Agrobacterium-mediated transformation of Guignardia citricarpa: An efficient tool to gene transfer and random mutagenesis

• Published in: Fungal biology 2013-07, Vol.117 (7-8), p.556-568
• Main Authors: Rodrigues, Maria Beatriz Calderan, Fávaro, Léia Cecília de Lima, Pallu, Ana Paula de Souza, Ferreira, Anderson, Sebastianes, Fernanda de Souza, Rodrigues, Maria Juliana Calderan, Spósito, Marcel Bellato, de Araújo, Welington Luiz, Pizzirani-Kleiner, Aline Aparecida
• Format: Article
• Language: English
• Subjects: acetosyringone; Agrobacterium radiobacter; Agrobacterium tumefaciens; Agrobacterium tumefaciens - genetics; Agrobacterium tumefaciens - physiology; Agrobacterium-mediated transformation; Ascomycota - genetics; Ascomycota - growth & development; Ascomycota - metabolism; cellulose; Citrus; Citrus - microbiology; Citrus Black Spot; fungi; gene transfer; Gene Transfer Techniques; genes; Genetic transformation; Genetic Vectors - genetics; Genetic Vectors - metabolism; green fluorescent protein; Green Fluorescent Proteins - genetics; Guignardia; Guignardia citricarpa; insertional mutagenesis; microscopy; Mutagenesis, Insertional - methods; mycelium; Mycelium - genetics; Mycelium - growth & development; Mycelium - metabolism; pathogenesis; pathogens; Plant Diseases - microbiology; Plant–pathogen interaction; polymerase chain reaction; Transformation, Genetic
• ISSN: 1878-6146; 1878-6162
• DOI: 10.1016/j.funbio.2013.06.005

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone – AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant–pathogen interaction. •We describe a genetic transformation of Guignardia citricarpa via A. tumefaciens.•We can observe the interaction of the transformants fungi with citrus plant.•The method facilitates the study of insertional mutagenesis and genetic knockouts.•The method allows identifying genes involved in the pathogenesis mechanisms.•The method is important to the study of the plant pathogen interaction.