Catanzariti Ann-Maree, Mago Rohit, Ellis Jeff, Dodds Peter
Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.
Methods Mol Biol. 2011;712:79-87. doi: 10.1007/978-1-61737-998-7_8.
The haustorium is a distinguishing feature of biotrophic plant pathogens. Several highly diverged -pathogen classes have independently evolved haustoria, suggesting that they represent an effective adaptation for growing within living plant tissue. Despite their clear importance in biotrophy, they have been difficult to study due to the close association of biotrophic pathogens with their host and the inability to produce haustoria in vitro. These drawbacks have been circumvented in the study of rust fungi by the development of a haustoria isolation technique. The strong binding of the lectin concanavalin A (ConA) to rust haustoria allows these structures to be purified from infected plant tissue by affinity chromatography on a ConA-Sepharose macrobead column. The isolation process results in substantial yields of intact haustoria that retain their cytoplasmic contents, making them amenable to experimentation. The construction of cDNA libraries from isolated rust haustoria and their subsequent sequence analysis have provided significant insight into haustoria function at a molecular level, revealing important roles in nutrient acquisition and the delivery of pathogenicity effector proteins. The generation of a rust haustorium-specific cDNA library is described in this chapter.
吸器是活体营养型植物病原体的一个显著特征。几个高度分化的病原体类别已经独立进化出吸器，这表明吸器是在活的植物组织内生长的一种有效适应方式。尽管吸器在活体营养过程中具有明显的重要性，但由于活体营养型病原体与其宿主紧密相关，且无法在体外产生吸器，因此对其进行研究一直很困难。在锈菌研究中，通过开发一种吸器分离技术，克服了这些缺点。凝集素伴刀豆球蛋白A（ConA）与锈菌吸器的强结合能力，使得这些结构能够通过在ConA-琼脂糖大珠柱上进行亲和层析，从受感染的植物组织中纯化出来。分离过程可获得大量完整的吸器，这些吸器保留了其细胞质内容物，便于进行实验。从分离出的锈菌吸器构建cDNA文库并随后进行序列分析，在分子水平上为吸器功能提供了重要见解，揭示了吸器在养分获取和致病性效应蛋白传递中的重要作用。本章描述了锈菌吸器特异性cDNA文库的构建。
