Agrobacterium-mediated transformation of Fusarium proliferatum.
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 Agrobacterium-mediated 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.
Laboratório de Biotecnologia Microbiana, Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá, Maringá, PR, Brasil.
J
J
Bernardi-Wenzel
Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brasil.
M C
MC
Quecine
Laboratório de Biotecnologia Microbiana, Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá, Maringá, PR, Brasil.
J L
JL
Azevedo
Laboratório de Biotecnologia Microbiana, Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá, Maringá, PR, Brasil.
J A
JA
Pamphile
D060054:Agrobacterium / Q000235:genetics; D060467:Disease Resistance / Q000235:genetics; D005670:Fusarium / Q000235:genetics* / Q000472:pathogenicity; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006921:Hygromycin B / Q000633:toxicity; D035261:Plant Components, Aerial / Q000382:microbiology; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology*; D013025:Glycine max / Q000235:genetics* / Q000382:microbiology; D014170:Transformation, Genetic
D049452:Green Fluorescent Proteins; D006921:Hygromycin B