Bidirectional promoter trapping T-DNA for insertional mutagenesis in Verticillium dahliae
Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for path...
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| Published in | Canadian journal of microbiology Vol. 60; no. 7; pp. 445 - 454 |
|---|---|
| Main Authors | , , , |
| Format | Journal Article |
| Language | English |
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Canada
NRC Research Press
01.07.2014
Canadian Science Publishing NRC Research Press |
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| Online Access | Get full text |
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| Abstract | Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek–Dox and on Czapek–Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek–Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional promoter-trapping Ti vector, combined with induction medium that contains root exudates, can be useful for identification of pathogenicity-related and functional genes in V. dahliae. |
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| AbstractList | Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek–Dox and on Czapek–Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek–Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional promoter-trapping Ti vector, combined with induction medium that contains root exudates, can be useful for identification of pathogenicity-related and functional genes in V. dahliae. Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bis-GFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transsformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional promoter-trapping Ti vector, combined with induction medium that contains root exudates, can be useful for identification of pathogenicity-related and functional genes in V. dahliae. Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional promoter-trapping Ti vector, combined with induction medium that contains root exudates, can be useful for identification of pathogenicity-related and functional genes in V. dahliae.Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional promoter-trapping Ti vector, combined with induction medium that contains root exudates, can be useful for identification of pathogenicity-related and functional genes in V. dahliae. Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bis-GFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transsformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional promoter-trapping Ti vector, combined with induction medium that contains root exudates, can be useful for identification of pathogenicity-related and functional genes in V. dahliae. Key words: Verticillium dahliae, pathogenicity-related genes, promoter trapping, Agrobacterium tumefaciens-mediated transformation. La mutagenese par insertion aleatoire d'ADN de transfert (ADN-T) est une approche universelle de genetique non dirigee permettant l'identification et le clonage de genes chez plusieurs champignons phytopathogenes. Le criblage de mutants presentant un phenotype donne au sein d'un vaste ensemble de transformants choisis au hasard est une etape laborieuse, surtout si l'on recherche des mutants ayant perdu de leur pathogenicite. Dans le but d'accelerer le criblage de mutants et l'identification de genes, on a construit et mis en application un vecteur de piegeage binaire Ti, 1300-bisGFP-hyg, pour les besoins de la presente etude. Plus de 6000 transformants de Verticillium dahliae ont ete obtenus des suites du transfert du vecteur par Agrobacterium tumefaciens. On a cultive mille transformants choisis au hasard sur des geloses Czapek-Dox et Czapek-Dox enrichies d'extrait de racine de coton. On a selectionne les transformants cultives qui exprimaient la proteine fluorescente verte ou presentaient un phenotype modifie, et ceux-ci ont ete soumis a des analyses de virulence et de piegeage de promoteur. Sur la foi de l'analyse de virulence de 60 transformants, la pathogenicite de 17 de ces mutants etait compromise. On a decele l'expression de la GFP chez 10 mutants a pathogenicite deficiente, dont 6 uniquement en milieu Czapek-Dox enrichi d'extrait de racine de coton. Au moyen du TAIL-PCR (<< thermal asymmetric interlaced polymerase chain reactio>>), on a su identifier les sites d'insertion de l'ADN-T chez 8 transformants exprimant la GFP, dont 5 mutants a pathogenicite deficiente et 3 transformants non affectes. Grace a la presente methode de l'ADN-T, on a su pieger le promoteur de 6 genes. Le transformant non pathogene 24C9 a fait l'objet d'une etude plus poussee. Il a exprime fortement la GFP en milieu aqueux gelifie enrichi d'extrait de racine de coton et sur des tiges de semis de coton. Les resultats d'analyses d'hybridation de type Southern et de PCR quantitatif en temps reel ont confirme que le taux de transcription du VdUGPU (codant l'UTP-glucose-1-phosphate uridylyltransferase) etait sensiblement attenuee en raison de l'insertion de l'ADN-T dans la zone du promoteur genique. Ces resultats indiquent que le vecteur Ti bidirectionnel a piegeage de promoteur, jumele a un milieu d'induction renfermant des exsudats racinaires, peut s'averer utile dans l'identification de genes fonctionnels et lies a la pathogenicite chez V. dahliae. [Traduit par la Redaction] Mots-cles: Verticillium dahliae, genes lies a la pathogenicite, piegeage de promoteurs, transformation via Agrobacterium tumefaciens. Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional promoter-trapping Ti vector, combined with induction medium that contains root exudates, can be useful for identification of pathogenicity-related and functional genes in V. dahliae.Original Abstract: La mutagenese par insertion aleatoire d'ADN de transfert (ADN-T) est une approche universelle de genetique non dirigee permettant l'identification et le clonage de genes chez plusieurs champignons phytopathogenes. Le criblage de mutants presentant un phenotype donne au sein d'un vaste ensemble de transformants choisis au hasard est une etape laborieuse, surtout si l'on recherche des mutants ayant perdu de leur pathogenicite. Dans le but d'accelerer le criblage de mutants et l'identification de genes, on a construit et mis en application un vecteur de piegeage binaire Ti, 1300-bisGFP-hyg, pour les besoins de la presente etude. Plus de 6000 transformants de Verticillium dahliae ont ete obtenus des suites du transfert du vecteur par Agrobacterium tumefaciens. On a cultive mille transformants choisis au hasard sur des geloses Czapek-Dox et Czapek-Dox enrichies d'extrait de racine de coton. On a selectionne les transformants cultives qui exprimaient la proteine fluorescente verte ou presentaient un phenotype modifie, et ceux-ci ont ete soumis a des analyses de virulence et de piegeage de promoteur. Sur la foi de l'analyse de virulence de 60 transformants, la pathogenicite de 17 de ces mutants etait compromise. On a decele l'expression de la GFP chez 10 mutants a pathogenicite deficiente, dont 6 uniquement en milieu Czapek-Dox enrichi d'extrait de racine de coton. Au moyen du TAIL-PCR (<< thermal asymmetric interlaced polymerase chain reaction >>), on a su identifier les sites d'insertion de l'ADN-T chez 8 transformants exprimant la GFP, dont 5 mutants a pathogenicite deficiente et 3 transformants non affectes. Grace a la presente methode de l'ADN-T, on a su pieger le promoteur de 6 genes. Le transformant non pathogene 24C9 a fait l'objet d'une etude plus poussee. Il a exprime fortement la GFP en milieu aqueux gelifie enrichi d'extrait de racine de coton et sur des tiges de semis de coton. Les resultats d'analyses d'hybridation de type Southern et de PCR quantitatif en temps reel ont confirme que le taux de transcription du VdUGPU (codant l'UTP-glucose-1-phosphate uridylyltransferase) etait sensiblement attenuee en raison de l'insertion de l'ADN-T dans la zone du promoteur genique. Ces resultats indiquent que le vecteur Ti bidirectionnel a piegeage de promoteur, jumele a un milieu d'induction renfermant des exsudats racinaires, peut s'averer utile dans l'identification de genes fonctionnels et lies a la pathogenicite chez V. dahliae. [Traduit par la Redaction] |
| Abstract_FL | La mutagenèse par insertion aléatoire d’ADN de transfert (ADN-T) est une approche universelle de génétique non dirigée permettant l’identification et le clonage de gènes chez plusieurs champignons phytopathogènes. Le criblage de mutants présentant un phénotype donné au sein d’un vaste ensemble de transformants choisis au hasard est une étape laborieuse, surtout si l’on recherche des mutants ayant perdu de leur pathogénicité. Dans le but d’accélérer le criblage de mutants et l’identification de gènes, on a construit et mis en application un vecteur de piégeage binaire Ti, 1300-bisGFP-hyg, pour les besoins de la présente étude. Plus de 6000 transformants de Verticillium dahliae ont été obtenus des suites du transfert du vecteur par Agrobacterium tumefaciens. On a cultivé mille transformants choisis au hasard sur des géloses Czapek–Dox et Czapek–Dox enrichies d’extrait de racine de coton. On a sélectionné les transformants cultivés qui exprimaient la protéine fluorescente verte ou présentaient un phénotype modifié, et ceux-ci ont été soumis à des analyses de virulence et de piégeage de promoteur. Sur la foi de l’analyse de virulence de 60 transformants, la pathogénicité de 17 de ces mutants était compromise. On a décelé l’expression de la GFP chez 10 mutants à pathogénicité déficiente, dont 6 uniquement en milieu Czapek–Dox enrichi d’extrait de racine de coton. Au moyen du TAIL-PCR (« thermal asymmetric interlaced polymerase chain reaction »), on a su identifier les sites d’insertion de l’ADN-T chez 8 transformants exprimant la GFP, dont 5 mutants à pathogénicité déficiente et 3 transformants non affectés. Grâce à la présente méthode de l’ADN-T, on a su piéger le promoteur de 6 gènes. Le transformant non pathogène 24C9 a fait l’objet d’une étude plus poussée. Il a exprimé fortement la GFP en milieu aqueux gélifié enrichi d’extrait de racine de coton et sur des tiges de semis de coton. Les résultats d’analyses d’hybridation de type Southern et de PCR quantitatif en temps réel ont confirmé que le taux de transcription du VdUGPU (codant l’UTP-glucose-1-phosphate uridylyltransférase) était sensiblement atténuée en raison de l’insertion de l’ADN-T dans la zone du promoteur génique. Ces résultats indiquent que le vecteur Ti bidirectionnel à piégeage de promoteur, jumelé à un milieu d’induction renfermant des exsudats racinaires, peut s’avérer utile dans l’identification de gènes fonctionnels et liés à la pathogénicité chez V. dahliae. [Traduit par la Rédaction] |
| Audience | Academic |
| Author | Zhang, Xin Deng, Sheng Lin, Ling Wang, Cai-yue |
| Author_xml | – sequence: 1 fullname: Deng, Sheng – sequence: 2 fullname: Wang, Cai-yue – sequence: 3 fullname: Zhang, Xin – sequence: 4 fullname: Lin, Ling |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/24993736$$D View this record in MEDLINE/PubMed |
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| CitedBy_id | crossref_primary_10_3390_biology11101535 crossref_primary_10_3390_jof9111066 crossref_primary_10_1371_journal_pone_0145190 crossref_primary_10_1016_j_funbio_2020_03_007 crossref_primary_10_1007_s11240_015_0825_0 |
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| Copyright | COPYRIGHT 2014 NRC Research Press Copyright Canadian Science Publishing NRC Research Press Jul 2014 |
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| Keywords | Verticillium dahliae transformation via Agrobacterium tumefaciens Agrobacterium tumefaciens-mediated transformation promoter trapping pathogenicity-related genes gènes liés à la pathogénicité piégeage de promoteurs |
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| Snippet | Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic... |
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| SubjectTerms | agar Agrobacterium radiobacter Agrobacterium tumefaciens Agrobacterium tumefaciens - physiology Agrobacterium tumefaciens-mediated transformation Cloning Cotton Deoxyribonucleic acid DNA DNA sequencing DNA, Bacterial - genetics Fungi Genes Genetic research Genetic Vectors Gossypium - microbiology Gram-negative bacteria green fluorescent protein gènes liés à la pathogénicité insertional mutagenesis Microbiological research Microbiology Mutagenesis Mutagenesis, Insertional - methods mutants Nucleotide sequencing pathogenicity-related genes Pathogens phenotype piégeage de promoteurs plant pathogenic fungi promoter regions Promoter Regions, Genetic promoter trapping Promoters (Genetics) Proteins quantitative polymerase chain reaction Real-Time Polymerase Chain Reaction root exudates screening Seedlings Seedlings - microbiology Southern blotting stems transfer DNA transformation via Agrobacterium tumefaciens Transformation, Genetic Trapping UTP-glucose-1-phosphate uridylyltransferase Verticillium - genetics Verticillium - pathogenicity Verticillium dahliae virulence Virulence - genetics |
| Title | Bidirectional promoter trapping T-DNA for insertional mutagenesis in Verticillium dahliae |
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