02846naa a2200301 a 450000100080000000500110000800800410001902400380006010000240009824501500012226000090027252018720028165000220215365000270217565000260220265300270222865300330225565300190228870000240230770000230233170000170235470000260237170000240239770000190242170000210244070000290246177300540249019709912018-12-26       2013    bl uuuu     u00u1 u    #d7 a10.1016/j.funbio.2013.06.0052DOI1 aRODRIGUES, M. B. C.  aAgrobacterium-mediated transformation of Guignardia citricarpaban efficient tool to gene transfer and random mutagenesis.h[electronic resource]  c2013  aGuignardia 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 con-ditions (type of membrane; inductor agent concentration [acetosyringone e AS] and cocul-tivation time). Results demonstrated that the best condition occurred with the utilization of cellulose?s ester membrane; 200 m M 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 muta-genesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plante pathogen interaction.  acitrus black spot  agenetic transformation  aGuignardia citricarpa  aAgrobacterium-mediated  aPlant e pathogen interaction  aTransformation1 aFAVARO, L. C. de L.1 aPALUU, A. P. de S.1 aFERREIRA, A.1 aSEBASTIANES, F. de S.1 aRODRIGUES, M. J. C.1 aSPOSITO, M. B.1 aARAUJO, W. L. de1 aPIZZIRANI-KLEINER, A. A.  tFungal Biologygv. 117, n. 7-8, p. 463-576, 2013.