Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum
Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum
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DOI：
10.1111/j.1364-3703.2008.00483.x
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发表时间：
2008-09-01
影响因子：
4.9
通讯作者：
Yarden, O.
中科院分区：
文献类型：
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作者：
Levy, M.;Erental, A.;Yarden, O.
同源重组需要基因靶向程序，如基因破坏和基因替代。在许多生物体中，Ku80是非同源末端连接DNA修复机制的一部分。我们鉴定并破坏了核菌核菌（Sclerotinia sclerotiorum）的Ku80同源基因，产生了富含Ku80缺陷核的异核突变体（ssku80）。ssku80突变体在番茄果实上的硬化形成和致病性正常。当用cna1（编码钙调磷酸酶）替代构建体转化时，这些菌株的同源重组频率远高于野生型。我们将同源重组的增加与直接bim - lab介导的转化过程结合起来，该转化过程利用压缩空气帮助转化DNA穿透菌丝体。我们发现这种方法是有效的和可重复的，并且它不改变突变体的适应度。我们还展示了第一个直接转化硬核的案例。介绍了诺氏菌素作为菌核菌的选择性标记。所描述的工具和程序将提高我们研究菌核菌基因功能的能力，并且最有可能适用于其他植物病原体。
Homologous recombination is required for gene-targeted procedures such as gene disruption and gene replacement. Ku80 is part of the non-homologous end-joining DNA repair mechanism in many organisms. We identified and disrupted the Ku80 homologue in Sclerotinia sclerotiorum and generated heterokaryon mutants enriched with Ku80-deficient nuclei (ssku80). Sclerotial formation and pathogenicity of ssku80 mutants were normal on tomato fruits. The frequencies of homologous recombination in these strains were much higher than those of the wild type when transformed with a cna1 (encoding calcineurin) replacement construct. We coupled the increase in homologous recombination with a direct BIM-LAB-mediated transformation procedure, which utilizes compressed air to assist the transforming DNA in penetrating fungal hyphae of S. sclerotiorum. We found this method to be efficient and reproducible, and it did not alter the fitness of the mutants. We also demonstrated the first case of direct transformation of sclerotia. Nourseothricin was introduced as a selectable marker in S. sclerotiorum. The tools and procedures described will improve our ability to study gene function in S. sclerotiorum and are most likely to be adaptable for use in other plant pathogens.