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.
Fungal Biol. 2013 Jul-Aug;117(7-8):556-68. doi: 10.1016/j.funbio.2013.06.005. Epub 2013 Jul 1.
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.
胶孢炭疽菌是柑橘黑斑病（CBS）的病原体，这是柑橘种植业中的一种重要病害。由于该病原体在全球范围内的重要性，尤其是在巴西，因此深入了解这种真菌的功能至关重要，这可以帮助我们阐明其感染机制，并提供控制 CBS 的工具。本工作首次描述了一种有效且成功的胶孢炭疽菌菌丝体遗传转化方法，该方法可生成表达 GFP（绿色荧光蛋白）基因的转化体，并可观察其与柑橘植物的相互作用。通过根癌农杆菌介导的方法，将携带质粒 pFAT-gfp 的质粒转化到胶孢炭疽菌的菌丝体中，该质粒包含潮霉素抗性基因（hph）和 GFP 基因。通过测试不同的条件（膜的类型；诱导剂浓度[乙酰丁香酮-AS]和共培养时间）来优化农杆菌介导的转化方法。结果表明，最好的条件是使用纤维素酯膜；AS 的浓度为 200μM，共培养时间为 96 小时。通过聚合酶链反应（PCR）技术证实 hph 基因插入后，转化体显示出高的有丝分裂稳定性（82%）。此外，通过荧光显微镜观察到 GFP 存在于菌丝体内。该技术首次实现了对病原菌与植物相互作用的直观可视化，为进一步研究该真菌的发病机制提供了可能。建立胶孢炭疽菌的转化方法为研究插入诱变和基因敲除提供了更多的可能性，以鉴定与发病机制和植物-病原菌相互作用相关的最重要基因。
