Efficient Genetic Transformation System for the Ochratoxigenic Fungus Aspergillus carbonarius

Morioka, Luiz Rodrigo Ito; Furlaneto, Márcia Cristina; Bogas, Andréa Cristina; Pompermayer, Patrícia; Duarte, Rubens Tadeu Delgado; Vieira, Maria Lúcia Carneiro; Watanabe, Maria Angélica Ehara; Fungaro, Maria Helena Pelegrinelli

doi:10.1007/s00284-005-0402-6

Cited by 18 publications

(8 citation statements)

References 21 publications

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“…In the case of F. avenaceum pre-induction of the used A. tumefaciens strain, for 16 hours, did not result in a statistically significant higher number of colonies (Figure 6). Similar results have been reported for Aspergillus carbonarius [41] and Magnaporthe grisea [42]. Mullins and co-workers found that pre-induction was not essential for the success of ATMT in the case of Fusarium oxysporum, but that the transformation process was delayed compared to experiments including pre-induction [43].…”

Section: Resultssupporting

confidence: 81%

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

et al. 2014

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BackgroundThe plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed.ResultsThe new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A.ConclusionThe new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.

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Section: Resultssupporting

confidence: 81%

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

et al. 2014

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“…Este nível de sensibilidade foi similarmente observado para outras espécies de Aspergillus (PUNT et al, 1987) e já havia sido relatada para a linhagem ITAL 99 de A. carbonarius por Morioka et al (2006) Dentre os 238 transformantes analisados em meio agar-coco para a produção de ocratoxina A, nove (3,7%) possíveis mutantes com redução da produção desta toxina foram identificados baseado na redução e/ou ausência de fluorescência azul esverdeada na colônia sob luz ultravioleta. Por outro lado, dois (0,8%) supostos mutantes com aumento na produção desta toxina também foram identificados.…”

Section: Resultsunclassified

“…A transformação genética da linhagem ITAL 187 foi realizada como descrito por Morioka et al (2006) com algumas modificações. Rapidamente, após cinco dias de crescimento da linhagem ITAL 187 em MC a 28ºC, foi preparada uma suspensão contendo 1x10 6 conídios/mL de meio de indução.…”

Section: Transformação Genética Mediada Por a Tumefaciensunclassified

Obtenção de mutantes de Aspergillus carbonarius via transformação genética mediada por Agrobacterium tumefaciens

Lunardi

Fier

Ashikaga

et al. 2006

Semin. Cienc. Biol. Saude

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ResumoAspergillus carbonarius é um potente produtor de ocratoxina A, uma toxina que apresenta efeitos nefrotóxico e carcinogênico. O conhecimento de genes envolvidos em sua biossíntese pode contribuir para o desenvolvimento de medidas de controle. O método de transformação genética mediado por Agrobacterium tumefaciens tem sido demonstrado como uma importante ferramenta para a obtenção de mutantes insercionais visando à caracterização de novos genes. O objetivo deste trabalho foi adequar o método de transformação genética via A. tumefaciens para a linhagem ITAL187 de A. carbonarius e obter mutantes alterados para a produção de ocratoxina A. Conídios foram transformados para resistência a higromicina B usando a linhagem AGL-1 de A. tumefaciens. A freqüência média de transformação foi de 20,04 transformantes por 10 5 conídios-alvo. A prova física da transformação foi obtida pela detecção do gene hph por PCR e Southern Blot. Esta última demonstrou que a integração do DNA exógeno ocorreu de forma aleatória no genoma fúngico. Dentre 238 transformantes, 12 (5,042%) mostraram variações morfológicas. Três mutantes (T44, T47 e T188) com significativa redução e dois mutantes (T238 e T162) com aumento da capacidade de produção de ocratoxina A foram obtidos. A identificação dos genes nocauteados contribuirá para a compreensão da biossíntese desta toxina. Palavras-chave: Aspergillus carbonarius. Ocratoxina A. Agrobacterium tumefaciens. Transformação genética. Mutantes insercionais. AbstractAspergillus carbonarius is a potent ochratoxin A producer, a mycotoxin that has nephrotoxic and carcinogenic effects. The knowledge of genes involved in biosynthesis of this toxin may be useful for the development of detection and control methods. The Agrobacterium tumefaciens-mediated transformation method has been demonstrated as a powerful tool to obtain insertional mutants for the characterization of new genes. The aim of this work was to adapt the A. tumefaciens-mediated transformation method for ITAL187 strain of A. carbonarius and to obtain transformants with alteration in ochratoxin A production. Conidia were transformed to hygromicin B resistance using AGL-1 strain of A. tumefaciens. The transformation frequency was 20,04 transformants per 10 5 target conidia. The transformation evidence was obtained by PCR and Southern Blot analysis. The last one showed us that 1 Bolsista PIBIC/CNPq/UEL -UEL

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“…The ATMT via germinated conidia could avoid the preparation of protoplast, which is usually expensive and timeconsuming (Song et al, 2004). Random integration of a single copy into the fungal genome is another important character for functional genomic study (Morioka et al, 2006). In this study, the hph was demonstrated to be randomly integrated in P. digitatum genome with single copy by Southern blot and sequencing of 14 randomly selected transformants.…”

Section: Analysis Of Fungal Transformantsmentioning

confidence: 91%

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

Wang

2008

J. Zhejiang Univ. Sci. B

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Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 10(6) conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.

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Efficient Genetic Transformation System for the Ochratoxigenic Fungus Aspergillus carbonarius

Cited by 18 publications

References 21 publications

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

Obtenção de mutantes de Aspergillus carbonarius via transformação genética mediada por Agrobacterium tumefaciens

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

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