Insertion Pool Sequencing for Insertional Mutant Analysis in Complex Host-Microbe Interactions

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2019-07-03 DOI:10.1002/cppb.20097

Simon Uhse, Florian G. Pflug, Arndt von Haeseler, Armin Djamei

引用次数: 2

Abstract

Insertional mutant libraries of microorganisms can be applied in negative depletion screens to decipher gene functions. Because of underrepresentation in colonized tissue, one major bottleneck is analysis of species that colonize hosts. To overcome this, we developed insertion pool sequencing (iPool-Seq). iPool-Seq allows direct analysis of colonized tissue due to high specificity for insertional mutant cassettes. Here, we describe detailed protocols for infection as well as genomic DNA extraction to study the interaction between the corn smut fungus Ustilago maydis and its host maize. In addition, we provide protocols for library preparation and bioinformatic data analysis that are applicable to any host-microbe interaction system. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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插入池测序用于复杂宿主-微生物相互作用的插入突变分析

微生物的插入突变文库可以应用于负耗尽筛选来破译基因功能。由于在定植组织中的代表性不足，一个主要的瓶颈是对定植宿主的物种进行分析。为了克服这个问题，我们开发了插入池测序(iPool-Seq)。iPool-Seq由于插入突变体的高特异性，可以直接分析定植组织。本文详细介绍了侵染玉米黑穗病菌及其寄主玉米间相互作用的研究方案和基因组DNA提取方法。此外，我们还提供了适用于任何宿主-微生物相互作用系统的文库制备和生物信息学数据分析的协议。©2019作者。这是一篇基于知识共享署名许可协议的开放获取文章，该协议允许在任何媒体上使用、分发和复制，前提是正确引用原始作品。

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来源期刊

Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.