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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 among the PthA4 interactors detected by yeast two-hybrid and mass spectrometry.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 Table 2 for details). The molecular markers (MM) are shown on the left.
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pone-0032305-g002: Protein-protein interactions among the PthA4 interactors detected by yeast two-hybrid and mass spectrometry.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 Table 2 for details). The molecular markers (MM) are shown on the left.

Mentions: Considering that the newly-identified PthA interactors are functionally related (Table 1), we tested whether they would interact with each other. As shown in Fig. 2A, a substantial number of protein-protein associations were detected relating for instance CsTRAX with CsSMC, CsPABP1, CsRRMP1 and CsVIP2. In addition CsTRAX self-interacted in yeast two-hybrid assays (Fig. 2A). Accordingly, CsSMC used as bait also interacted with CsTRAX, CsPABP1, CsPABP2 and CsVIP2, and it self-interacted (Fig. 2B), corroborating literature data [25]. CsVIP2 and CsPABP2 interacted with each other in reciprocal yeast two-hybrid assays and they also self-interacted (Fig. 2C). Moreover, CsVIP2 associated with CsPCBP, whereas CsHMG showed weak interactions with PABP1 and PABP2 (Fig. 2D).


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 among the PthA4 interactors detected by yeast two-hybrid and mass spectrometry.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 Table 2 for details). The molecular markers (MM) are shown on the left.
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Related In: Results  -  Collection

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pone-0032305-g002: Protein-protein interactions among the PthA4 interactors detected by yeast two-hybrid and mass spectrometry.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 Table 2 for details). The molecular markers (MM) are shown on the left.
Mentions: Considering that the newly-identified PthA interactors are functionally related (Table 1), we tested whether they would interact with each other. As shown in Fig. 2A, a substantial number of protein-protein associations were detected relating for instance CsTRAX with CsSMC, CsPABP1, CsRRMP1 and CsVIP2. In addition CsTRAX self-interacted in yeast two-hybrid assays (Fig. 2A). Accordingly, CsSMC used as bait also interacted with CsTRAX, CsPABP1, CsPABP2 and CsVIP2, and it self-interacted (Fig. 2B), corroborating literature data [25]. CsVIP2 and CsPABP2 interacted with each other in reciprocal yeast two-hybrid assays and they also self-interacted (Fig. 2C). Moreover, CsVIP2 associated with CsPCBP, whereas CsHMG showed weak interactions with PABP1 and PABP2 (Fig. 2D).

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