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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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PthA binds to CsHMG in vivo through its invariable LRR region.(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.
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pone-0032305-g004: PthA binds to CsHMG in vivo through its invariable LRR region.(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.

Mentions: To confirm the interactions between PthAs and CsHMG in vivo, PthAs 2 and 4 fused to GST were immobilized in glutathione resins and allowed to interact with proteins from citrus cell extracts. As shown in Fig. 4A, both PthAs 2 and 4, but not GST bound to CsHMG, confirming that CsHMG is an interacting partner of PthAs.


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)

PthA binds to CsHMG in vivo through its invariable LRR region.(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.
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Related In: Results  -  Collection

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pone-0032305-g004: PthA binds to CsHMG in vivo through its invariable LRR region.(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.
Mentions: To confirm the interactions between PthAs and CsHMG in vivo, PthAs 2 and 4 fused to GST were immobilized in glutathione resins and allowed to interact with proteins from citrus cell extracts. As shown in Fig. 4A, both PthAs 2 and 4, but not GST bound to CsHMG, confirming that CsHMG is an interacting partner of PthAs.

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