Roles of Dicer-Like Proteins 2 and 4 in Intra- and Intercellular Antiviral Silencing

RNA silencing is an innate antiviral mechanism conserved in organisms across kingdoms. Such cellular defense involves DICER or DICER-LIKEs (DCLs) that process viral RNAs into small interfering (vsi)RNAs. Plants encode four DCLs which play diverse roles in cell-autonomous virus-induced RNA silencing (known as VIGS) against viral invasion. However, intracellular VIGS can spread between cells, and the genetic basis and involvement of vsiRNAs in non-cell autonomous VIGS remains poorly understood. Here using GFP as a reporter gene together with a suite of DCL RNAi transgenic lines, we show that in addition to well-established activities of DCLs in intracellular VIGS and vsiRNA biogenesis, DCL4 inhibits intercellular VIGS whilst DCL2 is required, likely along with DCL2-processed/dependent vsiRNAs and their precursor RNAs, for efficient VIGS trafficking from epidermal to adjacent cells. DCL4 imposed an epistatic effect on DCL2 to impede cell-to-cell spread of VIGS. Our results demonstrate previously unknown functions for DCL2 and DCL4 which may form a dual defensive frontier for intra- and intercellular silencing to double-protect cells from virus infection in Nicotiana benthamiana

Molecular and functional characterization of SISPL-CNR in tomato fruit ripening and cell death

SlSPL-CNR, an SBP-box transcription factor (TF) gene residing at the epimutant Colourless non-ripening (Cnr) locus, is involved in tomato ripening. This epimutant provides a unique model to investigate the (epi)genetic basis of fruit ripening. Here we report that SlSPL-CNR is a nucleus-localized protein with a distinct monopartite nuclear localization signal (NLS). It consists of four consecutive residues ‘30KRKR33’ at the N-terminal of the protein. Mutation of the NLS abolishes SlSPL-CNR to localize into nucleus. SlSPL-CNR comprises two zinc-finger motifs (ZFMs) within the C-terminal SBP-box domain. Both ZFMs contribute to zinc-binding activity. SlSPL-CNR can induce cell death in tomato and tobacco. Induction of cell death by SlSPL-CNR is dependent on its nuclear localization. However, the two ZFMs have differential impacts on SlSPL-CNR to induce severe necrosis or mild necrotic ringspot. NLS and ZFM mutants cannot complement Cnr fruits to ripen. SlSPL-CNR interacts with SlSnRK1. Virus-induced SlSnRK1 silencing leads to reduction in expression of ripening-related genes and inhibits ripening in tomato. We conclude that SlSPL-CNR is a multifunctional protein that consists of a distinct monopartite NLS, binds to zinc and interacts with SlSnRK1 to affect cell death and tomato fruit ripening

Functional analysis of SlEZ1 a tomato Enhancer of zeste (E(z)) gene demonstrates a role in flower development

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceThe Enhancer of Zeste (E(z)) Polycomb group (PcG) proteins, which are encoded by a small gene family in Arabidopsis thaliana, have been shown to participate to the control of flowering and seed development. For the time being, little is known about the function of these proteins in other plants. In tomato E(z) proteins are encoded by at least two genes namely SlEZ1 and SlEZ2 while a third gene, SlEZ3, is likely to encode a truncated non-functional protein. The analysis of the corresponding mRNA demonstrates that these two genes are differentially regulated during plant and fruit development. We also show that SlEZ1 and SlEZ2 are targeted to the nuclei. These results together with protein sequence analysis makes it likely that both proteins are functional E(z) proteins. The characterisation of SlEZ1 RNAi lines suggests that although there might be some functional redundancy between SlEZ1 and SlEZ2 in most plant organs, the former protein is likely to play specific function in flower development

Characterisation of epigenetic modifications during plant development in wild and cultivated tomato species

Tomato is currently used as a model system to study fruit development and quality. Tomato fruit development can be divided in 3 distinct phases, namely cell division, cell elongation and fruit ripening. These developmental phases lead to massive changes of gene expression pattern. To analyse the potential role of epigenetic mechanisms during the development of tomato plant and fruit, genomic DNA methylation analysis were performed demonstrating global and locus specific variations of CG and CXG methylation pattern in fruits of the cultivated tomato. The comparative analysis of DNA methylation in wild and cultivated tomato species indicate contrasted situations, characterized by different pattern of DNA methylation at repetitive loci such as the 5s rDNA and various retrotransposons. In addition, the molecular analysis of genes encoding the Polycomb (PcG) proteins demonstrates a genetic variability at these loci. The functional consequences of these observations are currently investigated

Analysis of DNA methylation levels in wild and cultivated tomato species

Tomato plants are used as a model system to study fruit development and quality. Tomato fruit development can be divided into three main phases: cell division, cell elongation and fruit ripening. All these phases are associated with deep changes at the gene expression as well as at the epigenetic level. Cytosine methylation is an epigenetic mark associated with genetic inactivity and heterochromatin state in higher eukaryotes, including plants. In plants cytosines are mainly methylated at CG and CXG contexts and, at low levels, at CXX. These methylated sequences are widespread in the whole genome, but are present at higher density at LTR-retrotransposons. Using genomic DNA methylation assays, we analysed the global and locus specific variations of CG and CXG methylation patterns to understand the potential role of epigenetic mechanisms during the development of tomato plants. The analysis showed changes in the cytosine methylation pattern at CG and CXG sites in fruits of the cultivated tomato Ailsa Craig (Teyssier et al., 2008, Planta 228: 391-9). Domesticated and wild tomato species do show remarkable differences in terms of shape, colour, timing and development of fruits, therefore it is very interesting to compare these species also in terms of epigenetic variations. The preliminary comparative analysis of DNA methylation in wild and cultivated tomato species indicated contrasting situations, characterized by different patterns of DNA methylation at repetitive loci such as the 5S rDNA, various retrotransposons and at single loci. Furthermore, the analysis of the abundance of retrotransposons between species revealed differences in distribution and amount between wild and cultivated tomato species. A specific transposon-related case will be described, suggesting that the domestication process of tomato cultivated species might have been associated with DNA epigenetic variations