Agrobacterium-mediated transformation of Fusarium proliferatum

Abstract

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.

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

“…This method has been succeed in transforming DNA in many different fungi, including A. niger, Aspergillus awamori, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa etc. The Agrobacterium transformation method has also been successfully applied in some Fusarium species, such as F. oxysporum (Islam et al, 2012), Fusarium venenatum (de Groot et al, 1998), and F. proliferatum (Bernardi-Wenzel et al, 2016). Zhu et al (2009) tested the PEG and Agrobacterium-mediated transformations of a plasmid in F. fujikuroi using respectively protoplast and conidia as the competent cells.…”

Agrobacterium-Mediated Transformation of Diaporthe schini Endophytes Associated with Vitis labrusca L. and Its Antagonistic Activity Against Grapevine Phytopathogens

“…ATMT was performed according to Bernardi-Wenzel et al [14], with modifications. A. tumefaciens strains with vector pFAT-GFP or pCAM-DsRed were prepared as previously described [14]; except that 10 mL (instead of 25 mL) of YEP medium was used.…”

“…ATMT was performed according to Bernardi-Wenzel et al [14], with modifications. A. tumefaciens strains with vector pFAT-GFP or pCAM-DsRed were prepared as previously described [14]; except that 10 mL (instead of 25 mL) of YEP medium was used. For strain with pFAT-GFP, YEP medium was supplemented with 300 lg/mL of streptomycin (Sigma-Aldrich, St. Louis, USA) and 100 lg/mL rifampicin (Sigma); for strain with pCAM-DsRed, 100 lg/ mL of kanamycin (Sigma) and 100 lg/mL of rifampicin were used.…”

“…For strain with pFAT-GFP, YEP medium was supplemented with 300 lg/mL of streptomycin (Sigma-Aldrich, St. Louis, USA) and 100 lg/mL rifampicin (Sigma); for strain with pCAM-DsRed, 100 lg/ mL of kanamycin (Sigma) and 100 lg/mL of rifampicin were used. Fragments of mycelial growth (1 cm 2 ) from seven-day-old cultures of Diaporthe endophytes (previously grown in PDA) were transferred to potato dextrose broth (PDB; pH 6.6), incubated at 28°C for 6 h, and then centrifuged (4000 rpm, 7 min) and diluted in liquid inducing medium (IM) [14] until the OD 600 reached 0.6.…”

Agrobacterium tumefaciens isolated from hemodialysis water

“…Agrobacterium tumefaciens causes a crown gall disease in a wide range of dicotyledonous (broad-leaved) plants, especially members of the rose family and grapevine. This bacterium is known for its remarkable biology, as it is capable to transfer a part of its DNA, known as a tumor inducing plasmid of 200 base pair to the plant, integrating into a plant's genome, and, consequently, causing tumorous changes in plants 1 . Due to its ability to cause such changes in plants it should be considered as a potential pathogen for humans also, especially in an immunosuppressed host 2,3 .…”