Allorecognition Triggers Autophagy and Subsequent Necrosis in the Cnidarian <em>Hydractinia symbiolongicarpus</em>

<div><p>Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid <em>Hydractinia symbiolongicarpus</em> when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous <em>in vivo</em> digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion <em>in vivo</em> in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.</p> </div

Transmission electron micrographs of isogeneic fusions.

<p>(A) Colony assay (3000X). (B) Polyp assay (4400X). Arrows point to mesoglea, BEc, basal ectoderm; Ec, ectoderm; En, endoderm; GVC, gastrovascular cavity, N, nucleus.</p

Transmission electron micrographs of allogeneic colony fusion.

<p>(A) Ectodermal margin of separated <i>fr/ff</i> versus <i>rr/rr</i> fusion zone after separation (3,000X). Arrows to glycocalyx, double-headed arrows to points of apparent loss of membrane integrity. (B) Contact zone between <i>rf/ff</i> versus <i>rr/rr</i> encounter (3,000X). Arrows point to multivestivular bodies (MVB), double-headed arrow designates point of contact. (C) MVB (12,000X). (D) Multilamellar body (MLB) (20,000X). Gastroderm of (E) <i>rf/ff</i> versus <i>rr/rr</i> encounter (20,000X) and of (F) <i>rf/ff</i> versus <i>rr/rr</i> (12,000X), showing numerous phagophores and autophagosomes (arrows). MVB, multivesticular body; N, nucleus; NC, necrotic cell; Ph, phagophores; T, tear in section.</p

(A) Allogeneic colony fusion 22 hours post-contact (42X).

<p>(B) Same encounter as (A) at 55 hours post-contact. Arrowheads point to separation. (C) Scanning electron micrograph of colonies undergoing transitory fusion (80X). (D) Allogeneic polyp fusion showing necrotic cells shed at the graft boundary (108X).</p

Transmission electron micrographs of allogeneic polyp fusions.

<p>(A) Ectoderm at graft margin (4400X) 24 hours post-fusion, arrows to multivesticular bodies, double-shafted arrows to sites of apparent loss of membrane integrity. (B) Ectodermal surface at graft margin 24 hours post-fusion showing vacuolar ghosts (20000X) including (C) mitochondria remnants (85000X). (D) Ectoderm at graft margin at 48 hours post-fusion (12000X). (E) Ectoderm at graft margin at 48 hours post-fusion (20000X). MLB, multilamellar body; N, nucleus; NC, necrotic cell.</p

Localization of LA1.

<p>LA1-Citrine localizes to the nucleus (arrows) and cell membrane in (A) <i>N. benthamiana</i> epidermal cells and (B) <i>N. benthamiana</i> mesophyll cells. Inset shows detail of the mesophyll cell membrane. (C) Deletion of the putative NLS in LA1ΔNLS-Citrine abrogates nuclear localization. Scale bars are 20 uM. (D) Western blotting confirms expression of full length LA1-Citrine (lane 1) and LA1ΔNLS-Citrine (lane 2).</p

Manhattan plot of <i>la1-mu1 Taq</i>^αI sequencing.

<p>(<b>A</b>) Manhattan plot showing the distribution of reads from <i>la1-mu1</i> genomic DNA mapped throughout the B73 genome. Alternating colors represent each of the ten maize chromosomes. Each x-axis pixel represents a bin of 1 Mb and the logarithmic y-axis denotes the number of reads mapping to each bin. The red line represents the known genetic map position for the <i>la1</i> reference mutation. Each triangle below the plot represents the approximate location of mapped MFS. (<b>B</b>) Expanded Manhattan plot of a 1 Mb interval corresponding to the approximate map position of <i>la1</i>. Same as top with each x-axis pixel representing a bin of 1 kb. Filtered genes in the 1 Mb interval are shown as black rectangles. MFS mapping to this interval are shown as inverted red triangles.</p

Alignment of <i>Mu</i> Terminal Inverted Repeats (TIRs).

<p>ClustalW was used to align all known active and potentially active <i>Mu</i> elements. Strings of four or more consecutive bases that are entirely conserved among all elements are shaded in red. The conserved <i>Taq</i>^αI site is shaded in blue. <i>Mu4, 5,</i> and <i>6</i> are inactive and are not included [reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087053#pone.0087053-Walbot1" target="_blank">[5]</a>]. <i>Mu9</i> is MuDR. Only the first 39 bp of the <i>Mu10, 11,</i> and <i>12</i> TIRs have been sequenced <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087053#pone.0087053-Dietrich1" target="_blank">[62]</a>. However, the primer used to amplify the TIR ended in a 3′ GTC, allowing for the assumption that the sequence continues as GAC (shown as small case) and that the <i>Taq</i>^αI site remains intact. Of the most recently discovered <i>Mu</i> elements (13–19), only <i>Mu13</i> has been confirmed to actively move and create new mutations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087053#pone.0087053-Tan1" target="_blank">[58]</a>. TIR sequences obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087053#pone.0087053-Barker1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087053#pone.0087053-Tan1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087053#pone.0087053-Dietrich1" target="_blank">[62]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087053#pone.0087053-James1" target="_blank">[68]</a>.</p

Identification of <i>la1</i>.

<p>The <i>la1</i> mutant plants (<b>A</b>) exhibit prostrate growth habit caused by a lack of a negative gravitropic response. Wild type maize plants (<b>B</b>) purposefully grow away from gravity (negative gravitropism). (<b>C</b>) Gene structure of <i>la1</i> with the site of the <i>Mu</i> insertion in the <i>la1</i>-<i>mu1</i> mutant allele and the CACTA family insertion in the <i>la1-cacta</i> allele. Exons are shown as empty boxes and UTRs are shown as filled boxes. The <i>Mu</i> insertion has not been characterized and is not to scale. (<b>D</b>) Amino acid ClustalW alignment of the maize and rice predicted LAZY1 proteins. The pair shares 60% identity. The predicted transmembrane domain is shaded in blue, the predicted NLS domain is shaded in orange.</p