Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). ...Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.
Plant genomes contain large numbers of cell surface leucine-rich repeat (LRR) and intracellular nucleotide binding (NB)-LRR immune receptors encoded by resistance (R) genes that recognize specific ...pathogen effectors and trigger resistance responses. The unregulated expression of NB-LRR genes can trigger autoimmunity in the absence of pathogen infection and inhibit plant growth. Despite the potential serious consequence on agricultural production, the mechanisms regulating R-gene expression are not well understood. We identified microRNA (miRNA) progenitor genes precursor transcripts, and two miRNAs nta-miR6019 (22-nt) and nta-miR6020 (21-nt) that guide cleavage of transcripts of the Toll and Interleukin-1 receptor-NB-LRR immune receptor N from tobacco that confers resistance to tobacco mosaic virus (TMV). We further showed that cleavage by nta-miR6019 triggers RNA-dependent RNA polymerase 6- and ribonuclease Dicer-like 4-dependent biogenesis of 21-nt secondary siRNAs "in phase" with the 22-nt miR6019 cleavage site. Furthermore, we found that processing of the 22-nt nta-miR6019 depended on an asymmetric bulge caused by mismatch in the nta-miR6019 precursor. Interestingly, coexpression of N with nta-miR6019 and nta-miR6020 resulted in attenuation of N-mediated resistance to TMV, indicating that these miRNAs have functional roles in NB-LRR regulation. Using a bioinformatics approach, we identified six additional 22-nt miRNA and two 21-nt miRNA families from three Solanaceae species—tobacco, tomato, and potato. We show that members of these miRNA families cleave transcripts of predicted functional R genes and trigger production of phased secondary 21-nt siRNAs. Our results demonstrate a conserved role for miRNAs and secondary siRNAs in NB-LRR/LRR immune receptor gene regulation and pathogen resistance in SOLANACEAE:
The contribution of epigenetic variation to phenotypic variation is unclear. Imprinted genes, because of their strong association with epigenetic modifications, represent an opportunity for the ...discovery of such phenomena. In mammals and flowering plants, a subset of genes are expressed from only one parental allele in a process called gene imprinting. Imprinting is associated with differential DNA methylation and chromatin modifications between parental alleles. In flowering plants imprinting occurs in a seed tissue - endosperm. Proper endosperm development is essential for the production of viable seeds. We previously showed that in Arabidopsis thaliana intraspecific imprinting variation is correlated with naturally occurring DNA methylation polymorphisms. Here, we investigated the mechanisms and function of allele-specific imprinting of the class IV homeodomain leucine zipper (HD-ZIP) transcription factor HDG3. In imprinted strains, HDG3 is expressed primarily from the methylated paternally inherited allele. We manipulated the methylation state of endogenous HDG3 in a non-imprinted strain and demonstrated that methylation of a proximal transposable element is sufficient to promote HDG3 expression and imprinting. Gain of HDG3 imprinting was associated with earlier endosperm cellularization and changes in seed weight. These results indicate that epigenetic variation alone is sufficient to explain imprinting variation and demonstrate that epialleles can underlie variation in seed development phenotypes.
Imprinted gene expression occurs during seed development in plants and is associated with differential DNA methylation of parental alleles, particularly at proximal transposable elements (TEs). ...Imprinting variability could contribute to observed parent-of-origin effects on seed development. We investigated intraspecific variation in imprinting, coupled with analysis of DNA methylation and small RNAs, among three Arabidopsis strains with diverse seed phenotypes. The majority of imprinted genes were parentally biased in the same manner among all strains. However, we identified several examples of allele-specific imprinting correlated with intraspecific epigenetic variation at a TE. We successfully predicted imprinting in additional strains based on methylation variability. We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation. Our data demonstrate that epiallelic variation and genomic imprinting intersect to produce novel gene expression patterns in seeds.
...because of the transposition, some p-values in Figure 1—source data 5 were calculated incorrectly (a side-by-side comparison is shown in Supplementary file 1, file held on figshare under ...http://dx.doi.org/10.6084/m9.figshare.1409445). The correction does not change any of the conclusions that we drew from these data, which were ‘Imprinting calls based on whole-genome mRNA-seq were validated by sequencing or performing CAPs digestion on RT-PCR amplicons of 29 genes from independently isolated embryo and endosperm RNA samples (Figure 1—figure supplement 2, Figure 1—source data 5); results were mostly consistent with the mRNA-seq data’.
Two fundamental types of polyploids are known: allopolyploids, in which different parental chromosome sets were combined by ancestral hybridization and duplication; and autopolyploids, which derive ...from multiplication of the same chromosome set. In autopolyploids, changes to the nuclear environment are not as profound as in allopolyploids, and therefore the effects of genome doubling on gene regulation remain unclear. To investigate the consequences of autopolyploidization per se, we performed a microarray analysis in three equivalent lineages of matched diploids and autotetraploids of Arabidopsis thaliana. Additionally, we compared the expression levels of GFP transgenes driven by endogenous enhancer elements (enhancer traps) in diploids and autotetraploid of 16 transgenic lines. We expected that true ploidy-dependent changes should occur in independently derived autopolyploid lineages. By this criterion, our microarray analysis detected few changes associated with polyploidization, while the enhancer-trap analysis revealed altered GFP expression at multiple plant life stages for 25% of the lines tested. Genes on individual traps were coordinately regulated while endogenous gene expression was not affected except for one line. The unique sensitivity of enhancer traps to ploidy, in contrast to the observed stability of genes, could derive from lower complexity of regulatory pathways acting on traps versus endogenous genes.
► Hundreds of novel imprinted genes have been identified through genomic approaches. ► Imprinted genes can regulate nutrient transfer to the seed. ► Studying imprinting is an entry point into diverse ...biological processes.
Until recently, only a handful of imprinted genes, or genes with parent-of-origin dependent expression patterns, were known in plants. Study of these genes yielded key insights into mechanisms of monoallelic expression and imprinted gene function. The recent application of high throughput sequencing to the study of imprinting has confirmed that many previous findings are relevant on a genome-wide scale. The catalogue of imprinted genes in monocots and dicots now includes a large number of transcription factors, chromatin related genes, and metabolic or hormone biosynthesis enzymes. Interpretation of allele specific expression data remains a challenge, with careful validation of candidate imprinted genes necessary.
This protocol describes whole genome bisulfite-sequencing library preparation from plant tissue and subsequent data analysis. Allele-specific methylation analysis and genome-wide identification of ...differentially methylated regions are additional features of the analysis procedure.
Tomato bushy stunt virus (TBSV) coat protein (CP) replacement vectors have been used previously to silence transgenes (e.g., the green fluorescent protein gene) but have not been effective for ...silencing endogenous plant genes. New TBSV vectors which retained the CP gene were developed by engineering an XhoI restriction site in three positions (3f, CEB, and CEA) of the pTBSV-100 infectious clone. Magnesium chelatase (ChlH) and phytoene desaturase (PDS) were chosen as targets for endogenous gene silencing. Initial experiments using CP replacement vectors with a 230-bp sense or antisense ChlH insert gave a silencing phenotype prominent only in the first new leaves above those inoculated. No silencing phenotype was apparent beyond these leaves whereas, for PDS, no silencing phenotype was observed. When plants were inoculated with the XhoI insert vectors containing ChlH and PDS sequences, plants showed a silencing phenotype extensively throughout the challenged plant, indicating an improved ability for virus movement and silencing in Nicotiana benthamiana host plants. Silencing efficiencies were quantified using real-time reverse-transcription polymerase chain reaction, indicating specific silencing effects of each individual silencing vector. Only one recombinant vector (pPD-3f5), where the XhoI insert was at the 3' end of the CP gene, failed to give effective silencing. Here, we show that our new CP-retaining TBSV vectors (CEA-CEB) form typical TBSV virions, retain silencing inserts of variable lengths (110 to 260 nucleotides), and can systemically silence endogenous genes in N. benthamiana.
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