Blaise Françoise, Rémy Estelle, Meyer Michel, Zhou Ligang, Narcy Jean-Paul, Roux Jacqueline, Balesdent Marie-Hélène, Rouxel Thierry
Institut National de la Recherche Agronomique, Phytopathologie et Méthodologies de la Détection, Route de St-Cyr, F-78026 Versailles Cedex, France.
Fungal Genet Biol. 2007 Feb;44(2):123-38. doi: 10.1016/j.fgb.2006.07.006. Epub 2006 Sep 18.
We evaluated the usefulness and robustness of Agrobacterium tumefaciens-mediated transformation (ATMT) as a high-throughput transformation tool for pathogenicity gene discovery in the filamentous phytopathogen Leptosphaeria maculans. Thermal asymmetric interlaced polymerase chain reaction allowed us to amplify the left border (LB) flanking sequence in 135 of 400 transformants analysed, and indicated a high level of preservation of the T-DNA LB. In addition, T-DNA preferentially integrated as a single copy in gene-rich regions of the fungal genome, with a probable bias towards intergenic and/or regulatory regions. A total of 53 transformants out of 1388 (3.8%) showed reproducible pathogenicity defects when inoculated on cotyledons of Brassica napus, with diverse altered phenotypes. Co-segregation of the altered phenotype with the T-DNA integration was observed for 6 of 12 transformants crossed. If extrapolated to the whole collection, this indicates that 1.9% of the collection actually corresponds to tagged pathogenicity mutants. The preferential insertion into gene-rich regions along with the high ratio of tagged mutants renders ATMT a tool of choice for large-scale gene discovery in L. maculans.
我们评估了根癌农杆菌介导的转化（ATMT）作为一种高通量转化工具在丝状植物病原菌大斑壳针孢致病性基因发现中的实用性和稳健性。热不对称交错聚合酶链反应使我们能够在分析的400个转化体中的135个中扩增出左边界（LB）侧翼序列，并表明T-DNA LB具有高度的保守性。此外，T-DNA优先以单拷贝形式整合到真菌基因组的基因丰富区域，可能偏向于基因间和/或调控区域。在接种到甘蓝型油菜子叶上时，1388个转化体中有53个（3.8%）表现出可重复的致病性缺陷，具有多种改变的表型。在杂交的12个转化体中有6个观察到改变的表型与T-DNA整合的共分离。如果外推到整个群体，这表明1.9%的群体实际上对应于标记的致病性突变体。T-DNA优先插入基因丰富区域以及标记突变体的高比例使得ATMT成为大斑壳针孢大规模基因发现的首选工具。
