Limits...
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.

Show MeSH

Related in: MedlinePlus

CsHMG shows DNA and RNA-binding activities in vitro.(A) EMSA using the recombinant 6xHis-CsHMG protein (5 µg) and DNA probes (200 ng) derived from the citrus pr5 promoter and the multiple-cloning site of the pBluescript vector. The DNA-protein complexes and free probes detected by ethidium bromide staining are indicated by the arrows and asterisks, respectively. (B) Upper panel, EMSA using 32P-labelled single strand RNA probes at a final concentration of 12.5 nM and increased amounts of purified CsHMG (0.1 and 0.5 µg). Shifted bands corresponding to CsHMG:RNA complexes observed with the poly(U) RNA and the free probes are indicated by the arrow and asterisk, respectively. Bottom panel, SDS-PAGE of UV-crosslink EMSA showing the selective binding of CsHMG to the poly(U) RNA probe (arrow). (C) EMSA using the 32P-labelled poly(U) RNA as probe at a final concentration of 12.5 nM, 100 ng of CsHMG and increasing amounts of the double strand DNA (multiple-cloning site of the pBluescript vector) as competitor. Shifted bands corresponding to CsHMG:RNA complexes and the free probe are indicated by the arrow and asterisk, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3285215&req=5

pone-0032305-g005: CsHMG shows DNA and RNA-binding activities in vitro.(A) EMSA using the recombinant 6xHis-CsHMG protein (5 µg) and DNA probes (200 ng) derived from the citrus pr5 promoter and the multiple-cloning site of the pBluescript vector. The DNA-protein complexes and free probes detected by ethidium bromide staining are indicated by the arrows and asterisks, respectively. (B) Upper panel, EMSA using 32P-labelled single strand RNA probes at a final concentration of 12.5 nM and increased amounts of purified CsHMG (0.1 and 0.5 µg). Shifted bands corresponding to CsHMG:RNA complexes observed with the poly(U) RNA and the free probes are indicated by the arrow and asterisk, respectively. Bottom panel, SDS-PAGE of UV-crosslink EMSA showing the selective binding of CsHMG to the poly(U) RNA probe (arrow). (C) EMSA using the 32P-labelled poly(U) RNA as probe at a final concentration of 12.5 nM, 100 ng of CsHMG and increasing amounts of the double strand DNA (multiple-cloning site of the pBluescript vector) as competitor. Shifted bands corresponding to CsHMG:RNA complexes and the free probe are indicated by the arrow and asterisk, respectively.

Mentions: HMGBs are highly abundant chromosomal proteins known to bind DNA in a non-specific manner [26], [27]. Thus, we examined the ability of the recombinant CsHMG to bind DNA in gel-shift assays by testing its interaction with two unrelated double-strand DNA probes, one derived from the citrus pr5 promoter [28] and another derived from a bacterial cloning vector. As shown in Fig. 5A, CsHMG bound to the two DNA probes, indicating that the citrus protein does not display DNA sequence specificity, a general feature of HMGs. Although this result is in line with the literature data and with our observation that CsHMG made in E. coli co-purifies with bacterial DNA (not shown), we also noticed that the removal all traces of nucleic acids from our recombinant CsHMG preparations required an RNase treatment, which indicated that CsHMG had affinity for RNA as well. To test this hypothesis, we performed gel-shift assays to probe the binding of CsHMG to single-strand RNAs. Surprisingly, we found that CsHMG not only binds to single-strand RNA in vitro but it shows specificity to poly(U) RNAs (Fig. 5B, upper panel). The specific binding of CsHMG to poly(U) RNA was further confirmed by an UV-crosslinking gel-shift assay (Fig. 5B, bottom panel).


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)

CsHMG shows DNA and RNA-binding activities in vitro.(A) EMSA using the recombinant 6xHis-CsHMG protein (5 µg) and DNA probes (200 ng) derived from the citrus pr5 promoter and the multiple-cloning site of the pBluescript vector. The DNA-protein complexes and free probes detected by ethidium bromide staining are indicated by the arrows and asterisks, respectively. (B) Upper panel, EMSA using 32P-labelled single strand RNA probes at a final concentration of 12.5 nM and increased amounts of purified CsHMG (0.1 and 0.5 µg). Shifted bands corresponding to CsHMG:RNA complexes observed with the poly(U) RNA and the free probes are indicated by the arrow and asterisk, respectively. Bottom panel, SDS-PAGE of UV-crosslink EMSA showing the selective binding of CsHMG to the poly(U) RNA probe (arrow). (C) EMSA using the 32P-labelled poly(U) RNA as probe at a final concentration of 12.5 nM, 100 ng of CsHMG and increasing amounts of the double strand DNA (multiple-cloning site of the pBluescript vector) as competitor. Shifted bands corresponding to CsHMG:RNA complexes and the free probe are indicated by the arrow and asterisk, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285215&req=5

pone-0032305-g005: CsHMG shows DNA and RNA-binding activities in vitro.(A) EMSA using the recombinant 6xHis-CsHMG protein (5 µg) and DNA probes (200 ng) derived from the citrus pr5 promoter and the multiple-cloning site of the pBluescript vector. The DNA-protein complexes and free probes detected by ethidium bromide staining are indicated by the arrows and asterisks, respectively. (B) Upper panel, EMSA using 32P-labelled single strand RNA probes at a final concentration of 12.5 nM and increased amounts of purified CsHMG (0.1 and 0.5 µg). Shifted bands corresponding to CsHMG:RNA complexes observed with the poly(U) RNA and the free probes are indicated by the arrow and asterisk, respectively. Bottom panel, SDS-PAGE of UV-crosslink EMSA showing the selective binding of CsHMG to the poly(U) RNA probe (arrow). (C) EMSA using the 32P-labelled poly(U) RNA as probe at a final concentration of 12.5 nM, 100 ng of CsHMG and increasing amounts of the double strand DNA (multiple-cloning site of the pBluescript vector) as competitor. Shifted bands corresponding to CsHMG:RNA complexes and the free probe are indicated by the arrow and asterisk, respectively.
Mentions: HMGBs are highly abundant chromosomal proteins known to bind DNA in a non-specific manner [26], [27]. Thus, we examined the ability of the recombinant CsHMG to bind DNA in gel-shift assays by testing its interaction with two unrelated double-strand DNA probes, one derived from the citrus pr5 promoter [28] and another derived from a bacterial cloning vector. As shown in Fig. 5A, CsHMG bound to the two DNA probes, indicating that the citrus protein does not display DNA sequence specificity, a general feature of HMGs. Although this result is in line with the literature data and with our observation that CsHMG made in E. coli co-purifies with bacterial DNA (not shown), we also noticed that the removal all traces of nucleic acids from our recombinant CsHMG preparations required an RNase treatment, which indicated that CsHMG had affinity for RNA as well. To test this hypothesis, we performed gel-shift assays to probe the binding of CsHMG to single-strand RNAs. Surprisingly, we found that CsHMG not only binds to single-strand RNA in vitro but it shows specificity to poly(U) RNAs (Fig. 5B, upper panel). The specific binding of CsHMG to poly(U) RNA was further confirmed by an UV-crosslinking gel-shift assay (Fig. 5B, bottom panel).

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