RESUMO
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.
Assuntos
Citrus , Xanthomonas , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Citrus/genética , Citrus/microbiologia , Xanthomonas/genética , Regiões Promotoras Genéticas/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologiaRESUMO
The development of a new crop variety is a time-consuming and costly process due to the reliance of plant breeding on gene shuffling to introduce desired genes into elite germplasm, followed by backcrossing. Here, we propose alternative technology that transiently targets various regulatory circuits within a plant, leading to operator-specified alterations of agronomic traits, such as time of flowering, vernalization requirement, plant height or drought tolerance. We redesigned techniques of gene delivery, amplification and expression around RNA viral transfection methods that can be implemented on an industrial scale and with many crop plants. The process does not involve genetic modification of the plant genome and is thus limited to a single plant generation, is broadly applicable, fast, tunable and versatile, and can be used throughout much of the crop cultivation cycle. The RNA-based reprogramming may be especially useful in plant pathogen pandemics but also for commercial seed production and for rapid adaptation of orphan crops.
Assuntos
Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/genética , Edição de Genes , Melhoramento Vegetal/métodos , Sementes/crescimento & desenvolvimento , Sementes/genética , Regulação da Expressão Gênica de Plantas , Genoma de PlantaRESUMO
Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs31EBE ), and fused this engineered promoter with multiple EBEs (ProBs314EBE ) to either the ß-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs31EBE and ProBs314EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs314EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs314EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants.
Assuntos
Citrus/microbiologia , Genes Bacterianos , Engenharia Genética , Regiões Promotoras Genéticas , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Xanthomonas/genética , Xanthomonas/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsicum/genética , Morte Celular , Reações Cruzadas , Regulação da Expressão Gênica de Plantas , Mutação/genética , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Virulência/genética , Xanthomonas/crescimento & desenvolvimentoRESUMO
Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.
Assuntos
Proteínas de Bactérias/metabolismo , Capsicum/genética , Resistência à Doença/genética , Perfilação da Expressão Gênica/métodos , Genes de Plantas/genética , Doenças das Plantas/microbiologia , Xanthomonas/fisiologia , Proteínas de Bactérias/química , Capsicum/efeitos dos fármacos , Capsicum/imunologia , Capsicum/microbiologia , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , Cicloeximida/farmacologia , Resistência à Doença/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Estudos de Associação Genética , Doenças das Plantas/genética , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , Efetores Semelhantes a Ativadores de Transcrição , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Transcriptoma/genética , Xanthomonas/efeitos dos fármacosRESUMO
In this study, we present a method for transient expression of the type III effector AvrGf1 from Xanthomonas citri subsp. citri strain A(w) in grapefruit leaves (Citrus paradisi) via Agrobacterium tumefaciens. The coding sequence of avrGf1 was placed under the control of the constitutive CaMV 35S promoter in the binary vectors pGWB2 and pGWB5. Infiltration of grapefruit leaves with A. tumefaciens carrying these constructs triggered a hypersensitive response (HR) in grapefruit 4 days after inoculation. When transiently expressed in grapefruit leaves, two mutants, AvrGf1ΔN116 and AvrGf1ΔC83, failed to induce an HR. Moreover, using bioinformatics tools, a chloroplast transit signal was predicted at the N terminus of AvrGf1. We demonstrated chloroplast localization by using an AvrGf1::GFP fusion protein, where confocal images revealed that GFP fluorescence was accumulating in the stomatal cells that are abundant in chloroplasts. Transient expression in citrus has the potential for aiding in the development of new disease defense strategies in citrus.
Assuntos
Citrus/genética , Genes Bacterianos , Engenharia Genética/métodos , Folhas de Planta/genética , Cloroplastos/metabolismo , Citrus/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folhas de Planta/metabolismo , Estômatos de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rhizobium/genética , Análise de Sequência de Proteína , Deleção de Sequência , Nicotiana/genética , Nicotiana/metabolismo , Transformação Genética , Xanthomonas/genética , Xanthomonas/metabolismoRESUMO
Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNA-binding specificity of transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of user-defined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated the desired promoters in a sequence-specific manner as mutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NK mediates specific interaction with G nucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes.
Assuntos
Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Fatores de Transcrição/metabolismo , Xanthomonas/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/microbiologia , Polimorfismo Genético , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Xanthomonas/patogenicidadeRESUMO
*Plant pathogenic bacteria of the genus Xanthomonas inject transcription activator-like effector (TALe) proteins that bind to and activate host promoters, thereby promoting disease or inducing plant defense. TALes bind to corresponding UPT (up-regulated by TALe) promoter boxes via tandemly arranged 34/35-amino acid repeats. Recent studies uncovered the TALe code in which two amino acid residues of each repeat define specific pairing to UPT boxes. *Here we employed the TALe code to predict potential UPT boxes in TALe-induced host promoters and analyzed these via beta-glucuronidase (GUS) reporter and electrophoretic mobility shift assays (EMSA). *We demonstrate that the Xa13, OsTFX1 and Os11N3 promoters from rice are induced directly by the Xanthomonas oryzae pv. oryzae TALes PthXo1, PthXo6 and AvrXa7, respectively. We identified and functionally validated a UPT box in the corresponding rice target promoter for each TALe and show that box mutations suppress TALe-mediated promoter activation. Finally, EMSA demonstrate that code-predicted UPT boxes interact specifically with corresponding TALes. *Our findings show that variations in the UPT boxes of different rice accessions correlate with susceptibility or resistance of these accessions to the bacterial blight pathogen.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Oryza/genética , Doenças das Plantas/microbiologia , Xanthomonas/patogenicidade , Aminoácidos , Glucuronidase/metabolismo , Interações Hospedeiro-Patógeno/genética , Oryza/metabolismo , Doenças das Plantas/genética , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição , Ativação Transcricional , Regulação para CimaRESUMO
Plant pathogenic bacteria of the genus Xanthomonas inject transcription-activator like (TAL) effector proteins that manipulate the hosts' transcriptome to promote disease. However, in some cases plants take advantage of this mechanism to trigger defense responses. For example, transcription of the pepper Bs3 and rice Xa27 resistance (R) genes are specifically activated by the respective TAL effectors AvrBs3 from Xanthomonas campestris pv. vesicatoria (Xcv), and AvrXa27 from X. oryzae pv. oryzae (Xoo). Recognition of AvrBs3 was shown to be mediated by interaction with the corresponding UPT (UPregulated by TAL effectors) box UPT(AvrBs3) present in the promoter R gene Bs3 from the dicot pepper. In contrast, it was not known how the Xoo TAL effector AvrXa27 transcriptionally activates the matching R gene Xa27 from the monocot rice. Here we identified a 16-bp UPT(AvrXa27) box present in the rice Xa27 promoter that when transferred into the Bs3 promoter confers AvrXa27-dependent inducibility. We demonstrate that polymorphisms between the UPT(AvrXa27) box of the AvrXa27-inducible Xa27 promoter and the corresponding region of the noninducible xa27 promoter account for their distinct inducibility and affinity, with respect to AvrXa27. Moreover, we demonstrate that three functionally distinct UPT boxes targeted by separate TAL effectors retain their function and specificity when combined into one promoter. Given that many economically important xanthomonads deliver multiple TAL effectors, the engineering of R genes capable of recognizing multiple TAL effectors provides a potential approach for engineering broad spectrum and durable disease resistance.
Assuntos
Proteínas de Bactérias/metabolismo , Imunidade Inata/genética , Oryza/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Transativadores/genética , Xanthomonas/genética , Agrobacterium tumefaciens , Proteínas de Bactérias/genética , Sequência de Bases , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oryza/microbiologia , Doenças das Plantas/microbiologia , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNARESUMO
The pepper (Capsicum annuum) bacterial spot (Bs) resistance gene Bs3 and its allelic variant Bs3-E mediate recognition of the Xanthomonas campestris pv vesicatoria type III effector protein AvrBs3 and its deletion derivative AvrBs3Deltarep16. Recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by a defined promoter region, the UPA (for up-regulated by AvrBs3) box. Using site-directed mutagenesis, we defined the exact boundaries of the UPA(AvrBs3) box of the Bs3 promoter and the UPA(AvrBs3Deltarep16) box of the Bs3-E promoter and show that both boxes overlap by at least 11 nucleotides. Despite partial sequence identity, the UPA(AvrBs3) box and the UPA(AvrBs3Deltarep16) box were bound specifically by the corresponding AvrBs3 and AvrBs3Deltarep16 proteins, respectively, suggesting that selective promoter binding of AvrBs3-like proteins is the basis for promoter activation specificity. We also demonstrate that the UPA(AvrBs3) box retains its functionality at different positions within the pepper Bs3 promoter and confers AvrBs3 inducibility in a novel promoter context. Notably, the transfer of the UPA(AvrBs3) box to different promoter locations is always correlated with a new transcriptional start site. The analysis of naturally occurring Bs3 alleles revealed many pepper accessions that encode a nonfunctional Bs3 variant. These accessions showed no apparent abnormalities, supporting the supposition that Bs3 functions only in disease resistance and not in other developmental or physiological processes.
Assuntos
Alelos , Proteínas de Bactérias/metabolismo , Capsicum/genética , Capsicum/microbiologia , Genes de Plantas , Regiões Promotoras Genéticas , Pareamento de Bases , Sequência de Bases , Capsicum/imunologia , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mutagênese Insercional , Mapeamento Físico do Cromossomo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Xanthomonas campestris/metabolismoRESUMO
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 AvrBs3Deltarep16. 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 AvrBs3Deltarep16, respectively. We found that recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by binding of AvrBs3 or AvrBs3Deltarep16 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.
Assuntos
Proteínas de Bactérias/metabolismo , Capsicum/genética , Capsicum/microbiologia , Genes de Plantas , Oxigenases de Função Mista/genética , Regiões Promotoras Genéticas , Xanthomonas campestris/patogenicidade , Alelos , Proteínas de Bactérias/genética , Sequência de Bases , Cromossomos Artificiais Bacterianos , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/química , Dados de Sequência Molecular , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Nicotiana/genética , Transcrição Gênica , Transformação Genética , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismoRESUMO
The pepper (Capsicum annuum) Bs3 gene confers resistance to avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. To physically delimit Bs3, a pepper YAC library was screened with two flanking DNA markers that are separated from Bs3 by 1.0 and 1.2 cM, respectively resulting in the identification of three YAC clones. Genetic mapping of the corresponding YACends revealed however, that these YACs do not cover Bs3 and subsequent screens with newly developed YACend markers failed to identify new YAC clones. Marker saturation at the Bs3 locus was carried out by amplified fragment length polymorphism (AFLP). The analysis of 1,024 primer combinations resulted in the identification of 47 new Bs3-linked AFLPs. High-resolution linkage mapping of Bs3 was accomplished by inspecting more than 4,000 F(2) segregants resulting in a genetic resolution of 0.01 cM. Using tightly Bs3-linked YACend- and AFLP-derived markers we established a Bs3-spanning BAC contig and physically delimited the target gene within one BAC clone. The analysis of the Bs3-containing genomic region revealed substantial local variation in the correlation of genetic and physical distances.
Assuntos
Proteínas de Bactérias/genética , Capsicum/genética , Imunidade Inata/genética , Xanthomonas campestris/patogenicidade , Xanthomonas vesicatoria/patogenicidade , Proteínas de Bactérias/metabolismo , Capsicum/microbiologia , Cromossomos Artificiais Bacterianos , Cromossomos Artificiais de Levedura , DNA Bacteriano , Biblioteca Gênica , Variação Genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase , Efetores Semelhantes a Ativadores de Transcrição , Xanthomonas campestris/genética , Xanthomonas vesicatoria/genéticaRESUMO
Plant disease resistance (R) genes mediate specific recognition of pathogens via perception of cognate avirulence (avr) gene products. The numerous highly similar AvrBs3-like proteins from the bacterial genus Xanthomonas provide together with their corresponding R proteins a unique biological resource to dissect the molecular basis of recognition specificity. A central question in this context is if R proteins that mediate recognition of structurally similar Avr proteins are themselves functionally similar or rather dissimilar. The recent isolation of rice xa5, rice Xa27 and tomato Bs4, R genes that collectively mediate recognition of avrBs3-like genes, provides a first clue to the molecular mechanisms that plants employ to detect AvrBs3-like proteins. Their initial characterization suggests that these R proteins are structurally and functionally surprisingly diverge. This review summarizes the current knowledge on R-protein-mediated recognition of AvrBs3-like proteins and provides working models on how recognition is achieved at the molecular level.