(Tissue) P Systems with Vesicles of Multisets

We consider tissue P systems working on vesicles of multisets with the very simple operations of insertion, deletion, and substitution of single objects. With the whole multiset being enclosed in a vesicle, sending it to a target cell can be indicated in those simple rules working on the multiset. As derivation modes we consider the sequential mode, where exactly one rule is applied in a derivation step, and the set maximal mode, where in each derivation step a non-extendable set of rules is applied. With the set maximal mode, computational completeness can already be obtained with tissue P systems having a tree structure, whereas tissue P systems even with an arbitrary communication structure are not computationally complete when working in the sequential mode. Adding polarizations (-1, 0, 1 are sufficient) allows for obtaining computational completeness even for tissue P systems working in the sequential mode.Comment: In Proceedings AFL 2017, arXiv:1708.0622

Specific Tandem Repeats Are Sufficient for Paramutation-Induced Trans-Generational Silencing

<div><p>Paramutation is a well-studied epigenetic phenomenon in which <i>trans</i> communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the <i>b1</i> locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the <i>b1</i> tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the <i>mediator of paramutation1</i> gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous <i>b1</i> alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous <i>b1</i> repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele.</p></div

DNA methylation patterns in transgenic and endogenous <i>b1</i> repeats.

<p>Fragment sizes are indicated by numbers and are in kb. All DNA blots were hybridized with the full <i>b1</i> repeat probe (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen-1003773-g003" target="_blank">Figure 3A</a>). For all blots, genomic leaf DNA was cut with the methylation insensitive enzyme <i>Eco</i>RI to release the ∼7 kb fragment within which DNA methylation was assayed, and with the methylation sensitive enzymes indicated above each blot. Fragments resulting from complete digestion are indicated by open arrows, bands resulting from partial digestion (indicating partial DNA methylation) are indicated by gray arrowheads, while fragments that are the result of no digestion by methylation sensitive enzymes (indicating complete DNA methylation) are indicated by black arrowheads. Representative examples of DNA methylation patterns are shown. <b>A</b>. The progeny of two independent paramutagenic pBΔ transgenic events (3-39 and 3-46) were examined. The plants analyzed were the direct progeny of the primary transgenic plants and the transgenes were not yet exposed to <i>B'</i> or <i>B-I</i>. Results for representative <i>b-N/b-N; TG/-</i> plants (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen-1003773-g002" target="_blank">Figure 2A</a>) are shown. <b>B</b>. <i>B-I</i> was exposed to the pBΔ transgenic event 3-39 for one generation, resulting in light pigmented plants, and then the transgene and the newly induced <i>B'</i>^# were segregated away from each other. The transgene, <i>B'</i>^# and control samples (<i>B'</i> and <i>B-I</i>) were assayed. Circles at the bottom of the lanes indicate plant pigment phenotypes. <b>C</b>. The pBΔ transgenic locus 3-39 was propagated for four generations in a neutral <i>b-N</i> background. <b>D</b>. Summary of the DNA methylation data for the transgenic 3-39 and 3-46 lines, plants in which <i>B-I</i> spontaneously paramutated to <i>B'</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s004" target="_blank">Figure S4</a>), and the previously determined <i>B'</i> and <i>B-I</i> patterns <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773-Haring1" target="_blank">[21]</a>. One and four generations with <i>b-N</i> indicates the 3-39 allele was propagated for one and four generations in the presence of neutral <i>b-N</i> alleles, respectively. The one repeat shown represents all seven repeats. Subscripts indicate specific recognition sites present more than once in each repeat. <i>Alu</i>I (A), <i>Hpa</i>I (H), <i>Pst</i>I (P), <i>Sau</i>3AI (U) and <i>Sau</i>96I (S).</p

DNA methylation of maize <i>b1</i> repeats in <i>Arabidopsis</i>.

<p><b>A</b>. Drawings of constructs used to transform <i>Arabidopsis</i>. The indicated <i>b1</i> fragments were fused to the minimal 35S promoter, the luciferase reporter gene and nopaline synthase (nos) polyadenylation signal. The numbers above the diagrams indicate the genomic location from where the <i>b1</i> sequences are derived relative to the <i>b1</i> transcription start site in kb. Every independent transgenic event with an intact insertion was tested for luciferase activity; the numbers are indicated. The number of these events also tested for DNA methylation is indicated as well. <b>B</b>. DNA methylation analyses of <i>b1</i> repeats in primary <i>Arabidopsis</i> transgenic plants. Genomic DNA was digested with <i>Eco</i>RI, which cuts on both sides of the repeats, and one of three methylation sensitive enzymes, <i>Sau</i>3AI, <i>Sau</i>96I or <i>Alu</i>I. Representative examples are shown for each enzyme combination. Additional examples are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s007" target="_blank">Figure S7A</a>. Open arrows indicate fragments derived from complete digestion (no DNA methylation), while gray arrows indicate fragments containing one or more undigested, cytosine methylated restriction sites. Fragment sizes are indicated on the right of the blots. <b>C</b>. Summary of the DNA methylation pattern of the <i>b1</i> tandem repeats in transgenic events, and <i>B'</i> and <i>B-I</i> for comparison <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773-Haring1" target="_blank">[21]</a>. The single repeat shown represents all repeats present in each transgenic event or allele; the <i>Sau</i>3A (A), <i>Sau</i>96A (S), and <i>Alu</i>I (A) restriction sites are indicated. Subscripts indicate individual recognition sites present more than once in each repeat. The methylation levels at each site are indicated by the gray-scale shown. The multiple independent pEN-MS1/2 transgenic events had similar levels of DNA methylation (solid shading). The pEN-MS3/4 transgenic events showed different DNA methylation levels at some sites in independent transgenic events (hatched shading). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s007" target="_blank">Figure S7</a> shows additional DNA blots and summarizes reporter gene assays for <i>b1</i> repeat transgenes in different <i>Arabidopsis</i> mutant backgrounds.</p

Ability of non-paramutagenic or weakly paramutagenic events to become more paramutagenic upon exposure to <i>B'</i>.

a<p>Frequencies are as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s010" target="_blank">Table S2</a>.</p>b<p>Crossing scheme used to derive these plants is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s003" target="_blank">Figure S3</a>.</p

DNA blot analysis of maize transgenic events.

<p>The numbers above the arrows indicate approximate fragment sizes in kb. <b>A</b>. DNA blot analysis of the paramutagenic, paramutable, and neutral pB and pBΔ transgenic loci. Genomic DNA from transgenic plants was digested with <i>Eco</i>RI (E) and blots were hybridized with the tandem repeat probe shown as a bar below the map. The <i>B'</i> allele was used as a control to indicate the ∼7 kb <i>Eco</i>RI fragment containing the seven tandem repeats (black arrow). All transgenic plants were heterozygous for two different neutral <i>b1</i> alleles, each containing a single copy of the repeat unit, together producing a ∼6 kb doublet upon digestion (open arrow). Transgenic event number and construct names are shown above the lanes, while the approximate repeat copy number, estimated using phosphor imaging analysis, is shown below each lane. <b>B</b>. DNA blot analyses of plants carrying the pFA or pFB transgenes; genomic DNA was digested with <i>Bam</i>HI and <i>Bgl</i>II, which cut on either side of the tandem repeat array. The FA and FB fragments diagrammed below the blots were used as probes. Bands corresponding to <i>B-I</i> (∼7 kb) and the neutral <i>b1</i> alleles (1.4 kb and 1.3 kb) are indicated by black and open arrows, respectively. The 2.9 and 3.3 kb bands corresponding to the intact FA and FB tandem arrays, respectively, are indicated by dotted arrows.</p

Transgenic constructs used for maize transformation.

<p><b>A</b>. Schematic drawing of the upstream <i>b1</i> region and the two BAC clones used for plant transformation, pB and pBΔ. The scale at the top shows positions in kilobases (kb) relative to the ATG of the <i>b1</i> gene. The 107.8 kb insert in pB contains the <i>b1</i> repeats, the first two exons of <i>b1</i>, and all the sequences in between. The pBΔ clone is a deletion derivative of the pB clone that lacks 91.6 kb of internal sequences as indicated. The tandem repeats are indicated by arrowheads. <b>B</b>. Schematic representation of the pFA and pFB tandem repeat constructs. FA corresponds to one half of the repeat sequence and FB corresponds to the other half. The pFA and pFB tandem repeat constructs contain seven tandem copies of either FA or FB, respectively.</p

Northern blot analysis of repeat siRNAs in transgenic plants.

<p><b>A</b>. The levels of the <i>b1</i> tandem repeat siRNAs were detected with the indicated oligo probe (VC1657), which hybridizes to the FA part of the repeat. <b>B</b>. Genotypes of the plants used for the analysis are shown above the blot with −/− denoting the absence of the transgene and <i>TG/-</i> indicating the presence of one copy of the transgene locus. b1IR stands for 35S::b1IR. The RNA levels were detected by hybridization with ³²P labelled DNA/LNA oligonucleotide probes as described by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773-ArteagaVazquez2" target="_blank">[24]</a>. The levels of the <i>b1</i> tandem repeat siRNAs detected with the VC1657 probe were normalized to U6 RNA levels, which served as a loading control <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773-ArteagaVazquez2" target="_blank">[24]</a>. The average abundances of <i>b1</i> repeat siRNAs are presented relative to the levels in their non-transgenic sibling plants, which were set to 1.0; +/− indicates the standard deviation. A 35S::b1IR transgene that produces high levels of siRNAs was used as a positive control for hybridization and is described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773-ArteagaVazquez2" target="_blank">[24]</a>. Transgenes pBΔ 3-39, pFA::GUS and pFB::GUS are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen-1003773-g001" target="_blank">Figure 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s006" target="_blank">Figure S6</a>, respectively.</p

Heritability and paramutagenicity of transgene-induced <i>B'^#</i> silencing^a.

a<p>Crossing scheme to test <i>B'^#</i> heritability and paramutagenicity is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s002" target="_blank">Figure S2</a>; the data are summarized in this table.</p>b<p>Heritability of <i>B'^#</i> after one generation in the presence of the transgene.</p>c<p>Heritability of <i>B'^#</i> after two generations in the presence of the transgene.</p>d<p>Paramutagenicity of <i>B'^#</i> after one generation in the presence of the transgene.</p>e<p>Paramutagenicity of <i>B'^#</i> after two generations in the presence of the transgene.</p><p>Nt, not tested because paramutation was already 100% after one generation with the transgene.</p

Silencing of the <i>B-I</i> allele by transgenes carrying tandem repeat sequences integrated in non-allelic locations.

<p><b>A</b>. Crossing strategy for testing the ability of transgenes to induce silencing of the endogenous <i>B-I</i> allele. Regenerated transgenic plants, <i>b-N/b-N</i>; <i>TG/-</i>, were crossed with plants carrying the paramutable <i>B-I</i> allele and a neutral <i>b-N</i> allele. Silencing of the <i>B-I</i> allele was assessed by analyzing the pigmentation of the <i>B-I/b-N</i> progeny plants. If <i>B-I</i> is silenced by the transgene, indicated by <i>B'^#</i>, transgenic plants should be light. If a transgene is not able to silence <i>B-I</i>, all plants, transgenic and non-transgenic, should be dark, unless spontaneous paramutation endogenous of <i>B-I</i> to <i>B'</i> occurred. Non-transgenic <i>B-I/b-N</i> siblings served as controls for spontaneous paramutation of <i>B-I</i> to <i>B'</i> and should remain dark if spontaneous paramutation does not occur. Any families that showed spontaneous paramutation of <i>B-I</i> to <i>B'</i> in non-transgenic siblings were removed from further analysis. <b>B</b>. Results from the experiments indicated in Panel A for the four constructs diagrammed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen-1003773-g001" target="_blank">Figure 1</a>. Detailed information on each transgenic event is in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s009" target="_blank">Table S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s010" target="_blank">Table S2</a>. The indicated frequencies of transgenic plants with a light phenotype are a compilation of the data obtained for transgenic loci maintained up to six generations in the presence of a neutral <i>b1</i> allele (outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003773#pgen.1003773.s001" target="_blank">Figure S1</a>). Frequency of light plants was calculated by diving the number of light transgenic plants over the total number of transgenic plants. The designation <i>b-N</i> is used to represent the neutral alleles used in the crosses; <i>b-N</i> alleles carry a single 853 bp repeat unit, do not participate in paramutation and produce no plant pigment.</p