RESUMEN
Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes.
Asunto(s)
Escherichia coli , Regulación Bacteriana de la Expresión Génica , Represoras Lac , Regiones Promotoras Genéticas , Represoras Lac/metabolismo , Represoras Lac/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Operón Lac , Efectores Tipo Activadores de la Transcripción/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Ingeniería de Proteínas/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Multimerización de Proteína , Conformación de Ácido Nucleico , ADN/metabolismo , ADN/genética , ADN/química , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/química , TermodinámicaRESUMEN
Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.
Asunto(s)
Proteínas Bacterianas , Regulación de la Expresión Génica de las Plantas , Oryza , Enfermedades de las Plantas , Proteínas de Plantas , Xanthomonas , Oryza/microbiología , Oryza/genética , Xanthomonas/patogenicidad , Xanthomonas/fisiología , Xanthomonas/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Virulencia/genética , Regiones Promotoras Genéticas/genética , Resistencia a la Enfermedad/genética , Sistemas CRISPR-Cas , Edición Génica , Plantas Modificadas GenéticamenteRESUMEN
One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.
Asunto(s)
Citosina , Edición Génica , Timina , Edición Génica/métodos , Humanos , Citosina/metabolismo , Citosina/química , Timina/metabolismo , Timina/química , Efectores Tipo Activadores de la Transcripción/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , ADN/metabolismo , ADN/genética , Células HEK293RESUMEN
Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.
Asunto(s)
Muerte Celular , Regulación de la Expresión Génica de las Plantas , Nicotiana , Muerte Celular/genética , Nicotiana/genética , Nicotiana/microbiología , Xanthomonas/fisiología , Xanthomonas/patogenicidad , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Efectores Tipo Activadores de la Transcripción/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Genes de Plantas , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Regiones Promotoras Genéticas/genética , Perfilación de la Expresión Génica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Xanthomonas species infect many important crops and cause huge yield loss. These pathogens deliver transcription activator-like (TAL) effectors into the cytoplasm of plant cells. TAL effectors move to host nuclei, directly bind to the promoters of host susceptible genes, and activate their transcription. However, the molecular mechanisms by which TAL effectors induce host transcription remain unclear. We herein demonstrated that TAL effectors interacted with the SIMILAR TO RCD ONE (SRO) family proteins OsSRO1a and OsSRO1b in nuclei. A transactivation assay using rice protoplasts indicated that OsSRO1a and OsSRO1b enhanced the activation of the OsSWEET14 promoter by the TAL effector AvrXa7. The AvrXa7-mediated expression of OsSWEET14 was significantly reduced in ossro1a mutants. However, the overexpression of OsSRO1a increased disease resistance by up-regulating the expression of defense-related genes, such as WRKY62 and PBZ1. This was attributed to OsSRO1a and OsSRO1b also enhancing the transcriptional activity of WRKY45, a direct regulator of WRKY62 expression. Therefore, OsSRO1a and OsSRO1b appear to positively contribute to transcription mediated by bacterial TAL effectors and rice transcription factors.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Oryza , Enfermedades de las Plantas , Proteínas de Plantas , Efectores Tipo Activadores de la Transcripción , Xanthomonas , Oryza/genética , Oryza/microbiología , Xanthomonas/fisiología , Xanthomonas/patogenicidad , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Efectores Tipo Activadores de la Transcripción/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Resistencia a la Enfermedad/genética , Regiones Promotoras Genéticas/genética , Interacciones Huésped-Patógeno/genéticaRESUMEN
BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze Câ¢G-to-Tâ¢A editing. This technology has not been optimized for use in plant cells. RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a ß-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with Câ¢G-to-Tâ¢A conversions in their chloroplast genome, as well as N. benthamiana with Câ¢G-to-Tâ¢A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.
Asunto(s)
Edición Génica , Nicotiana , Edición Génica/métodos , Nicotiana/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Citosina/metabolismo , Oryza/genéticaRESUMEN
DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.
Asunto(s)
ADN Mitocondrial , Efectores Tipo Activadores de la Transcripción , Animales , Humanos , Ratones , Adenina , Citosina , ADN Mitocondrial/genética , Edición Génica , ARN , Efectores Tipo Activadores de la Transcripción/metabolismo , Ingeniería de ProteínasRESUMEN
Xanthomonas oryzae pv. oryzae (Xoo) secretes transcription activator-like effectors (TALEs) to activate rice susceptibility (S) genes, causing bacterial blight (BB), as well as resistance (R) genes, leading to defense against BB. This activation follows a gene-for-gene paradigm that results in an arms race between the TALE of the pathogen and effector-binding elements (EBEs) in the promoters of host genes. In this study, we characterized a novel TALE, designated Tal6b/AvrXa27A, that activates the rice S gene OsSWEET11a and the rice R gene Xa27. Tal6b/AvrXa27A is a member of the AvrXa27/TalAO class and contains 16 repeat variable diresidues (RVDs); one RVD is altered and one is deleted in Tal6b/AvrXa27A compared with AvrXa27, a known avirulence (avr) effector of Xa27. Tal6b/AvrXa27A can transcriptionally activate the expression of Xa27 and OsSWEET11a via EBEs in their corresponding promoters, leading to effector-triggered immunity and susceptibility, respectively. The 16 RVDs in Tal6b/AvrXa27A have no obvious similarity to the 24 RVDs in the effector PthXo1, but EBETal6b and EBEPthXo1 are overlapped in the OsSWEET11a promoter. Tal6b/AvrXa27A is prevalent among Asian Xoo isolates, but PthXo1 has only been reported in the Philippine strain PXO99A. Genome editing of EBETal6b in the OsSWEET11a promoter further confirmed the requirement for OsSWEET11a expression in Tal6b/AvrXa27A-dependent susceptibility to Xoo. Moreover, Tal6b/AvrXa27A resulted in higher transcription of Xa27 than of OsSWEET11a, which led to a strong, rapid resistance response that blocked disease development. These findings suggest that Tal6b/AvrXa27A has a dual function: triggering resistance by activating Xa27 gene expression as an avirulence factor and inducing transcription of the S gene OsSWEET11a, resulting in virulence. Intriguingly, Tal6b/AvrXa27A, but not AvrXa27, can bind to the promoter of OsSWEET11a. The underlying recognition mechanism for this binding remains unclear but appears to deviate from the currently accepted TALE code.
Asunto(s)
Oryza , Xanthomonas , Oryza/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Regiones Promotoras Genéticas/genética , Edición Génica , Virulencia , Xanthomonas/genéticaRESUMEN
Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator-like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc-TALE-trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death-inducing executor gene, avrGf2, was strictly TALE-dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc-TALE-trap mediates resistance to this global panel of Xcc isolates. We also studied in planta-evolved TALEs (eTALEs) with novel DNA-binding domains and found that these eTALEs also activate the Xcc-TALE-trap, suggesting that the Xcc-TALE-trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc-TALE-trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc-TALE-trap offer a promising sustainable approach to control CBC.
Asunto(s)
Citrus , Xanthomonas , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Citrus/genética , Citrus/microbiología , Xanthomonas/genética , Regiones Promotoras Genéticas/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
Using genetic resistance against bacterial blight (BB) caused by Xanthomonas oryzae pathovar oryzae (Xoo) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm against Xoo. Here, we use an improved prime-editing system to implement two new strategies for BB resistance. Knock-in of TAL effector binding elements (EBE) derived from the BB susceptible gene SWEET14 into the promoter of a dysfunctional executor R gene xa23 reaches 47.2% with desired edits including biallelic editing at 18% in T0 generation that enables an inducible TALE-dependent BB resistance. Editing the transcription factor TFIIA gene TFIIAγ5 required for TAL effector-dependent BB susceptibility recapitulates the resistance of xa5 at an editing efficiency of 88.5% with biallelic editing rate of 30% in T0 generation. The engineered loci provided resistance against multiple Xoo strains in T1 generation. Whole-genome sequencing detected no OsMLH1dn-associated random mutations and no off-target editing demonstrating high specificity of this PE system. This is the first-ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock-in of 30-nucleotides cis-regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolving Xoo strains and protect it from epidemics.
Asunto(s)
Oryza , Xanthomonas , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Oryza/metabolismo , Fitomejoramiento , Regiones Promotoras Genéticas , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/prevención & control , Enfermedades de las Plantas/microbiologíaRESUMEN
Xanthomonas oryzae pv. oryzae (Xoo) is a major rice pathogen, and its genome harbors extensive inter-strain and inter-lineage variations. The emergence of highly virulent pathotypes of Xoo that can overcome major resistance (R) genes deployed in rice breeding programs is a grave threat to rice cultivation. The present study reports on a long-read Oxford nanopore-based complete genomic investigation of Xoo isolates from 11 pathotypes that are reported based on their reaction toward 10 R genes. The investigation revealed remarkable variation in the genome structure in the strains belonging to different pathotypes. Furthermore, transcription activator-like effector (TALE) proteins secreted by the type III secretion system display marked variation in content, genomic location, classes, and DNA-binding domain. We also found the association of tal genes in the vicinity of regions with genome structural variations. Furthermore, in silico analysis of the genome-wide rice targets of TALEs allowed us to understand the emergence of pathotypes compatible with major R genes. Long-read, cost-effective sequencing technologies such as nanopore can be a game changer in the surveillance of major and emerging pathotypes. The resource and findings will be invaluable in the management of Xoo and in appropriate deployment of R genes in rice breeding programs.
Asunto(s)
Oryza , Xanthomonas , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Enfermedades de las Plantas/genética , Fitomejoramiento , Xanthomonas/genética , Oryza/genéticaRESUMEN
Transcription activator-like effectors (TALEs) are bacterial proteins that are injected into the eukaryotic nucleus to act as transcriptional factors and function as key virulence factors of the phytopathogen Xanthomonas. TALEs are translocated into plant host cells via the type III secretion system and induce the expression of host susceptibility (S) genes to facilitate disease. The unique modular DNA binding domains of TALEs comprise an array of nearly identical direct repeats that enable binding to DNA targets based on the recognition of a single nucleotide target per repeat. The very nature of TALE structure and function permits the proliferation of TALE genes and evolutionary adaptations in the host to counter TALE function, making the TALE-host interaction the most dynamic story in effector biology. The TALE genes appear to be a relatively young effector gene family, with a presence in all virulent members of some species and absent in others. Genome sequencing has revealed many TALE genes throughout the xanthomonads, and relatively few have been associated with a cognate S gene. Several species, including Xanthomonas oryzae pv. oryzae and X. citri pv. citri, have near absolute requirement for TALE gene function, while the genes appear to be just now entering the disease interactions with new fitness contributions to the pathogens of tomato and pepper among others. Deciphering the simple and effective DNA binding mechanism also has led to the development of DNA manipulation tools in fields of gene editing and transgenic research. In the three decades since their discovery, TALE research remains at the forefront of the study of bacterial evolution, plant-pathogen interactions, and synthetic biology. We also discuss critical questions that remain to be addressed regarding TALEs.
Asunto(s)
Oryza , Xanthomonas , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Virulencia , Xanthomonas/genética , Oryza/microbiologíaRESUMEN
INTRODUCTION: Xa23 as an executor mediates broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), which contains a matching avirulence gene avrXa23, in rice for bacterial leaf blight (BLB). avrXa23 encodes a transcription activator-like effector (TALE) protein which binds to the EBE (effector-binding element) of the Xa23 promoter. It is unclear whether the considerable pressure of Xa23 leads to an emerging Xoo strain that overcomes Xa23 resistance. OBJECTIVES: This study aimed to uncover new Xoo isolate(s) that overcome Xa23-mediated resistance and to investigate how the pathogen evades the resistance. METHODS: Totally 185 Xoo isolates were used to screen possibly compatible strain(s) with Xa23-containing rice CBB23 by pathogenicity test. Genome Sequencing, Southern blot, tal gene cloning, Western blot, qRT-PCR and electrophoretic mobility shift assays (EMSA) were conducted to determine the mechanism of one Xoo isolate being compatible with Xa23-containing rice. RESULTS: One isolate AH28 from Anhui province is compatible with CBB23. AH28 strain contains an ortholog of avrXa23, tal7b and has 17 tal genes. The 4th RVD (repeat-variable diresidue) in Tal7b are missed and the 5th and 8th RVDs changed from NG and NS to NS and S*, respectively. These alternations made Tal7b unable to bind to the EBE of Xa23 promoter to activate the expression of Xa23 in rice. The ectopic expression of tal7b in a tal-free mutant PH of PXO99A did not alter the virulence of the strain PH, whereas avrXa23 made AH28 from compatibility to incompatibility with Xa23 rice. CONCLUSION: Best to our knowledge, this is the first insight of a naturally-emerging Xoo isolate that overcomes the broad-spectrum resistance of Xa23 by the variable AvrXa23-like TALE Tal7b. The RVD alteration in AvrXa23 may be a common strategy for the pathogen evolution to avoid being "trapped" by the executor R gene.
Asunto(s)
Oryza , Oryza/genética , Oryza/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Regulación de la Expresión Génica de las PlantasRESUMEN
Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.
Asunto(s)
Sistemas CRISPR-Cas , Nucleósido Desaminasas , Sistemas CRISPR-Cas/genética , ADN/genética , Roturas del ADN de Doble Cadena , Edición Génica/métodos , Nucleósido Desaminasas/genética , Nucleósido Desaminasas/metabolismo , Nucleótidos , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
Bacterial blight (BB) and bacterial leaf streak (BLS), caused by phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively, are the most serious bacterial diseases of rice, while blast, caused by Magnaporthe oryzae (M. oryzae), is the most devastating fungal disease in rice. Generating broad-spectrum resistance to these diseases is one of the key approaches for the sustainable production of rice. Executor (E) genes are a unique type of plant resistance (R) genes, which can specifically trap transcription activator-like effectors (TALEs) of pathogens and trigger an intense defense reaction characterized by a hypersensitive response in the host. This strong resistance is a result of programed cell death induced by the E gene expression that is only activated upon the binding of a TALE to the effector-binding element (EBE) located in the E gene promoter during the pathogen infection. Our previous studies revealed that the E gene Xa23 has the broadest and highest resistance to BB. To investigate whether the Xa23-mediated resistance is efficient against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of BLS, we generated a new version of Xa23, designated as Xa23p1.0, to specifically trap the conserved TALEs from multiple Xoc strains. The results showed that the Xa23p1.0 confers broad resistance against both BB and BLS in rice. Moreover, our further experiment on the Xa23p1.0 transgenic plants firstly demonstrated that the E-gene-mediated defensive reaction is also effective against M. oryzae, the causal agent of the most devastating fungal disease in rice. Our current work provides a new strategy to exploit the full potential of the E-gene-mediated disease resistance in rice.
Asunto(s)
Oryza , Xanthomonas , Resistencia a la Enfermedad/genética , Expresión Génica Ectópica , Oryza/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Efectores Tipo Activadores de la Transcripción/metabolismo , Xanthomonas/genéticaRESUMEN
Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.
Asunto(s)
Oryza , Efectores Tipo Activadores de la Transcripción , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Resistencia a la Enfermedad/genética , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica de las Plantas , Oryza/metabolismo , Fitomejoramiento , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Talco/metabolismo , Efectores Tipo Activadores de la Transcripción/metabolismo , XanthomonasRESUMEN
Xanthomonas species colonize many host plants and cause huge losses worldwide. Transcription activator-like effectors (TALEs) are secreted by Xanthomonas and translocated into host cells to manipulate the expression of target genes, especially by Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, which cause bacterial blight and bacterial leaf streak, respectively, in rice. In this review, we summarize the progress of studies on the interaction between Xanthomonas and hosts, covering both rice and other plants. TALEs are not only key factors that make plants susceptible but are also essential components of plant resistance. Characterization of TALEs and TALE-like proteins has improved our understanding of TALE evolution and promoted the development of gene editing tools. In addition, the interactions between TALEs and hosts have also provided strategies and possibilities for genetic engineering in crop improvement.
Asunto(s)
Interacciones Huésped-Patógeno , Oryza , Efectores Tipo Activadores de la Transcripción , Xanthomonas , Interacciones Huésped-Patógeno/genética , Oryza/metabolismo , Oryza/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismoRESUMEN
The present study aimed to evaluate transcriptional activator-like effector (TALE) genes in 86 Xanthomonas oryzae pv. oryzicola strains collected from 8 rice-growing regions in Yunnan, and to examine the relationship between TALE genotypes and virulence in 6 differential rice lines. Besides, the geographical areas, distribution of these genotypes were studied in detail. Genetic diversity was analyzed through the number and size of putative TALE genes based on TALE gene avrXa3 as a probe. We found that X. oryzae pv. oryzicola strains consist of variable number (13-27) of avrXa3-hybridizing fragments (putative TALE genes). Test strains were classified into 8 genotypes (G1-G8) with major genotypes G3 and G7 widely distributed in Yunnan. Pathogenicity of X. oryzae pv. oryzicola was evaluated by inoculating 6 differential rice lines with a single resistance gene into 9 pathotypes clusters (I-IX), the dominant Genotypes G3 and G7 consist of pathotypes I, II, and IV. Furthermore, we also detected the known TALE target genes expression in susceptible rice cultivar (cv. nipponbare) after inoculating 8 genotypes-representative X. oryzae pv. oryzicola strain. Correlation between the numbers of putative TALE genes of X. oryzae pv. oryzicola and relevant target genes in nipponbare confirmed up-regulation. Altogether, this study has given insights into the population structure of X. oryzae pv. oryzicola that may inform strategies to control BLS in rice.
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Oryza , Xanthomonas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , China , ADN Bacteriano/genética , Oryza/genética , Enfermedades de las Plantas , Hojas de la Planta/metabolismo , Efectores Tipo Activadores de la Transcripción/metabolismo , Virulencia/genética , Xanthomonas/genéticaRESUMEN
The molecular mechanism of pomegranate susceptibility to bacterial blight, a serious threat to pomegranate production in India, is largely unknown. In the current study, we have used PacBio and Illumina sequencing of Xanthomonas citri pv. punicae (Xcp) strain 119 genome to identify tal genes and RNA-Seq analysis to identify putative host targets in the susceptible pomegranate variety Bhagwa challenged with Xcp119. Xcp119 genome encodes seven transcription activator-like effectors (TALEs), three of which are harbored by a plasmid. RVD-based phylogenetic analysis of TALEs of Xanthomonas citri pathovars indicate the TALEs of Xcp as evolutionarily and functionally close to Xanthomonas citri pv. malvacearum and Xanthomonas citri pv. glycines. Comparative RNA-Seq of Xcp and mock-inoculated leaf tissues revealed Xcp-induced pomegranate transcription modulation. The prediction of TALE binding elements (EBEs) in the promoters of up-regulated genes identified a set of TALE-targeted candidate genes in pomegranate-Xcp interaction. The predicted candidate susceptibility genes include two oxoglutarate-dependent dioxygenase gene, ethylene-responsive transcription factor and flavanone 3-hydroxylase-like gene, and the further characterization of these would enable blight resistance engineering in pomegranate.
Asunto(s)
Oryza , Granada (Fruta) , Xanthomonas , Oryza/microbiología , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismoRESUMEN
Executor (E) genes comprise a new type of plant resistance (R) genes, identified from host-Xanthomonas interactions. The Xanthomonas-secreted transcription activation-like effectors (TALEs) usually function as major virulence factors, which activate the expression of the so-called "susceptibility" (S) genes for disease development. This activation is achieved via the binding of the TALEs to the effector-binding element (EBE) in the S gene promoter. However, host plants have evolved EBEs in the promoters of some otherwise silent R genes, whose expression directly causes a host cell death that is characterized by a hypersensitive response (HR). Such R genes are called E genes because they trap the pathogen TALEs in order to activate expression, and the resulting HR prevents pathogen growth and disease development. Currently, deploying E gene resistance is becoming a major component in disease resistance breeding, especially for rice bacterial blight resistance. Currently, the biochemical mechanisms, or the working pathways of the E proteins, are still fuzzy. There is no significant nucleotide sequence homology among E genes, although E proteins share some structural motifs that are probably associated with the signal transduction in the effector-triggered immunity. Here, we summarize the current knowledge regarding TALE-type avirulence proteins, E gene activation, the E protein structural traits, and the classification of E genes, in order to sharpen our understanding of the plant E genes.