<i>Tol-MirrorTree</i> analyses reveal the co-evolution of the MAGO and Y14 families at protein levels.

<p>(<b>a</b>) Phylogenetic tree of the MAGO protein family. (<b>b</b>) Phylogenetic tree of the Y14 family. These ML trees include sequences from algae (yellow), animals (blue), dicots (orange), monocots: grass (green) and others (pink), and gymnosperm and moss (purple). The red lines represent the branches with less than 50% bootstrap in the trees. (<b>c–d</b>) Evaluation of tree similarity. Genetic distances (GD) between MAGO and Y14 sequences were corrected by 18s rRNA to avoid a contribution of speciation. (<b>c</b>) Similarity of the ML trees with the whole set of sequences shown in <b>a</b> and <b>b</b>. (<b>d</b>) Tree similarity when single sequence for each family in one organism. Similarity evaluation of the ML trees with an inclusion of <i>MAGO1</i> and <i>Y14a</i> sequences is presented in (<b>d</b>). Pearson correlation coefficient is 0.61 (<i>P</i> = 0.00) when <i>MAGO2</i> and <i>Y14b</i> sequences were included.</p

Evolutionary rates and purifying selection of the <i>MAGO</i> and <i>Y14</i> families.

<p>(<b>a</b>) Evolutionary rates of the MAGO and Y14 families inferred from all included homologous sequences. <i>dN</i>: non-synonymous substitution rate; <i>dS</i>: synonymous substitution rate. ω = <i>dN</i>/<i>dS</i>. (<b>b</b>) Comparison of the evolutionary rate. <i>dN</i> and <i>dS</i> values were determined with different inputs. 1, <i>Y14</i> genes excluding <i>Y14b</i> copies; 2, <i>Y14</i> genes excluding <i>Y14a</i> copies; 3, <i>MAGO</i> genes excluding <i>MAGO2</i> copies. 4, <i>MAGO</i> genes excluding <i>MAGO1</i> copies; 5, all <i>Y14</i> genes and 6, all <i>MAGO</i> genes.</p

Determination of minimal interaction domains (MIDs) of rice MAGO and Y14 in yeast.

<p>(<b>a</b>) The MID of ΔOsMAGO1 to interact with OsY14a. (<b>b</b>) The MID of ΔOsY14a to interact with OsMAGO1. (<b>c</b>) The MID of ΔOsMAGO2 to interact with OsY14b. (<b>d</b>) The MID of ΔOsY14b to interact with OsMAGO2. The deletion versions of the proteins were generated as indicated in <b>a</b>-<b>d</b>. The defined MIDs of the OsMAGO1/2 and OsY14a/b are boxed. The log for each protein family is presented. (<b>e</b>) Confirmation of the MID in yeast. Further deleted versions (ΔΔ) did not maintain the capability to interact, while the MID (Δ) formed heterodimer similar to their native proteins. Non-lethal β-galactosidase assays were performed suggest their interactions.</p

Slow Co-Evolution of the MAGO and Y14 Protein Families Is Required for the Maintenance of Their Obligate Heterodimerization Mode

<div><p>The exon junction complex (EJC) plays important roles in RNA metabolisms and the development of eukaryotic organisms. MAGO (short form of MAGO NASHI) and Y14 (also Tsunagi or RBM8) are the EJC core components. Their biological roles have been well investigated in various species, but the evolutionary patterns of the two gene families and their protein-protein interactions are poorly known. Genome-wide survey suggested that the <i>MAGO</i> and <i>Y14</i> two gene families originated in eukaryotic organisms with the maintenance of a low copy. We found that the two protein families evolved slowly; however, the MAGO family under stringent purifying selection evolved more slowly than the Y14 family that was under relative relaxed purifying selection. MAGO and Y14 were obliged to form heterodimer in a eukaryotic organism, and this obligate mode was plesiomorphic. Lack of binding of MAGO to Y14 as functional barrier was observed only among distantly species, suggesting that a slow co-evolution of the two protein families. Inter-protein co-evolutionary signal was further quantified in analyses of the <i>Tol-MirroTree</i> and co-evolution analysis using protein sequences. About 20% of the 41 significantly correlated mutation groups (involving 97 residues) predicted between the two families was clade-specific. Moreover, around half of the predicted co-evolved groups and nearly all clade-specific residues fell into the minimal interaction domains of the two protein families. The mutagenesis effects of the clade-specific residues strengthened that the co-evolution is required for obligate MAGO-Y14 heterodimerization mode. In turn, the obliged heterodimerization in an organism serves as a strong functional constraint for the co-evolution of the MAGO and Y14 families. Such a co-evolution allows maintaining the interaction between the proteins through large evolutionary time scales. Our work shed a light on functional evolution of the EJC genes in eukaryotes, and facilitates to understand the co-evolutionary processes among protein families.</p></div

The clade-specific sites in the MAGO and Y14 families.

<p>The clade-specific amino acids are arranged that the small nonpolar residues (G, A, S and T) are highlighted in yellow, the hydrophobic residues (C, V, I, L, P, F, Y, M and W) in green, the polar residues (N, Q and H) in magenta, the negatively charged residues (D and E) in red, and the positively charged residues (K and R) in blue. The residues between the two protein families that were predicted to be correlated mutation groups in CAPS were connected by color lines. The black, red, green, blue, yellow, purple, pink, orange and gray line respectively represents the G4, G5, G6, G12, G18, G24, G33, G35 and G40 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084842#pone-0084842-g005" target="_blank">Figure 5</a>; Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084842#pone.0084842.s004" target="_blank">File S1</a>). The given position of the residues in the MAGO and Y14 families corresponds to OsMAGO2 and OsY14b, respectively. The red numbers showed that these sites were predicted to be co-evolved in CAPS, the white was not.</p

Crucial roles of the clade-specific residues in the MAGO-Y14 heterodimerization.

<p>(<b>a</b>) Protein-protein interactions in yeast. The combination of the bait proteins (BD, horizontal arrows) and the prey proteins (AD, vertical arrow) is indicated. Left panel: The growth of the same amounts of the co-transformed yeast cells on the highest stringent conditions of the SD/Leu-Trp-His-Ade plates. Right panel: The result of the non-lethal β-galactosidase assay. The clade-specific residues 64 and 142 of AtMAGO and the residue 154 of AtY14 in <i>Arabidopsis</i> (a dicot) were mutated to the corresponding sites in the rice proteins, the representatives from monocots (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084842#pone-0084842-g006" target="_blank">Figure 6</a>), and the resultant proteins were AtMAGOm64, AtMAGO142, AtMAGOm64m142 and AtY14m154. The combinations of the BD proteins and pGADT7 or the AD proteins and pGBKT7 were included as negative controls. (<b>b</b>) Quantification of the heterodimerization strength. The relative β-galactosidase activity was normalized with the interaction strength of AtMAGO -AtY14 (AtMAGO as prey). The combination of the pGADT7 and pGBKT7 empty vectors was included as a negative control. The experiments were repeated three times. The average enzyme activity and the standard deviation are presented. The significance of the strength difference between interactions was evaluated using two-tailed <i>t</i>-tests (<i>P</i> = 0.000). The black stars (**) indicate the comparison to AtMAGO -AtY14 (AtMAGO as prey), while the blue stars show the comparison to AtY14-AtMAGO (AtY14 as prey).</p

Evolution of the MAGO-Y14 interaction mode.

<p>The experimental data from the corresponding references are listed. Star [*] indicates the data from the present work (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084842#pone-0084842-g004" target="_blank">Figure 4</a>). Filled circles indicate the presence of the MAGO-Y14 interactions that appeared to be a plesiomorphic trait.</p

Correlated mutation groups between the MAGO and Y14 families.

<p>Groups (G1–G41) of the correlated mutation residues were detected by CAPS. All residues included in one circle are predicted to co-evolve between each other. The correlated mutation residues of the Y14 and MAGO are represented in black and red hexagon, respectively. Functionally or structurally important sites in the MAGO-core or Y14-RBD are in bold box, while others sites are in thin box. The position of the correlated mutational residues in the MAGO and Y14 proteins is referred to as OsMAGO2 and OsY14b, respectively. For the details, see Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084842#pone.0084842.s004" target="_blank">File S1</a>.</p

Protein interaction matrices of the MAGO and Y14 proteins from different species.

<p>The combination of the bait proteins (BD) and the prey proteins (AD) is indicated. The name of MAGO and Y14 sequences started with the abbreviated name of the species such as <i>Arabidopsis thaliana</i> (At), <i>Solanum lycopersicum</i> (Sl), <i>Physalis philadelphica</i> (Pp), <i>Physalis floridana</i> (Pf), <i>Oryza sativa</i> (Os), <i>Caenothabditis elegans</i> (Ce), <i>Drosophila melanogaster</i> (Dm), <i>Homo sapiens</i> (Hs) and <i>Mus musculus</i> (Mm). The average genetic distance of MAGO and Y14 among dicots (Do), monocots (Mo), worm (Wo), fly (Fl) and mammals (Ma) was given. Dash (-) indicates no data due to single sequence was used in that clade. The survived cells of the same amounts of the co-transformed yeast cells on the highest stringent conditions (SD/Leu-Trp-His-Ade) were subjected to a non-lethal β-galactosidase assay.</p

Efficient Gene Silencing Mediated by Tobacco Rattle Virus in an Emerging Model Plant <i>Physalis</i>

<div><p>The fruit of <i>Physalis</i> has a berry and a novelty called inflated calyx syndrome (ICS, also named the ‘Chinese lantern’). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in <i>Physalis floridana</i>. First, we characterized the putative gene of a phytoene desaturase in <i>P. floridana</i> (<i>PfPDS</i>). Infecting the leaves of the <i>Physalis</i> seedlings with the <i>PfPDS</i>-<i>TRV</i> vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous <i>PfPDS</i>. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in <i>Physalis</i>. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, <i>MPF2</i> and <i>MPF3</i>, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both <i>MPF2</i> and <i>MPF3</i>. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in <i>P. floridana</i>. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in <i>Physalis</i>, thus facilitating understanding of the genetic basis of the evolution and development of the morphological diversities within the Solanaceae.</p></div