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1.
BMC Genomics ; 22(1): 914, 2021 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-34965853

RESUMO

BACKGROUND: The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to their target box and determine the specificity of a TALE.All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box. RESULTS: To this end, we extend our previously published tool PrediTALE by considering two epigenetic features: (i) chromatin accessibility of potentially bound regions and (ii) DNA methylation of cytosines within target boxes. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS data in rice.We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq data of infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both.Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets. CONCLUSIONS: Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xanthomonas TALEs.


Assuntos
Doenças das Plantas , Xanthomonas , Proteínas de Bactérias/genética , Epigênese Genética , Doenças das Plantas/genética , Efetores Semelhantes a Ativadores de Transcrição/genética , Xanthomonas/genética , Xanthomonas/metabolismo
2.
PLoS Comput Biol ; 15(7): e1007206, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31295249

RESUMO

Plant-pathogenic Xanthomonas bacteria secrete transcription activator-like effectors (TALEs) into host cells, where they act as transcriptional activators on plant target genes to support bacterial virulence. TALEs have a unique modular DNA-binding domain composed of tandem repeats. Two amino acids within each tandem repeat, termed repeat-variable diresidues, bind to contiguous nucleotides on the DNA sequence and determine target specificity. In this paper, we propose a novel approach for TALE target prediction to identify potential virulence targets. Our approach accounts for recent findings concerning TALE targeting, including frame-shift binding by repeats of aberrant lengths, and the flexible strand orientation of target boxes relative to the transcription start of the downstream target gene. The computational model can account for dependencies between adjacent RVD positions. Model parameters are learned from the wealth of quantitative data that have been generated over the last years. We benchmark the novel approach, termed PrediTALE, using RNA-seq data after Xanthomonas infection in rice, and find an overall improvement of prediction performance compared with previous approaches. Using PrediTALE, we are able to predict several novel putative virulence targets. However, we also observe that no target genes are predicted by any prediction tool for several TALEs, which we term orphan TALEs for this reason. We postulate that one explanation for orphan TALEs are incomplete gene annotations and, hence, propose to replace promoterome-wide by genome-wide scans for target boxes. We demonstrate that known targets from promoterome-wide scans may be recovered by genome-wide scans, whereas the latter, combined with RNA-seq data, are able to detect putative targets independent of existing gene annotations.


Assuntos
Modelos Biológicos , Oryza/microbiologia , Doenças das Plantas/microbiologia , Efetores Semelhantes a Ativadores de Transcrição/fisiologia , Xanthomonas/patogenicidade , Biologia Computacional , Genes de Plantas , Genoma de Planta , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Oryza/genética , Doenças das Plantas/genética , Sequências de Repetição em Tandem , Efetores Semelhantes a Ativadores de Transcrição/genética , Sítio de Iniciação de Transcrição , Virulência/genética , Virulência/fisiologia , Xanthomonas/genética , Xanthomonas/fisiologia
3.
Front Plant Sci ; 10: 162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30858855

RESUMO

Rice-pathogenic Xanthomonas oryzae bacteria cause severe harvest loss and challenge a stable food supply. The pathogen virulence relies strongly on bacterial TALE (transcription activator-like effector) proteins that function as transcriptional activators inside the plant cell. To understand the plant targets of TALEs, we determined the genome sequences of the Indian X. oryzae pv. oryzae (Xoo) type strain ICMP 3125T and the strain PXO142 from the Philippines. Their complete TALE repertoire was analyzed and genome-wide TALE targets in rice were characterized. Integrating computational target predictions and rice transcriptomics data, we were able to verify 12 specifically induced target rice genes. The TALEs of the Xoo strains were reconstructed and expressed in a TALE-free Xoo strain to attribute specific induced genes to individual TALEs. Using reporter assays, we could show that individual TALEs act directly on their target promoters. In particular, we show that TALE classes assigned by AnnoTALE reflect common target genes, and that TALE classes of Xoo and the related pathogen X. oryzae pv. oryzicola share more common target genes than previously believed. Taken together, we establish a detailed picture of TALE-induced plant processes that significantly expands our understanding of X. oryzae virulence strategies and will facilitate the development of novel resistances to overcome this important rice disease.

4.
Genome Biol Evol ; 9(6): 1599-1615, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28637323

RESUMO

Transcription activator-like effectors (TALEs) are secreted by plant-pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity.


Assuntos
Proteínas de Bactérias/genética , Evolução Molecular , Efetores Semelhantes a Ativadores de Transcrição/genética , Xanthomonas/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Códon/genética , Códon/metabolismo , Oryza/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Sequências Repetitivas de Ácido Nucleico , Efetores Semelhantes a Ativadores de Transcrição/química , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Xanthomonas/química , Xanthomonas/classificação , Xanthomonas/metabolismo
5.
Plant J ; 89(1): 155-168, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27579989

RESUMO

Genome editing facilitated by Cas9-based RNA-guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non-model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome-edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non-coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T0 and Arabidopsis T2 populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non-coding DNA for deletion by programmable nucleases.


Assuntos
Arabidopsis/genética , Aberrações Cromossômicas , Cromossomos de Plantas/genética , Edição de Genes/métodos , Nicotiana/genética , Alelos , Sequência de Bases , Sistemas CRISPR-Cas , Genoma de Planta/genética , Modelos Genéticos , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Polimorfismo Genético
6.
Plant Biotechnol J ; 15(3): 306-317, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27539813

RESUMO

As a key virulence strategy to cause bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo) injects into the plant cell DNA-binding proteins called transcription activator-like effectors (TALEs) that bind to effector-binding elements (EBEs) in a sequence-specific manner, resulting in host gene induction. TALEs AvrXa7, PthXo3, TalC and Tal5, found in geographically distant Xoo strains, all target OsSWEET14, thus considered as a pivotal TALE target acting as major susceptibility factor during rice-Xoo interactions. Here, we report the generation of an allele library of the OsSWEET14 promoter through stable expression of TALE-nuclease (TALEN) constructs in rice. The susceptibility level of lines carrying mutations in AvrXa7, Tal5 or TalC EBEs was assessed. Plants edited in AvrXa7 or Tal5 EBEs were resistant to bacterial strains relying on the corresponding TALE. Surprisingly, although indels within TalC EBE prevented OsSWEET14 induction in response to BAI3 wild-type bacteria relying on TalC, loss of TalC responsiveness failed to confer resistance to this strain. The TalC EBE mutant line was, however, resistant to a strain expressing an artificial SWEET14-inducing TALE whose EBE was also edited in this line. This work offers the first set of alleles edited in TalC EBE and uncovers a distinct, broader range of activities for TalC compared to AvrXa7 or Tal5. We propose the existence of additional targets for TalC beyond SWEET14, suggesting that TALE-mediated plant susceptibility may result from induction of several, genetically redundant, host susceptibility genes by a single effector.


Assuntos
Oryza/genética , Oryza/microbiologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Xanthomonas/patogenicidade , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/fisiologia
7.
Sci Rep ; 6: 21077, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26876161

RESUMO

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present 'AnnoTALE', a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.


Assuntos
Proteínas de Bactérias/genética , Evolução Molecular , Efetores Semelhantes a Ativadores de Transcrição/genética , Xanthomonas/genética , Biologia Computacional , Genoma , Anotação de Sequência Molecular , Análise de Sequência de DNA , Fatores de Virulência/genética
8.
Nat Commun ; 5: 3447, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24614980

RESUMO

Transcription activator-like effectors (TALEs) are important Xanthomonas virulence factors that bind DNA via a unique tandem 34-amino-acid repeat domain to induce expression of plant genes. So far, TALE repeats are described to bind as a consecutive array to a consecutive DNA sequence, in which each repeat independently recognizes a single DNA base. This modular protein architecture enables the design of any desired DNA-binding specificity for biotechnology applications. Here we report that natural TALE repeats of unusual amino-acid sequence length break the strict one repeat-to-one base pair binding mode and introduce a local flexibility to TALE-DNA binding. This flexibility allows TALEs and TALE nucleases to recognize target sequence variants with single nucleotide deletions. The flexibility also allows TALEs to activate transcription at allelic promoters that otherwise confer resistance to the host plant.


Assuntos
Proteínas de Bactérias/metabolismo , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mutação , Oryza/genética , Oryza/metabolismo , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Sequências Repetitivas de Aminoácidos/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transativadores/genética , Transativadores/metabolismo , Virulência/genética , Fatores de Virulência/genética , Xanthomonas/genética , Xanthomonas/metabolismo , Xanthomonas/patogenicidade
9.
PLoS Comput Biol ; 9(3): e1002962, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23526890

RESUMO

Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library Jstacs, and is freely available as a web-application and a command line program.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Ligação Proteica , Reprodutibilidade dos Testes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xanthomonas/genética , Xanthomonas/patogenicidade
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