Institut National de la Recherche Agronomique, Universite Pierre et Marie Curie/Universite Paris 06, AgroParisTech, UMR217, Interactions Plantes-Pathogènes, F-75005, Paris, France.
Plant J. 2015 Apr;82(2):352-62. doi: 10.1111/tpj.12812.
Transcriptome analysis of bacterial pathogens is a powerful approach to identify and study the expression patterns of genes during host infection. However, analysis of the early stages of bacterial virulence at the genome scale is lacking with respect to understanding of plant-pathogen interactions and diseases, especially during foliar infection. This is mainly due to both the low ratio of bacterial cells to plant material at the beginning of infection, and the high contamination by chloroplastic material. Here we describe a reliable and straightforward method for bacterial cell purification from infected leaf tissues, effective even if only a small amount of bacteria is present relative to plant material. The efficiency of this method for transcriptomic analysis was validated by analysing the expression profiles of the phytopathogenic enterobacterium Dickeya dadantii, a soft rot disease-causing agent, during the first hours of infection of the model host plant Arabidopsis thaliana. Transcriptome profiles of epiphytic bacteria and bacteria colonizing host tissues were compared, allowing identification of approximately 100 differentially expressed genes. Requiring no specific equipment, cost-friendly and easily transferable to other pathosystems, this method should be of great interest for many other plant-bacteria interaction studies.
细菌病原体的转录组分析是一种强大的方法，可以识别和研究宿主感染过程中基因的表达模式。然而，在理解植物-病原体相互作用和疾病方面，特别是在叶片感染期间，对于细菌毒力的早期阶段在基因组规模上的分析仍然缺乏。这主要是由于感染初期细菌细胞与植物材料的比例低，以及叶绿体物质的高度污染。在这里，我们描述了一种从感染叶片组织中纯化细菌细胞的可靠而直接的方法，即使相对于植物材料，细菌数量较少，该方法也非常有效。通过分析模式宿主植物拟南芥感染后的前几个小时，植物病原肠杆菌迪氏棒杆菌（Dickeya dadantii）的表达谱，验证了该方法进行转录组分析的效率。比较了附生细菌和定植于宿主组织的细菌的转录组谱，鉴定了大约 100 个差异表达基因。该方法不需要特定的设备，成本低廉，易于转移到其他病理系统，对于许多其他植物-细菌相互作用的研究应该具有很大的兴趣。
