A rapid and non-destructive method for spatial-temporal quantification of colonization by Pseudomonas syringae pv. tomato DC3000 in Arabidopsis and tomato.
A rapid and non-destructive method for spatial-temporal quantification of colonization by Pseudomonas syringae pv. tomato DC3000 in Arabidopsis and tomato.
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DOI：
10.1186/s13007-021-00826-2
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发表时间：
2021-12-13
期刊：
影响因子：
5.1
通讯作者：
Ton J
中科院分区：
文献类型：
--
作者：
Furci L;Pascual-Pardo D;Ton J
番茄细菌性叶病病原菌番茄假单胞菌（Pst）是植物病理学研究中最常用的模式病原菌。以前的方法来研究植物-Pst相互作用依赖于Pst定殖的破坏性定量，这可能是费时费力的，并且不允许对细菌定殖进行时空监测。在这里，我们描述了一种快速和非破坏性的方法来量化和可视化的时空殖民PST在拟南芥和番茄的完整叶片。本文提出的方法使用生物发光Pst DC 3000菌株，其组成型表达来自发光光杆菌（Pst：：LUX）的luxCDABE操纵子，并且需要具有灵敏的CCD/CMOS相机和成像软件（Photoshop或Image J）的普通凝胶记录（Gel Doc）系统。通过捕获亮场和生物发光图像从Pst：：LUX感染的叶子，我们成像的时空动态的Pst感染。拟南芥喷雾接种后5天的时间过程中，从活Pst细菌的生物发光分析揭示了从老叶到嫩叶和顶端分生组织的细菌存在的过渡。通过计算相对生物发光值从数码照片获得Pst：LUX生物发光的定殖，所述相对生物发光值针对生物发光强度进行调整并通过叶表面进行归一化。该方法检测到在基础抗性不同的拟南芥基因型之间Pst：：LUX定殖的统计学显著差异，以及在拟南芥和番茄中通过抗性诱导处理在Pst：：LUX定殖的统计学显著降低。选择性琼脂培养基上的相对生物发光值与常规菌落计数的比较显示了统计学显著相关性，这在不同Gel Doc系统之间具有重现性。我们提出了一种非破坏性的方法来量化殖民化的生物发光Pst：：LUX在植物中。使用一个共同的凝胶文件系统和成像软件，我们的方法需要更少的时间和劳动力比传统的方法，是基于受感染的叶片材料的破坏性采样。此外，与传统的策略相比，我们的方法提供了额外的信息的时空模式的Pst殖民。在线版本包含补充材料，可通过10.1186/s13007-021-00826-2获得。
The bacterial leaf pathogen Pseudomonas syringae pv tomato (Pst) is the most popular model pathogen for plant pathology research. Previous methods to study the plant-Pst interactions rely on destructive quantification of Pst colonisation, which can be labour- and time-consuming and does not allow for spatial–temporal monitoring of the bacterial colonisation. Here, we describe a rapid and non-destructive method to quantify and visualise spatial–temporal colonisation by Pst in intact leaves of Arabidopsis and tomato. The method presented here uses a bioluminescent Pst DC3000 strain that constitutively expresses the luxCDABE operon from Photorhabdus luminescens (Pst::LUX) and requires a common gel documentation (Gel Doc) system with a sensitive CCD/CMOS camera and imaging software (Photoshop or Image J). By capturing bright field and bioluminescence images from Pst::LUX-infected leaves, we imaged the spatiotemporal dynamics of Pst infection. Analysis of bioluminescence from live Pst bacteria over a 5-day time course after spray inoculation of Arabidopsis revealed transition of the bacterial presence from the older leaves to the younger leaves and apical meristem. Colonisation by Pst:LUX bioluminescence was obtained from digital photos by calculating relative bioluminescence values, which is adjusted for bioluminescence intensity and normalised by leaf surface. This method detected statistically significant differences in Pst::LUX colonisation between Arabidopsis genotypes varying in basal resistance, as well as statistically significant reductions in Pst::LUX colonisation by resistance-inducing treatments in both Arabidopsis and tomato. Comparison of relative bioluminescence values to conventional colony counting on selective agar medium revealed a statistically significant correlation, which was reproducible between different Gel Doc systems. We present a non-destructive method to quantify colonisation by bioluminescent Pst::LUX in plants. Using a common Gel Doc system and imaging software, our method requires less time and labour than conventional methods that are based on destructive sampling of infected leaf material. Furthermore, in contrast to conventional strategies, our method provides additional information about the spatial–temporal patterns of Pst colonisation. The online version contains supplementary material available at 10.1186/s13007-021-00826-2.