Correction: The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA

<p>Correction: The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA</p

Schematic model of the citrus multiprotein complex comprising the PthA4-interacting partners.

<p>Protein-protein and protein-RNA contacts involving the PthA4 interactors based on the yeast two-hybrid, GST-pulldown and gel-shift assays described here, and literature data. The citrus multiprotein complex is reminiscent of that of mammalian miRISC involved in miRNA-mediated deadenylation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Fabian1" target="_blank">[51]</a>. Importin-α, which interacts with all PthA variants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Domingues1" target="_blank">[16]</a> is also a component of the cap-binding complex (CBC) which inhibits mRNA deadenylation when in the presence of a poly(A)-specific ribonucleases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Balatsos1" target="_blank">[55]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone.0032305-Sato1" target="_blank">[58]</a>. It is suggested that by interacting with such proteins and with poly(U) RNA (not necessarily simultaneously), PthA proteins may displace some of the components of this complex thought to promote deadenylation and mRNA decay and thus increase mRNA stabilization and translation initiation. U-rich sequences found in both 5′ and 3′ ends of mRNAs could represent binding sites of CsHMG and PthA4.</p

Protein-protein interactions among the PthA4 interactors detected by yeast two-hybrid and mass spectrometry.

<p>Yeast cells double-transformed with the indicated prey-bait constructs were grown in SC -Trp -Leu -His -Ade in the presence of 5 mM 3AT. (A) Positive interactions observed between CsTRAX and CsSMC, CsPABP1, CsTRAX, CsRRMP1 and CsVIP2. (B) Protein-protein interactions observed between CsSMC and CsPABP2, CsSMC, CsPABP1, CsVIP2 and CsTRAX, but not between CsSMC and CsKH. (C) Interactions of CsVIP2 with CsKH, reciprocal interactions between CsPABP2 and CsVIP2, and self interactions of CsVIP2 and CsPABP2. (D) Weak interactions between CsHMG and the poly(A)-binding proteins CsPABP2 and CsPABP1. (E) A diagram illustrating the network of interactions observed among the citrus PthA targets. (F) Silver-stained SDS polyacrylamide gels of citrus proteins trapped in cobalt beads carrying the recombinant 6xHis-tagged CsSMC or CsTRAX as baits (bands 9 and 10, respectively). Protein bands excised from the gels, indicated by the numbers, were identified by mass spectrometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone-0032305-t002" target="_blank">Table 2</a> for details). The molecular markers (MM) are shown on the left.</p

CsHMG shows identity to plant HMGBs of group B.

<p>(A) Schematic representation of the CsHMG primary structure showing its central HMG-box domain flanked by the basic K-rich N-terminal and the acidic DE-rich C-terminal. (B) Phylogenetic analysis of plant HMGB proteins showing that CsHMG belongs to group B HMGBs. (C) Western-blot detection of the recombinant 6xHis-CsHMG (∼22 kDa) made in bacteria compared to bands detected in citrus cell extracts with the expected molecular size for the endogenous CsHMG (∼16 kDa). The anti-CsHMG serum also cross-reacted with a band of similar size in the cell extracts of <i>A. thaliana</i> wild-type and heterozygous <i>hmg-b1</i> mutant. This band, which has the expected molecular weight for AtHMGB1 (∼18 kDa), is less pronounced in the heterozygous <i>hmgb-1</i> mutant, thus indicating that CsHMG is structurally related to AtHMGB1.</p

<i>Citrus sinensis</i> proteins identified as targets of PthA4 are homologous to nuclear factors involved in chromatin remodeling and repair, transcription regulation and mRNA stabilization/modification.

<p><i>Citrus sinensis</i> proteins identified as targets of PthA4 are homologous to nuclear factors involved in chromatin remodeling and repair, transcription regulation and mRNA stabilization/modification.</p

PthA binds to CsHMG <i>in vivo</i> through its invariable LRR region.

<p>(A) PthA2-GST, PthA4-GST or GST alone bound to glutathione resins were incubated with a citrus cell lisate. Bound proteins were separated by gel electrophoresis and CsHMG was detected by the anti-CsHMG serum in the PthA samples only. (B) Western blot of GST-pulldown assay of immobilized GST or GST-CsHMG as baits and purified 6xHis-PthA5.5rep+CT2 as prey. The eluted 6xHis-PthA5.5rep+CT2 (∼63 kDa) was detected by the anti-PthA serum only when GST-CsHMG was used as bait. The purified 6xHis-PthA5.5rep+CT2 was added in the first lane of the gel as reference. (C) Western blot analysis of eluted fractions of GST-pulldown assay of immobilized GST or GST-PthALRR as baits and purified 6xHis-CsHMG as prey. The eluted 6xHis-CsHMG (∼22 kDa) was detected only when GST-PthALRR was used as bait. (D) Yeast two-hybrid assay showing the interaction between CsHMG and the PthA LRR domain. Yeast double-tranformants, including controls (GAL4AD+GAL4BD-PthALRR and GAL4BD+GAL4AD-CsHMG), were grown in SC -Trp -Leu -His -Ade in the presence of 5 mM 3AT.</p

<i>Citrus sinensis</i> proteins identified as binding partners of CsSMC and CsTRAX by mass spectrometry.

<p>Protein bands are numbered and they correspond to those depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone-0032305-g002" target="_blank">Fig. 2F</a>.</p

Protein-protein interactions between PthAs and citrus nuclear proteins.

<p>(A) Citrus preys fused to yeast GAL4-AD (GAL4AD-prey) or control plasmid (GAL4AD) were moved into yeast cells carrying one of the four PthA variants fused to GAL4-BD domain as shown in the diagram (1 to 4, respectively). Yeast double-transformants were grown on SC -Trp -Leu -His -Ade in the presence of 5 mM of 3AT. None of prey fusions transactivated the reporter genes when co-transformed with empty bait vector (5). The PthA baits also did not transactivate the reporter genes when co-transformed with the empty prey vector in the same growth conditions (GAL4AD). (B) Western blot detection of eluted fractions from GST pulldown assays using the purified 6xHis-PthAs 1–4 as prey and immobilized GST or GST-fusion proteins as baits. Arrows indicate bands corresponding to the expected size for the GST-fusion proteins CsHMG (∼45 kDa), CsTRAX (∼55 kDa), CsSMC (∼45 kDa), CsRRPMP1 (∼50 kDa), CsRRMP2 (∼46 kDa), CsPABP1 (∼53 kDa) and CsVIP2 (∼85 kDa) detected by the GST anti-serum. PthA proteins (∼116–122 kDa) were detected using the anti-PthA serum. Recombinant PthAs 3 and 4 were added as references in the first lanes of the gels shown in the middle and right panels, respectively.</p