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  • 1
    Language: English
    In: Nucleic acids research, June 2014, Vol.42(11), pp.7160-9
    Description: AvrBs3, the founding member of the Xanthomonas transcription-activator-like effectors (TALEs), is translocated into the plant cell where it localizes to the nucleus and acts as transcription factor. The DNA-binding domain of AvrBs3 consists of 17.5 nearly-identical 34 amino acid-repeats. Each repeat specifies binding to one base in the target DNA via amino acid residues 12 and 13 termed repeat variable diresidue (RVD). Natural target sequences of TALEs are generally preceded by a thymine (T0), which is coordinated by a tryptophan residue (W232) in a degenerated repeat upstream of the canonical repeats. To investigate the necessity of T0 and the conserved tryptophan for AvrBs3-mediated gene activation we tested TALE mutant derivatives on target sequences preceded by all possible four bases. In addition, we performed domain swaps with TalC from a rice pathogenic Xanthomonas because TalC lacks the tryptophan residue, and the TalC target sequence is preceded by cytosine. We show that T0 works best and that T0 specificity depends on the repeat number and overall RVD-composition. T0 and W232 appear to be particularly important if the RVD of the first repeat is HD ('rep1 effect'). Our findings provide novel insights into the mechanism of T0 recognition by TALE proteins and are important for TALE-based biotechnological applications.
    Keywords: Bacterial Proteins -- Chemistry ; DNA-Binding Proteins -- Chemistry ; Transcription Factors -- Chemistry
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 2
    In: Plant Journal, August 2017, Vol.91(3), pp.430-442
    Description: Many Gram‐negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type secretion system during infection. In , recognition of XopQ/HopQ1 proteins induces an effector‐triggered immunity () reaction which is not associated with strong cell death but renders plants immune against and pv. strains. Additionally, XopQ suppresses cell death in . when transiently co‐expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the ‐‐ class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed ‐mediated protein expression experiments in wild‐type and ‐deficient () . leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in plants. We conclude that XopQ‐mediated cell death suppression in . is due to the attenuation of ‐mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co‐expression assays for elucidation of effector activities, at least under conditions of induction. Many and pv. strains are not pathogenic on because recognition of the effector proteins HopQ1 and XopQ, respectively, induces effector‐triggered immunity. The comparison of XopQ/HopQ1‐family proteins and characterization of XopQ derivatives indicate that conserved structural features of the protein family are recognized. In transient expression assays, XopQ‐induced effector‐triggered immunity acts efficiently against . resulting in attenuated bacterial multiplication and transient protein accumulation.
    Keywords: Xopq ; Hopq1 ; Non‐Host Resistance ; Eti ; Nicotiana Benthamiana ; Xanthomonas ; Agrobacterium Tumefaciens ; Transient Protein Expression ; Eds 1
    ISSN: 0960-7412
    E-ISSN: 1365-313X
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  • 3
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(3), p.e0120214
    Description: AvrBs3, the archetype of the family of transcription activator-like (TAL) effectors from phytopathogenic Xanthomonas bacteria, is translocated by the type III secretion system into the plant cell. AvrBs3 localizes to the plant cell nucleus and activates the transcription of target genes. Crucial for this is the central AvrBs3 region of 17.5 34-amino acid repeats that functions as a DNA-binding domain mediating recognition in a "one-repeat-to-one base pair" manner. Although AvrBs3 forms homodimers in the plant cell cytosol prior to nuclear import, it binds DNA as a monomer. Here, we show that complex formation of AvrBs3 proteins negatively affects their DNA-binding affinity in vitro. The conserved cysteine residues at position 30 of each repeat facilitate AvrBs3 complexes via disulfide bonds in vitro but are also required for the gene-inducing activity of the AvrBs3 monomer, i.e., activation of plant gene promoters. Our data suggest that the latter is due to a contribution to protein plasticity and that cysteine substitutions to alanine or serine result in a different DNA-binding mode. In addition, our studies revealed that extended parts of both the N-terminal and C-terminal regions of AvrBs3 contribute to DNA binding and, hence, gene-inducing activity in planta.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 4
    In: PLoS ONE, 2015, Vol.10(8)
    Description: The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria ( Xcv ) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv -infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv -infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv -tomato and Xcv -pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens.
    Keywords: Research Article
    E-ISSN: 1932-6203
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  • 5
    Language: English
    In: PLoS ONE, 2011, Vol.6(5), p.e19509
    Description: TAL ( t ranscription a ctivator- l ike) effectors are translocated by Xanthomonas bacteria into plant cells where they activate transcription of target genes. DNA target sequence recognition occurs in a unique mode involving a central domain of tandem repeats. Each repeat recognizes a single base pair in a contiguous DNA sequence and a pair of adjacent hypervariable amino acid residues per repeat specifies which base is bound. Rearranging the repeats allows the design of novel TAL proteins with predictable DNA-recognition specificities. TAL protein-based transcriptional activation in plant cells is mediated by a C-terminal activation domain (AD). Here, we created synthetic TAL proteins with designed repeat compositions using a novel modular cloning strategy termed “Golden TAL Technology”. Newly programmed TAL proteins were not only functional in plant cells, but also in human cells and activated targeted expression of exogenous as well as endogenous genes. Transcriptional activation in different human cell lines was markedly improved by replacing the TAL-AD with the VP16-AD of herpes simplex virus. The creation of TAL proteins with potentially any desired DNA-recognition specificity allows their versatile use in biotechnology.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Plant Biology ; Biotechnology
    E-ISSN: 1932-6203
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  • 6
    In: Plant Journal, March 2018, Vol.93(5), pp.856-870
    Description: pv. type ‐secreted effectors were screened for candidates influencing plant cell processes relevant to the formation and maintenance of stromules in lower leaf epidermis. Transient expression of XopL, a unique type of E3 ubiquitin ligase, led to a nearly complete elimination of stromules and the relocation... Until now, stromule research has focused on actin as the central cytoskeletal element responsible for the extension and dynamics of stromules. This study has revealed the existence of two distinct populations of stromules: (i) stromules likely dependent on actin, and (ii) slower moving, longer lasting...
    Keywords: Actin ; E3 Ubiquitin Ligase ; Microtubules ; Nicotiana Benthamiana ; Stromule Dynamics ; Stromule Morphology ; Stromule Velocity ; Xanthomonas Campestris Pv. Vesicatoria ; Xopl
    ISSN: 0960-7412
    E-ISSN: 1365-313X
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  • 7
    Language: English
    In: New Phytologist, Sept, 2010, Vol.187, p.1058(17)
    Description: To authenticate to the full-text of this article, please visit this link: http://dx.doi.org/10.1111/j.1469-8137.2010.03346.x Byline: Robert Szczesny, Daniela Buttner, Lucia Escolar, Sebastian Schulze, Anja Seiferth, Ulla Bonas Keywords: AvrRxv; bacterial spot disease; effectors; pepper; protease; transacetylase; YopJ Abstract: Summary Pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) depends on a type III secretion system that translocates a cocktail of 〉 25 type III effector proteins into the plant cell. In this study, we identified the effector AvrBsT as a suppressor of specific plant defense. AvrBsT belongs to the YopJ/AvrRxv protein family, members of which are predicted to act as proteases and/or acetyltransferases. AvrBsT suppresses the hypersensitive response (HR) that is elicited by the effector protein AvrBs1 from Xcv in resistant pepper plants. HR suppression occurs inside the plant cell and depends on a conserved predicted catalytic residue of AvrBsT. Yeast two-hybrid based analyses identified plant interaction partners of AvrBs1 and AvrBsT, including a putative regulator of sugar metabolism, SNF1-related kinase 1 (SnRK1), as interactor of AvrBsT. Intriguingly, gene silencing experiments revealed that SnRK1 is required for the induction of the AvrBs1-specific HR. We therefore speculate that SnRK1 is involved in the AvrBsT-mediated suppression of the AvrBs1-specific HR. Article History: Received: 29 April 2010Accepted: 18 May 2010 Article note: Author for correspondence:, Ulla Bonas, Tel: +49 345 5526290, Email: ulla.bonas@genetik.uni-halle.de
    Keywords: Proteases -- Physiological Aspects ; Carbohydrate Metabolism -- Physiological Aspects ; Glucose Metabolism -- Physiological Aspects ; Bacterial Infections -- Physiological Aspects ; Cytological Research -- Physiological Aspects ; Proteins -- Physiological Aspects
    ISSN: 0028-646X
    Source: Cengage Learning, Inc.
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  • 8
    Language: English
    In: New Phytologist, Sept, 2010, Vol.187, p.1058(17)
    Description: To authenticate to the full-text of this article, please visit this link: http://dx.doi.org/10.1111/j.1469-8137.2010.03346.x Byline: Robert Szczesny, Daniela Buttner, Lucia Escolar, Sebastian Schulze, Anja Seiferth, Ulla Bonas Keywords: AvrRxv; bacterial spot disease; effectors; pepper; protease; transacetylase; YopJ Abstract: Summary Pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) depends on a type III secretion system that translocates a cocktail of 〉 25 type III effector proteins into the plant cell. In this study, we identified the effector AvrBsT as a suppressor of specific plant defense. AvrBsT belongs to the YopJ/AvrRxv protein family, members of which are predicted to act as proteases and/or acetyltransferases. AvrBsT suppresses the hypersensitive response (HR) that is elicited by the effector protein AvrBs1 from Xcv in resistant pepper plants. HR suppression occurs inside the plant cell and depends on a conserved predicted catalytic residue of AvrBsT. Yeast two-hybrid based analyses identified plant interaction partners of AvrBs1 and AvrBsT, including a putative regulator of sugar metabolism, SNF1-related kinase 1 (SnRK1), as interactor of AvrBsT. Intriguingly, gene silencing experiments revealed that SnRK1 is required for the induction of the AvrBs1-specific HR. We therefore speculate that SnRK1 is involved in the AvrBsT-mediated suppression of the AvrBs1-specific HR. Article History: Received: 29 April 2010Accepted: 18 May 2010 Article note: Author for correspondence:, Ulla Bonas, Tel: +49 345 5526290, Email: ulla.bonas@genetik.uni-halle.de
    Keywords: Proteases -- Physiological Aspects ; Carbohydrate Metabolism -- Physiological Aspects ; Glucose Metabolism -- Physiological Aspects ; Bacterial Infections -- Physiological Aspects ; Cytological Research -- Physiological Aspects ; Proteins -- Physiological Aspects
    ISSN: 0028-646X
    Source: Cengage Learning, Inc.
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  • 9
    Language: English
    In: Science, 2007, Vol.318(5850), pp.645-648
    Description: Plant disease resistance (R) proteins recognize matching pathogen avirulence proteins. Alleles of the pepper R gene Bs3 mediate recognition of the Xanthomonas campestris pv. vesicatoria (Xcv) type III effector protein AvrBs3 and its deletion derivative AvrBs3Δrep16. Pepper Bs3 and its allelic variant Bs3-E encode flavin monooxygenases with a previously unknown structure and are transcriptionally activated by the Xcv effector proteins AvrBs3 and AvrBs3Δrep16, respectively. We found that recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by binding of AvrBs3 or AvrBs3Δrep16 to a defined promoter region. Our data suggest a recognition mechanism in which the Avr protein binds and activates the promoter of the cognate R gene. ; Includes references ; p. 645-648.
    ISSN: 0036-8075
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  • 10
    Language: English
    In: Science (New York, N.Y.), 26 October 2007, Vol.318(5850), pp.648-51
    Description: Pathogenicity of many Gram-negative bacteria relies on the injection of effector proteins by type III secretion into eukaryotic cells, where they modulate host signaling pathways to the pathogen's benefit. One such effector protein injected by Xanthomonas into plants is AvrBs3, which localizes to the plant cell nucleus and causes hypertrophy of plant mesophyll cells. We show that AvrBs3 induces the expression of a master regulator of cell size, upa20, which encodes a transcription factor containing a basic helix-loop-helix domain. AvrBs3 binds to a conserved element in the upa20 promoter via its central repeat region and induces gene expression through its activation domain. Thus, AvrBs3 and likely other members of this family provoke developmental reprogramming of host cells by mimicking eukaryotic transcription factors.
    Keywords: Bacterial Proteins -- Physiology ; Basic Helix-Loop-Helix Transcription Factors -- Physiology ; Capsicum -- Genetics ; Plant Proteins -- Physiology ; Xanthomonas Campestris -- Metabolism
    ISSN: 00368075
    E-ISSN: 1095-9203
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