The repeat domain of the type III effector protein PthA shows a TPR‐like structure and undergoes conformational changes upon DNA interaction

Murakami, Mário Tyago; Sforça, Maurício L.; Neves, Jorge Luiz; Paiva, J.H.; Domingues, M.N.; Pereira, André Luiz Araújo; Zeri, Ana Carolina de Mattos; Benedetti, Celso Eduardo

doi:10.1002/prot.22846

Cited by 40 publications

(53 citation statements)

References 63 publications

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“…All binding isotherms displayed positive cooperativity ([XPCT1L] 0.9 / [XPCT1L] 0.1 ϳ10) consistent with Ref. 18. Apparent dissociation constants (K d ) were determined with the Simfit/Sffit programs using a cooperative ligand binding saturation function (19).…”

Section: Methodssupporting

confidence: 61%

Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation

Valton¹,

Dupuy²,

Daboussi³

et al. 2012

Journal of Biological Chemistry

166

142

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Background: TALE-based technologies are poised to revolutionize the field of biotechnology; however, their sensitivity to cytosine methylation may drastically restrict their ranges of applications. Results: TALE repeat N* proficiently accommodates 5-methylated cytosine. Conclusion: Sensitivity of TALE to cytosine methylation can be overcome by using TALE repeat N*. Significance: Utilization of TALE repeat N* enables broadening the scope of TALE-based technologies.Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.Transcription activator-like effectors (TALEs), 4 a group of bacterial plant pathogen proteins, have recently emerged as new engineerable scaffolds for production of tailored DNA binding domains with chosen specificities (1). Interest in these systems comes from the apparent simple cipher governing DNA recognition by their DNA binding domain (2, 3). The TALE DNA binding domain is composed of multiple TALE repeats that individually recognize one DNA base pair through specific amino acid di-residues (repeat variable di-residues or RVDs). The remarkably high specificity of TALE repeats and the apparent absence of context-dependent effects among repeats in an array allow modular assembly of TALE DNA binding domains able to recognize almost any DNA sequence of interest. Within the past 2 years, engineered TALE DNA binding domains have been fused to transcription activator (dTALEs) (4), repressor (5), or nuclease domains (TALENs) (6) and used to specifically regulate or modify genes of interest (1). Although successfully used in different cellular contexts, engineered TALE DNA binding domains have recently been reported to be affected by the presence of 5-methylated cytosine (5mC) in their endogenous cognate target (7). Often considered as the fifth base, 5mC is found in about 70% of CpG dinucleotides in mammalian and plant somatic/pluripotent cells (8, 9) and has also been reported in 5-cytosine-phosphoadenine, 5-cytosine-phosphothymine, and 5-cytosine-phosphocytosine dinucleotides (10). Moreover, 5mC has been identified in CpG islands embedded in many promoters (11) and, to a higher extent, in proximal exons of several genes (12). These two critical regulatory regions are generally chosen by investigators to knock out genes of therapeutic and biotechnological interest or to modulate their expression using TALE-based technologies. The ubiquity of 5mC in different cell types and genomic kingdoms, its particular localization, and its negative impact on dTALE activity reported in Ref. 7 make this epigenetic modification a major dra...

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Section: Methodssupporting

confidence: 61%

Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation

Valton¹,

Dupuy²,

Daboussi³

et al. 2012

Journal of Biological Chemistry

166

142

View full text Add to dashboard Cite

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“…The TPR domains in the PcrH chaperone of the Pseudomonas aeruginosa T3SS are required for translocation of PopB and PopD substrates (7). In Xanthomonas axonopodis, the causative agent of citrus canker, the PthA T3SS substrate is a TPRcontaining protein (35). The current work furthered understanding of the roles of L. pneumophila TPR-containing proteins LpnE and EnhC in T4SS-dependent virulence phenotypes in three areas.…”

Section: Discussionmentioning

confidence: 70%

Implication of Proteins Containing Tetratricopeptide Repeats in Conditional Virulence Phenotypes of Legionella pneumophila

et al. 2012

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Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/ Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ⌬lpnE or the ⌬enhC mutant required that the other TPR-containing protein be present. The single and double ⌬lpnE and ⌬enhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ⌬lpnE single and the ⌬lpnE ⌬enhC double mutant showed 3-to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates.

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“…TAL effectors recognize promoter elements of host genes through their internal repetitive DNA-binding domain, a superhelical structure that embraces the DNA target sequence and provides the specificity for the interactions (Murakami et al, 2010;Deng et al, 2012;Mak et al, 2012). Although the specificity, DNA-binding mode, and numerous targets of TAL effectors have been elucidated, the molecular mechanism by which TAL effectors modulate transcription in the host is still poorly understood.…”

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confidence: 99%

Citrus MAF1, a Repressor of RNA Polymerase III, Binds the Xanthomonas citri Canker Elicitor PthA4 and Suppresses Citrus Canker Development

et al. 2013

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Transcription activator-like (TAL) effectors from Xanthomonas species pathogens act as transcription factors in plant cells; however, how TAL effectors activate host transcription is unknown. We found previously that TAL effectors of the citrus canker pathogen Xanthomonas citri, known as PthAs, bind the carboxyl-terminal domain of the sweet orange (Citrus sinensis) RNA polymerase II (Pol II) and inhibit the activity of CsCYP, a cyclophilin associated with the carboxyl-terminal domain of the citrus RNA Pol II that functions as a negative regulator of cell growth. Here, we show that PthA4 specifically interacted with the sweet orange MAF1 (CsMAF1) protein, an RNA polymerase III (Pol III) repressor that controls ribosome biogenesis and cell growth in yeast (Saccharomyces cerevisiae) and human. CsMAF1 bound the human RNA Pol III and rescued the yeast maf1 mutant by repressing tRNAHis transcription. The expression of PthA4 in the maf1 mutant slightly restored tRNA His synthesis, indicating that PthA4 counteracts CsMAF1 activity. In addition, we show that sweet orange RNA interference plants with reduced CsMAF1 levels displayed a dramatic increase in tRNA transcription and a marked phenotype of cell proliferation during canker formation. Conversely, CsMAF1 overexpression was detrimental to seedling growth, inhibited tRNA synthesis, and attenuated canker development. Furthermore, we found that PthA4 is required to elicit cankers in sweet orange leaves and that depletion of CsMAF1 in X. citri-infected tissues correlates with the development of hyperplastic lesions and the presence of PthA4. Considering that CsMAF1 and CsCYP function as canker suppressors in sweet orange, our data indicate that TAL effectors from X. citri target negative regulators of RNA Pol II and Pol III to coordinately increase the transcription of host genes involved in ribosome biogenesis and cell proliferation.

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The repeat domain of the type III effector protein PthA shows a TPR‐like structure and undergoes conformational changes upon DNA interaction

Cited by 40 publications

References 63 publications

Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation

Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation

Implication of Proteins Containing Tetratricopeptide Repeats in Conditional Virulence Phenotypes of Legionella pneumophila

Citrus MAF1, a Repressor of RNA Polymerase III, Binds the Xanthomonas citri Canker Elicitor PthA4 and Suppresses Citrus Canker Development

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