Reversed <i>Ac</i> ends transposition generates direct duplication.

<p>The two lines indicate sister chromatids of maize chromosome 1, joined at the centromere (black). The blue boxes are exons 3, 2, and 1 (left to right) of the <i>p1</i> gene. Red lines with arrowhead(s) indicate <i>Ac/fAc</i> insertions, and the open and solid arrowheads indicate the 3′ and 5′ ends, respectively, of <i>Ac</i>/<i>fAc</i>. The short horizontal arrows show the orientations and approximate positions of PCR primers, and the numbers below are the primer names. The green/black triangles indicate the transposon target site sequences and target site duplications. (<i>A</i>) <i>Ac</i> transposase cleaves the lower chromatid at the 3′ end of <i>fAc</i> and the 5′ end of <i>Ac</i> (arrows). (<i>B</i>) Following transposase cleavage, the internal <i>p1</i> genomic sequences are joined to form a circle. Dotted lines indicate the insertion of the <i>fAc</i> and <i>Ac</i> termini into the a/b site on the sister chromatid. (<i>C</i>) Transposon ends insert into the upper sister chromatid at a proximal site. (<i>D</i>) The <i>Ac</i> 5′ end joins to the distal side (green) of the target site and the <i>fAc</i> 3′ end joins to the proximal side (black) of the target site to generate a proximal deletion (upper chromatid) and a direct duplication (lower chromatid). The shaded arrows encompass the duplicated segments. For animation, please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen.1003691.s006" target="_blank">Movie S1</a>.</p

Tandem direct duplications in maize generated by Reversed-Ends Transposition.

<p>Red lines with arrowhead(s) indicate the <i>dhAT</i> family elements associated with each duplication; solid and open arrowheads indicate the transposon 5′ and 3′ ends, respectively. The truncated solid arrowhead in <i>dhAT-Zm13</i> indicates a deletion of 12 bp from the 5′ TIR. The blue lines represent duplicated segments. The blue triangles indicate the transposon target site duplications. Numbers above each line indicate the length of that segment. Sequences and genomic positions are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen.1003691.s009" target="_blank">Text S2</a>.</p

Ears with twinned sectors T1 (left) and T481 (right).

<p>The white and red phenotypic twinned sectors are outlined. The remainder of the ear has the orange-variegated phenotype specified by the progenitor <i>P1-ovov454</i> allele.</p

The lengths of the duplications.

<p>The lengths of the duplications.</p

DNA gel blot analysis of the twinned alleles.

<p>(<i>A</i>) Structure of progenitor <i>P1-ovov454</i> allele (upper), and predicted structures of the <i>P1-rr-Twin</i> (duplication) and <i>p1-ww-Twin</i> (deletion) alleles (lower) generated via reversed <i>Ac</i> ends transposition. Blue lines and boxes indicate <i>p1</i> sequences, green lines indicate sequences proximal to <i>p1</i>, and gray boxes indicate sequences homologous to probe 15. The short vertical black lines indicate <i>Sal</i>I sites, and red asterisks (*) mark methylated <i>Sal</i>I sites. The other symbols have the same meaning as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen-1003691-g001" target="_blank">Figure 1</a>. (<i>B</i>) DNA gel blot. Genomic DNA was digested with <i>Sal</i>I and hybridized with <i>p1</i> genomic probe 15 (gray boxes in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen-1003691-g005" target="_blank">Figure 5<i>A</i></a>). Lane 1: <i>p1-ww[4Co63]</i>, Lane 2: <i>P1-ovov454</i>/<i>p1-ww[4Co63]</i>, Lane 3: <i>P1-rr-T1</i>, Lane 4: <i>p1-ww-T1/p1-ww[4Co63]</i>, Lane 5: <i>P1-rr-T481</i>.</p

RT-PCR Analysis of <i>P-oo</i> Transcripts

<p>RNA was extracted from kernel pericarp (20 DAP), reverse transcribed, and PCR-amplified using primers complementary to both <i>p1</i> and <i>p2</i> transcripts. The progenitor allele <i>(P1-rr11)</i> shows amplification of a 605-bp band from <i>p1.</i> The <i>p-ww2</i> and <i>P-oo</i> alleles show amplification of a 522-bp band characteristic of the 5′ region of the <i>p2</i> gene. The <i>p1-ww1112</i> allele has a deletion of <i>p1;</i> the native <i>p2</i> gene is intact in this allele, but is not expressed in kernel pericarp.</p

PCR primers.

<p>PCR primers.</p

Breakpoint sequences of reciprocal duplication/deletion alleles <i>P1-rr-T1</i> and <i>p1-ww-T1</i> generated by Reversed Ends Transposition.

<p>(<i>A</i>) Diagram shows the structure of the progenitor <i>P1-ovov454</i> allele prior to RET. Two sister chromatids are shown, with symbols as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003691#pgen-1003691-g001" target="_blank">Figure 1</a>. The dotted box shows the a/b target site region, whose sequence is indicated above. The color of the letters in the sequences matches the chromatid line color. (<i>B</i>) PCR amplification of the deletion/duplication breakpoints in <i>p1-ww-T1</i> and <i>P1-rr-T1</i> with primers 2+Ac3 (upper panel) or 1+Ac5 (lower panel). Lane 1: DNA ladder, lane 2: <i>p1-ww[4Co63]</i>, lane 3: <i>P1-ovov454/p1-ww[4Co63]</i>, lane 4: <i>p1-ww-T1/p1-ww[4Co63]</i>, lane 5: <i>P1-rr-T1/p1-ww[4Co63]</i>. (<i>C</i>) Sister chromatid structures of <i>p1-ww-T1</i> (upper) and <i>P1-rr-T1</i> (lower). Sequences of the deletion and duplication breakpoints (dotted boxes) are shown in color matching the chromatid line color. Note that each breakpoint has a copy of the 8 bp TSD GCGCTTTA which is present in a single copy at the a/b target site in the progenitor allele.</p

A simplified model for crosstalk between the circadian clock and plant innate immunity.

<p>(<b>A</b>) Timing of stomata-dependent and -independent defense in a day. At night, plants might rely more on closed stomata to provide physical constrains to limit pathogen invasion but have relatively lower levels of stomata-independent defense. Once pathogens bypass such constrains (i.e. via infiltration infection in the laboratory), they encounter a plant host that is more susceptible than during the day. During the day, most stomata are wide open. In the presence of pathogens, plants can only transiently reduce stomatal aperture for a few hours (this study and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Melotto1" target="_blank">[1]</a>). Thus, during the day plants might depend more on stomata-independent defense to restrict pathogen invasion. Stomata-dependent defense could be stronger at night while stomata-independent defense could be stronger during the day. (<b>B</b>) The circadian clock regulates both stomata-dependent and -independent defense pathways to restrict pathogen growth in Arabidopsis. In a stomata-dependent pathway, CCA1 and LHY act, at least in part, through GRP7 as a direct downstream target to regulate stomatal aperture and thereby defense. Other downstream targets of CCA1 and LHY and other components of the central oscillator of the circadian clock might also be involved in regulating stomata-dependent and –independent defense. On the other hand, pathogen infection can activate PTI, ETI and other defense signaling in the host. PTI induced by flg22 feeds back to regulate clock activity. In addition, flg22-triggered signaling is under circadian clock control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Bhardwaj1" target="_blank">[27]</a>. Thus, we conclude that the clock-defense crosstalk involves flg22-mediated signaling. Flg22 can affect stomatal aperture <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Zhang1" target="_blank">[91]</a>. However, whether this function of flg22 is through its regulation of the circadian clock or through a direct regulation of stomata is unclear. Other questions, such as whether additional PAMPs, effectors, and other defense signaling molecules are involved in clock-defense crosstalk, remain to be answered.</p

CCA1 and LHY contribute synergistically to resistance to <i>Hyaloperonospora arabidopsidis</i> (<i>Hpa</i>).

<p>(<b>A</b>) Infection with <i>Hpa</i> Emco5. (<b>B</b>) Infection with <i>Hpa</i> Emoy2. Seven-day-old seedlings were spray-infected at ZT7 in LD with the virulent strain <i>Hpa</i> Emco5 or the avirulent strain <i>Hpa Emoy2</i> (5×10⁴ spores/ml in water). Sporangiophore production in cotyledons of each genotype was counted at 7 dpi. Data represent the average number of sporangiophores from 20 seedlings for <i>CCA1ox</i> and 50 seedlings for other genotypes ± SEM. Letters indicate significant difference among the samples (P<0.01; Mann-Whitney test). These experiments were repeated three times with similar results.</p