Uncovering Divergence of Rice Exon Junction Complex Core Heterodimer Gene Duplication Reveals Their Essential Role in Growth, Development, and Reproduction

The exon junction complex (EJC) plays important developmental roles in animals; however, its role in plants is not well known. Here, we show various aspects of the divergence of each duplicated MAGO NASHI (MAGO) and Y14 gene pair in rice (Oryza sativa) encoding the putative EJC core subunits that form the obligate MAGO-Y14 heterodimers. OsMAGO1, OsMAGO2, and OsY14a were constitutively expressed in all tissues, while OsY14b was predominantly expressed in embryonic tissues. OsMAGO2 and OsY14b were more sensitive to different stresses than OsMAGO1 and OsY14a, and their encoded protein pair shared 93.8% and 46.9% sequence identity, respectively. Single MAGO down-regulation in rice did not lead to any phenotypic variation; however, double gene knockdowns generated short rice plants with abnormal flowers, and the stamens of these flowers showed inhibited degradation and absorption of both endothecium and tapetum, suggesting that OsMAGO1 and OsMAGO2 were functionally redundant. OsY14a knockdowns phenocopied OsMAGO1OsMAGO2 mutants, while down-regulation of OsY14b failed to induce plantlets, suggesting the functional specialization of OsY14b in embryogenesis. OsMAGO1OsMAGO2OsY14a triple down-regulation enhanced the phenotypes of OsMAGO1OsMAGO2 and OsY14a down-regulated mutants, indicating that they exert developmental roles in the MAGO-Y14 heterodimerization mode. Modified gene expression was noted in the altered developmental pathways in these knockdowns, and the transcript splicing of UNDEVELOPED TAPETUM1 (OsUDT1), a key regulator in stamen development, was uniquely abnormal. Concomitantly, MAGO and Y14 selectively bound to the OsUDT1 premessenger RNA, suggesting that rice EJC subunits regulate splicing. Our work provides novel insights into the function of the EJC locus in growth, development, and reproduction in angiosperms and suggests a role for these genes in the adaptive evolution of cereals

Exon junction complex (EJC) core genes play multiple developmental roles in Physalis floridana

Key message Molecular and functional characterization of four gene families of the Physalis exon junction complex (EJC) core improved our understanding of the evolution and function of EJC core genes in plants. Abstract The exon junction complex (EJC) plays significant roles in posttranscriptional regulation of genes in eukaryotes. However, its developmental roles in plants are poorly known. We characterized four EJC core genes from Physalis floridana that were named PFMAGO, PFY14, PFeIF4AIII and PFBTZ. They shared a similar phylogenetic topology and were expressed in all examined organs. PFMAGO, PFY14 and PFeIF4AIII were localized in both the nucleus and cytoplasm while PFBTZ was mainly localized in the cytoplasm. No protein homodimerization was observed, but they could form heterodimers excluding the PFY14-PFBTZ heterodimerization. Virus-induced gene silencing (VIGS) of PFMAGO or PFY14 aborted pollen development and resulted in low plant survival due to a leaf-blight-like phenotype in the shoot apex. Carpel functionality was also impaired in the PFY14 knockdowns, whereas pollen maturation was uniquely affected in PFBTZ-VIGS plants. Once PFeIF4AIII was strongly downregulated, plant survival was reduced via a decomposing root collar after flowering and Chinese lantern morphology was distorted. The expression of Physalis orthologous genes in the DYT1-TDF1-AMS-bHLH91 regulatory cascade that is associated with pollen maturation was significantly downregulated in PFMAGO-, PFY14- and PFBTZ-VIGS flowers. Intron-retention in the transcripts of P. floridana dysfunctional tapetum1 (PFDYT1) occurred in these mutated flowers. Additionally, the expression level of WRKY genes in defense-related pathways in the shoot apex of PFMAGO- or PFY14-VIGS plants and in the root collar of PFeIF4AIII-VIGS plants was significantly downregulated. Taken together, the Physalis EJC core genes play multiple roles including a conserved role in male fertility and newly discovered roles in Chinese lantern development, carpel functionality and defense-related processes. These data increase our understanding of the evolution and functions of EJC core genes in plants

Targeting MAGO proteins with a peptide aptamer reinforces their essential roles in multiple rice developmental pathways

Peptide aptamers are artificial short peptides that potentially interfere with the biological roles of their target proteins; however, this technology has not yet been applied to plant functional genomics. MAGO and Y14, the two core subunits of the exon junction complex (EJC), form obligate heterodimers in eukaryotes. In Oryza sativa L. (rice), each of the two genes has two homologs, designated OsMAGO1 and OsMAGO2, and OsY14a and OsY14b, respectively. Here, we characterized a 16-amino acida peptide aptamer (PAP) for the rice MAGO proteins. PAP and rice Y14 bound competitively to rice MAGO proteins. Specifically targeting the MAGO proteins by expressing the aptamer in transgenic rice plants did not affect the endogenous synthesis and accumulation of MAGO proteins; however, the phenotypic variations observed in multiple organs phenocopied those of transgenic rice plants harboring RNA interference (RNAi) constructs in which the accumulation of MAGO and/or OsY14a transcripts and MAGO proteins was downregulated severely. Morphologically, the aptamer transgenic plants were short with abnormally developed flowers, and the stamens exhibited reduced degradation and absorption of both the endothecium and tapetum, thus confirming that EJC core heterodimers play essential roles in rice development, growth and reproduction. This study reveals that as a complementary approach of RNAi, peptide aptamers are powerful tools for interfering with the function of proteins in higher plants

Peptide Aptamers to Inhibit Protein Function in Plants

Peptide aptamers - artificial short peptideswithspecific binding-affinity for target molecules - can be used to interfere with protein functions and protein-protein interactions in plant cells. Therefore, peptide aptamers have emerged as a new, powerful tool with high efficiency and specificity and wide applications in functional genomics and plant biotechnology

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

<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

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