Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa.
Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; Biotechnology Platform, Agricultural Research Council, Onderstepoort Campus, Pretoria, South Africa.
Microbiol Res. 2019 Sep;226:55-64. doi: 10.1016/j.micres.2019.05.004. Epub 2019 May 24.
Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 10 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.
在包括非洲树木病原体 Ceratocystis albifundus 在内的许多真菌中，基因组基因座与特定生物特征之间的功能关联仍然缺乏。这主要是因为缺乏合适的转化系统，无法对这种真菌和其他真菌进行遗传操作。在这里，我们提出了一种优化的 Ceratocystis albifundus 农杆菌介导转化的方案。根癌农杆菌 AGL-1 菌株和四个二元 T-DNA 载体（赋予潮霉素 B 或遗传霉素抗性和/或表达绿色荧光蛋白 [GFP]）用于转化 C. albifundus 的三个分离株的萌发孢子。通过在选择性和非选择性培养基上连续继代培养真菌转化体，以及使用 PCR 和序列分析，证实了这些 T-DNA 编码特性的稳定表达。通过 Southern 杂交分析证实了各自 T-DNA 在这些真菌基因组中的单拷贝整合。确定和优化的实验参数范围包括：（i）抑制野生型真菌生长所需的潮霉素 B 和遗传霉素的浓度，（ii）转化对乙酰丁香酮诱导细菌毒力基因的依赖性，以及（iii）真菌-细菌共培养时间的长短，以及（iv）用于后者的真菌孢子和细菌细胞的浓度。本研究中开发的系统稳定，效率高，每个 10 个孢子可产生多达 400 个转化体。这是 C. albifundus 转化方案的首次报道，它的可用性对于该重要真菌的功能研究将是非常宝贵的。
