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

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

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

Tissue dependent variations of DNA methylation and endoreduplication levels during tomato fruit development and ripening.

Tomato fruit cells are characterized by a strong increase in nuclear ploidy during fruit development. Average ploidy levels increased to similar levels (above 50C) in two distinct fruit tissues, pericarp and locular tissue. However, ploidy profiles differed significantly between these two tissues suggesting a tissue-specific control of endoreduplication in tomato fruit. To determine possible relationships between endoreduplication and epigenetic mechanisms, the methylation status of genomic DNA from pericarp and locular tissue of tomato fruit was analysed. Pericarp genomic DNA was characterized by an increase of CG and/or CNG methylation at the 5S and 18S rDNA loci and at gyspsy-like retrotransposon sequences during fruit growth. A sharp decrease of the global DNA methylation level together with a reduction of methylation at the rDNA loci was also observed in pericarp during fruit ripening. Inversely, no major variation of DNA methylation either global or locus-specific, was observed in locular tissue. Thus, tissue-specific variations of DNA methylation are unlikely to be triggered by the induction of endoreduplication in fruit tissues, but may reflect tissue-specific ploidy profiles. Expression analysis of eight putative tomato DNA methyltransferases encoding genes showed that one chromomethylase (CMT) and two rearranged methyltransferases (DRMs) are preferentially expressed in the pericarp during fruit growth and could be involved in the locus-specific increase of methylation observed at this developmental phase in the pericarp

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