Depto. Biología Celular, Genética y Fisiología, Instituto de Hortofruticultura Subtropical y Mediterránea, Campus de Teatinos, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC);
Depto. Biología Celular, Genética y Fisiología, Instituto de Hortofruticultura Subtropical y Mediterránea, Campus de Teatinos, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC).
J Vis Exp. 2022 Oct 6(188). doi: 10.3791/64614.
A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.
大量的致病微生物不断侵袭植物。丁香假单胞菌复合群包括革兰氏阴性植物病原菌，它们对许多宿主具有特殊的相关性。丁香假单胞菌从叶片表面进入植物，在质外体中迅速繁殖，形成微菌落，占据细胞间隙。细菌组成型表达荧光蛋白，可用于可视化微菌落，并在微观水平上监测感染的发展。单细胞分析的最新进展揭示了克隆同基因细菌群体所达到的巨大复杂性。这种复杂性被称为表型异质性，是细菌群落中基因表达（与遗传差异无关）的细胞间差异的结果。为了在单细胞水平上分析单个基因座的表达，已广泛使用荧光蛋白的转录融合。在应激条件下，例如在植物质外体定殖期间，丁香假单胞菌根据关键毒力基因（即 Hrp Ⅲ型分泌系统）的异质表达分化为不同的亚群。然而，由于接种和细菌提取过程中固有的机械破坏释放的细胞碎片，从植物组织中回收的任何给定丁香假单胞菌群体的单细胞分析都具有挑战性。本报告详细介绍了一种在拟南芥和豆类植物定殖过程中监测感兴趣的丁香假单胞菌基因在单细胞水平表达的方法。描述了使用真空室制备植物和用于接种的细菌悬浮液。还解释了通过质外体液提取从感染叶片中回收内生细菌的方法。细菌接种和细菌提取方法均经过经验优化，以最大程度地减少植物和细菌细胞的损伤，从而获得最适合显微镜和流式细胞术分析的细菌制剂。
