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Abstract：
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 106 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.
摘要：
在许多真菌中，基因组基因座与特定生物学性状之间的功能关联仍然缺乏，包括非洲树病原体Ceratocystisalbifundus。这主要是因为缺乏合适的转化系统来允许对这种真菌和其他真菌进行遗传操作。这里,我们提出了根癌农杆菌介导的C.albifundus转化的优化方案。使用根癌农杆菌的菌株AGL-1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]）转化三种分离株的发芽分生孢子。通过在选择性和非选择性培养基上连续传代培养真菌转化体并使用PCR和序列分析来证实这些T-DNA编码性状的稳定表达。使用Southern杂交分析确认了各个T-DNA到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和遗传霉素浓度，以及（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-细菌共培养期的持续时间和（iv）用于后者的真菌分生孢子和细菌细胞的浓度。本研究开发的系统运行稳定,效率高,每106分生孢子产生多达400个转化体。这是C.albifundus转化方案的第一份报告，其可用性对于这种重要真菌的功能研究将是无价的。
