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The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U) RNA.

de Souza TA, Soprano AS, de Lira NP, Quaresma AJ, Pauletti BA, Paes Leme AF, Benedetti CE - PLoS ONE (2012)

Bottom Line: Plant pathogenic bacteria utilize an array of effector proteins to cause disease.Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors.Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brazil.

ABSTRACT
Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

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Protein-protein interactions between PthAs and citrus nuclear proteins.(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.
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pone-0032305-g001: Protein-protein interactions between PthAs and citrus nuclear proteins.(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.

Mentions: As reported previously, some of the citrus proteins that were identified as targets of PthAs 2 and 3 also interacted with other PthA variants [16]. Thus, the identified PthA4 preys were tested for interactions with the four PthA variants in yeast two-hybrid assays. As shown in Fig. 1A, the majority of the preys interacted not only with PthA4, confirming the primary two-hybrid screening, but with PthAs 2 and 3, preferentially. Notably, CsVIP2 interacted specifically with PthA4, whereas CsPCBP, CsRRMP1 and CsHAP3 interacted with PthAs 2 and 3 under more stringent conditions (no adenine). By contrast, CsHMG was the only prey capable of interacting with all the PthA variants in the absence of adenine (Fig. 1A).


The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U) RNA.

de Souza TA, Soprano AS, de Lira NP, Quaresma AJ, Pauletti BA, Paes Leme AF, Benedetti CE - PLoS ONE (2012)

Protein-protein interactions between PthAs and citrus nuclear proteins.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285215&req=5

pone-0032305-g001: Protein-protein interactions between PthAs and citrus nuclear proteins.(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.
Mentions: As reported previously, some of the citrus proteins that were identified as targets of PthAs 2 and 3 also interacted with other PthA variants [16]. Thus, the identified PthA4 preys were tested for interactions with the four PthA variants in yeast two-hybrid assays. As shown in Fig. 1A, the majority of the preys interacted not only with PthA4, confirming the primary two-hybrid screening, but with PthAs 2 and 3, preferentially. Notably, CsVIP2 interacted specifically with PthA4, whereas CsPCBP, CsRRMP1 and CsHAP3 interacted with PthAs 2 and 3 under more stringent conditions (no adenine). By contrast, CsHMG was the only prey capable of interacting with all the PthA variants in the absence of adenine (Fig. 1A).

Bottom Line: Plant pathogenic bacteria utilize an array of effector proteins to cause disease.Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors.Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brazil.

ABSTRACT
Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

Show MeSH
Related in: MedlinePlus