A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans

被引：56

作者：

Blaise, Francoise ^{[1
]}

Remy, Estelle ^{[1
]}

Meyer, Michel ^{[1
]}

Zhou, Ligang ^{[1
]}

Narcy, Jean-Paul ^{[1
]}

Roux, Jacqueline ^{[1
]}

Balesdent, Marie-Helene ^{[1
]}

Rouxel, Thierry ^{[1
]}

机构：

[1] INRA, Phytopathol & Methodol Detect, Route St Cyr, F-78026 Versailles, France

来源：

FUNGAL GENETICS AND BIOLOGY | 2007年 / 44卷 / 02期

关键词：

Leptosphaeria maculans; plant-fungus interaction; pathogenicity gene; agrotransformation; formal genetics; TAIL-PCR; SON-PCR; gene tagging;

D O I：

10.1016/j.fgb.2006.07.006

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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. Cosegregation 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. (c) 2006 Elsevier Inc. All rights reserved.

引用

页码：123 / 138

页数：16

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