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首页> 外文学位 >Identification of new pathogenicity genes in Magnaporthe oryzae through the construction of an Agrobacterium tumefaciens-mediated insertion mutant library.
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Identification of new pathogenicity genes in Magnaporthe oryzae through the construction of an Agrobacterium tumefaciens-mediated insertion mutant library.

机译：通过根癌农杆菌介导的插入突变体文库的构建，鉴定稻米中新的致病基因。

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摘要

An understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens- mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.; The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contains a T-DNA insertion in the upstream region of two divergently transcribed genes that encode the vacuolar type-ATPase subunit c'' and the general transcription factor TFIIA subunit gamma. Genetic complementation demonstrated the insertion of the T-DNA in the promoter region of the general transcription factor TFIIA subunit gamma is responsible for observed defects in conidiation, appressorium morphogenesis, and appressorium function. This is the first report relating the function of TFIIA subunit gamma to pathogenicity.
机译：了解植物病原体与宿主之间的相互作用对于设计控制作物病害的有效策略至关重要。稻瘟病菌（Magnaporthe oryzae）是稻瘟病的一种无糖真菌和致病因子，是研究宿主-微生物相互作用的模型生物。本论文的总体目标是通过随机插入诱变文库的构建和鉴定来鉴定与致病性有关的基因。为了使DNA插入片段使基因组饱和，通过两种转化方法（PEG / CaCl2（聚乙二醇）介导的原生质体转化和根癌农杆菌介导的转化）产生了米曲霉70-15株> 54,000个插入株系。 。本文的第一部分描述了两种转化方法的优化，比较了它们的效率，并描述了插入线的高通量处理和表型分析。对12,000个T-DNA插入菌株进行的体外贴壁试验可鉴定135个品系，这些品系的形态或功能均不同于野生型。水稻感染分析表明，这些菌株中有112株表现出致病性缺陷。本论文的第二部分分析了致病性突变子集中的T-DNA整合模式。本部分旨在通过回收邻近T-DNA插入位点的米曲霉序列来鉴定被破坏的基因。通过反向PCR挽救米曲霉染色体T-DNA侧翼序列，在致病性突变体中61个T-DNA插入的基因组图谱鉴定出了22个具有预测功能的保守假设基因，11个没有GenBank显着匹配的预测开放阅读框，两个基因组装配的未注释区域和七个基因间区域。本文的最后一部分描述了米曲霉致病性突变体的特征，该突变体在两个不同转录基因的上游区域包含一个T-DNA插入，​​该基因编码液泡型ATPase亚基c''和一般转录因子TFIAA亚基伽玛遗传互补表明，T-DNA在一般转录因子TFIIA亚基γ的启动子区域中的插入负责观察到的分生孢子，Appressorium形态发生和Appressorium功能。这是有关TFIIA亚基γ功能与致病性相关的第一份报告。

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  • 作者

    Betts, Melania Figueroa.;

  • 作者单位

    The University of Arizona.$bPlant Pathology.;

  • 授予单位 The University of Arizona.$bPlant Pathology.;
  • 学科 Biology Molecular.; Agriculture Plant Pathology.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 437 p.
  • 总页数 437
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;植物病理学;
  • 关键词

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