Muniz C R, da Silva G F, Souza M T, Freire F C O, Kema G H J, Guedes M I F
Embrapa Agroindústria Tropical, Fortaleza, CE, Brasil
Laboratório de Biologia Molecular, Embrapa Amazônia Ocidental, Manaus, AM, Brasil.
Genet Mol Res. 2014 Feb 21;13(2):2906-13. doi: 10.4238/2014.February.21.8.
Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.
可可毛色二孢是包括腰果植物在内的许多不同作物栽培品种的主要病原体。本文描述了一种高效的根癌农杆菌介导的转化（ATMT）系统，用于成功递送T-DNA，将绿色荧光蛋白（gfp）基因和潮霉素B磷酸转移酶（hph）基因转移至可可毛色二孢。当真菌分生孢子器孢子与携带含有hph-gfp基因的二元载体的根癌农杆菌共培养时，只有在添加乙酰丁香酮的情况下才会产生抗潮霉素的真菌。对接种了表达GFP的真菌的腰果植株进行落射荧光显微镜观察，发现了典型的病原体定殖现象。在菌丝体培养物的所有延伸部分，均观察到转化体发出强烈而明亮的绿色菌丝。在接种后25天之前，观察到在表皮表面下方靠近接种部位的薄壁细胞被穿透。穿透之后，入侵的宿主细胞内会形成菌丝。这些发现为可可毛色二孢转化提供了一种快速且可重复的ATMT方法。
