Agrobacterium-mediated transformation of Guignardia citricarpa: an efficient tool to gene transfer and random mutagenesis.
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.
Universidade de São Paulo, Escola Superior de Agricultura 'Luiz de Queiroz', Departamento de Genética, Laboratório de Genética de Microrganismos Prof. Dr João Lúcio de Azevedo, Av. Pádua Dias, 11, Caixa Postal 83, CEP: 13400-970 Piracicaba, SP, Brazil. biacalderan@yahoo.com.br
Maria Beatriz Calderan
MB
Rodrigues
Léia Cecília de Lima
LC
Fávaro
Ana Paula de Souza
AP
Pallu
Anderson
A
Ferreira
Fernanda de Souza
Fde S
Sebastianes
Maria Juliana Calderan
MJ
Rodrigues
Marcel Bellato
MB
Spósito
Welington Luiz
WL
de Araújo
Aline Aparecida
AA
Pizzirani-Kleiner
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000502:physiology; D001203:Ascomycota / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D002957:Citrus / Q000382:microbiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D025282:Mycelium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology*; D014170:Transformation, Genetic
D049452:Green Fluorescent Proteins
Agrobacterium-mediated transformation; Citrus Black Spot; Genetic transformation; Guignardia citricarpa; Plant–pathogen interaction