNA silencing pathways in plants restrict viral infections and are suppressed by distinct viral proteins. Here we show that the endogenous trans-acting (ta)siRNA pathway, which depends on Dicer-like (DCL) 4 and RNA-dependent RNA polymerase (RDR) 6, is suppressed by infection of Arabidopsis with Cauliflower mosaic virus (CaMV). This effect was associated with overaccumulation of unprocessed, RDR6-dependent precursors of tasiRNAs and is due solely to expression of the CaMV transactivator/viroplasmin (TAV) protein. TAV expression also impaired secondary, but not primary, siRNA production from a silenced transgene and increased accumulation of mRNAs normally silenced by the four known tasiRNA families and RDR6-dependent secondary siRNAs. Moreover, TAV expression upregulated DCL4, DRB4 and AGO7 that mediate tasiRNA biogenesis. Our findings suggest that TAV is a general inhibitor of silencing amplification that impairs DCL4-mediated processing of RDR6-dependent double-stranded RNA to siRNAs. The resulting deficiency in tasiRNAs and other RDR6-/DCL4-dependent siRNAs appears to trigger a feedback mechanism that compensates for the inhibitory effects.     

Biomonitoring the genotoxicity of environmental factors with transgenic plants.

All organisms must react to constantly changing surroundings. Environmental factors are thus powerful forces continuously shaping the genomes of all species. Induced genetic changes can be followed using a biomonitor - a living organism that reacts to a given compound in the environment. A vital but challenging task is identifying organisms with which to study the influence of changing environmental conditions. Plants are especially valuable biomonitors. Here, we describe the use of transgenic plant systems to evaluate the genotoxicity of chemical and radiological compounds. We evaluate the potential of further transgene-based systems for studying somatic and germ-line mutations.     

Morphogenesis of bacteriophage Lambda: Electron microscopy of thin sections

Quantitative measurements on number, size, shape, location and time of appearance of heads and head-related structures in thin sections of induced bacteriophage λ lysogens were performed. Three types of particles can be distinguished: empty heads with a mean diameter of 39 nm (petit λ), heads partially filled with DNA with a mean diameter of 51 nm (grizzled particles) and particles filled with DNA, having a diameter of 47 nm (black particles). Some of the latter ones can be seen with a tail attached. The particles first to appear are the petit λ. A few minutes later grizzled and black particles can be seen. This sequence correspons to measurements of biological activities in lysates, i.e. to plaque-forming units, and to the number of particles which can be packaged with DNA and transformed in vitro to plaque-forming particles, respectively.DNA packaging seems to occur on the boundary area between cytoplasm and DNA plasm. Tails, on the other hand, accumulate near the cytoplasmic membrane.Two steps in DNA packaging can be distinguished, since one type of mutant blocked in DNA packaging (amber in gene A) produces paracrystalline agglomerations of petit λ and clusters of tails while another (amber in gene D) produces grizzled particles in addition.