Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis

Fitzgerald, Anna; Mudge, Agnieszka M.; Gleave, Andrew P.; Plummer, Kim M.

doi:10.1017/s0953756203008086

Cited by 71 publications

(82 citation statements)

References 27 publications

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“…Strains of A. flavus with either a tan or white genotype have been successfully induced to form protoplasts (35,49), suggesting that the pigments produced by strain NRRL6541 may inhibit protoplast formation. A hyaline mutant was also needed to obtain protoplasts from Venturia inaequalis (14). Agrobacterium-mediated transformation (9) has been successful for some filamentous fungi and might circumvent the present difficulties.…”

Section: Discussionmentioning

confidence: 99%

Indole-Diterpene Gene Cluster fromAspergillus flavus

Zhang

Monahan

Tkacz

et al. 2004

Appl Environ Microbiol

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Aflatrem is a potent tremorgenic mycotoxin produced by the soil fungus Aspergillus flavus and is a member of a large structurally diverse group of secondary metabolites known as indole-diterpenes. By using degenerate primers for conserved domains of fungal geranylgeranyl diphosphate synthases, we cloned two genes, atmG and ggsA (an apparent pseudogene), from A. flavus. Adjacent to atmG are two other genes, atmC and atmM. These three genes have 64 to 70% amino acid sequence similarity and conserved synteny with a cluster of orthologous genes, paxG, paxC, and paxM, from Penicillium paxilli which are required for indole-diterpene biosynthesis. atmG, atmC, and atmM are coordinately expressed, with transcript levels dramatically increasing at the onset of aflatrem biosynthesis. A genomic copy of atmM can complement a paxM deletion mutant of P. paxilli, demonstrating that atmM is a functional homolog of paxM. Thus, atmG, atmC, and atmM are necessary, but not sufficient, for aflatrem biosynthesis by A. flavus. This provides the first genetic evidence for the biosynthetic pathway of aflatrem in A. flavus.Aflatrem is a potent tremorgenic mycotoxin produced by the soil fungus Aspergillus flavus (17,18). This compound is a member of a large, structurally diverse group of secondary metabolites known as indole-diterpenes that includes paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines (34). These metabolites all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from either tryptophan or a tryptophan precursor. Different patterns of prenylation, hydroxylation, epoxidation, acetylation, and ring stereochemistry around the basic indole-diterpene ring structure define this structural diversity (34). The mechanism by which aflatrem and related indole-diterpenes cause tremorgenicity in mammals is not well defined, but biochemical and clinical studies indicate that these effects are due in part to effects on receptors and to interference with neurotransmitter release in the central and peripheral nervous systems (40).Very little is known about the pathways of indole-diterpene biosynthesis other than the origins of the indole and diterpene components (7, 10). Biosynthetic schemes based on the chemical identification of likely intermediates have been proposed, but until recently none of these steps had been validated by biochemical or genetic studies (25,30).Recently, a cluster of genes for paxilline biosynthesis was cloned from Penicillium paxilli (53). Key genes in this cluster include a GGPP synthase gene (paxG), a FAD-dependent monooxygenase gene (paxM), a prenyl transferase gene (paxC), and two cytochrome P450 monooxygenase genes, paxP and paxQ. The deletion of paxG, paxM, or paxC results in mutants that are defective in paxilline biosynthesis (53; B. Scott, L. McMillan, J. Astin, C. Young, A. Bryant, and E.Parker, unpublished results). PaxM and PaxC may catalyze the additi...

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Section: Discussionmentioning

confidence: 99%

Indole-Diterpene Gene Cluster fromAspergillus flavus

Zhang

Monahan

Tkacz

et al. 2004

Appl Environ Microbiol

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“…The binary transformation vector pFAT-GFP (Fitzgerald et al, 2003) was kindly provided by Kim M. Plummer (PlantIndustry CSIRO, Australia). This binary vector of 18,422 bp contains the T-DNA region, the hygromycin B resistance gene (hph) from Escherichia coli driven by the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) from Glomerella cingulata, and the gfp gene from the jellyfish Aequorea victoria.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

“…tumefaciens-mediated transformation of F. proliferatum was performed with modifications of the methods of De Groot et al (1998), Fitzgerald et al (2003), and Rodrigues et al (2013). The effect of two factors on the efficiency of transformation was assessed: the addition acetosyringone and the use of nitrocellulose membrane or filter paper.…”

Section: A Tumefaciens-mediated Transformation Of F Proliferatummentioning

confidence: 99%

Agrobacterium-mediated transformation of Fusarium proliferatum

Bernardi-Wenzel¹,

Quecine²,

Azevedo³

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacteriummediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.

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“…In recent years, efficient transformation systems have been developed for a wide range of filamentous fungi (reviewed in [1,4,5]). Successful transformation of fungi has been achieved by: CaCl 2 /polyethylene glycol [6][7][8], electroporation [9-15], particle bombardment [2,6,10,16,17], and Agrobacterium tumefaciens-mediated transformation (AMT) [1].…”

Section: Achieving Stable Homokaryotic Transformationmentioning

confidence: 99%

Approaches to functional genomics in filamentous fungi

et al. 2006

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The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.

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Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis

Cited by 71 publications

References 27 publications

Indole-Diterpene Gene Cluster fromAspergillus flavus

Indole-Diterpene Gene Cluster fromAspergillus flavus

Agrobacterium-mediated transformation of Fusarium proliferatum

Approaches to functional genomics in filamentous fungi

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