MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein-protein interactions.

Background: The bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein-protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial.

Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified Arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6.

Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA.

Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number.

An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions. Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant.

Biochemical evidence for translational repression by Arabidopsis microRNAs.

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally through RNA silencing, a mechanism conserved in eukaryotes. Prevailing models entail most animal miRNAs affecting gene expression by blocking mRNA translation and most plant miRNAs, triggering mRNA cleavage. Here, using polysome fractionation in Arabidopsis thaliana, we found that a portion of mature miRNAs and ARGONAUTE1 (AGO1) is associated with polysomes, likely through their mRNA target.

Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions.

The Erwinia chrysanthemi genome is predicted to encode three systems, Nif, Isc and Suf, known to assist Fe/S cluster biogenesis and the CsdAE cysteine desulphurase. Single iscU, hscA and fdx mutants were found sensitive to paraquat and exhibited reduced virulence on both chicory leaves and Arabidopsis thaliana. Depletion of the whole Isc system led to a pleiotropic phenotype, including sensitivity to both paraquat and 2,2'-dipyridyl, auxotrophies for branched-chain amino acids, thiamine, nicotinic acid, and drastic alteration in virulence.

Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.

Here we describe a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis, some of which direct the cleavage of endogenous mRNAs. These siRNAs correspond to both sense and antisense strands of a noncoding RNA (At2g27400) that apparently is converted to double-stranded RNA and then processed in 21 nt increments. These siRNAs differ from previously described regulatory small RNAs in two respects.

The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development.

MicroRNAs (miRNAs) are endogenous 21-24-nt RNAs that can down-regulate gene expression by pairing to the messages of protein-coding genes to specify mRNA cleavage or repression of productive translation. They act within the RNA-induced silencing complex (RISC), which in animals contains a member of the Argonaute family of proteins. In the present study, we show that Arabidopsis ago1 mutants have increased accumulation of mRNAs known to be targeted for cleavage by miRNAs.

The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing.

MicroRNAs (miRNAs) are 21-24 nucleotides long molecules processed from imperfect double-stranded RNAs (dsRNAs). They regulate gene expression by targeting complementary mRNA for cleavage or interfering with their translation. In Arabidopsis, point mutations in or short truncations of the nuclear DICER-LIKE1 (DCL1) or HEN1 protein reduce miRNA accumulation and increase uncleaved target mRNAs accumulation, resulting in developmental abnormalities.

cDNA-AFLP display for the isolation of differentially expressed genes during chicory root development.

To identify genes expressed during root enlargment during the early vegetative growth of chicory (Cichorium intybus L.), we used the cDNA-AFLP technology. The radial pattern of chicory roots was investigated by histological analysis to determine the spatial vascular cambium setting.

Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance.

In animals, double-stranded short interfering RNA (siRNA) and single-stranded microRNA (miRNA) regulate gene expression by targeting homologous mRNA for cleavage or by interfering with their translation, respectively. siRNAs are processed from injected or transgene-derived, long, perfect double-stranded RNA (dsRNA), while miRNAs are processed from short, imperfect dsRNA precursors transcribed from endogenous intergenic regions. In plants, both siRNAs and miRNAs activate cleavage of homologous RNA targets, but little is known about the genes controlling their production or action.

High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants.

Posttranscriptional gene silencing (PTGS) in transgenic plants is an epigenetic form of RNA degradation related to PTGS and RNA interference (RNAi) in fungi and animals. Evidence suggests that transgene loci and RNA viruses can generate double-stranded RNAs similar in sequence to the transcribed region of target genes, which then undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs. The silent state in transgenic plants and in Caenorhabditis elegans can spread systemically, implying that mobile silencing signals exist.