Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Japan.
J Biosci Bioeng. 2010 Feb;109(2):153-8. doi: 10.1016/j.jbiosc.2009.07.012. Epub 2009 Aug 22.
We monitored growth and movement of Ralstonia solanacearum harboring the plasmid pRSS12 in tomato seedlings. The plasmid contains a gene for green fluorescent protein (GFP) and is stably maintained in R. solanacearum cells without selection pressure. Bacteria harboring the plasmid can be tracked in planta by visualizing GFP fluorescence. Stems of seedlings were infected with R. solanacearum cells transformed with pRSS12, and bacterial growth and movement, particularly around the vascular bundles, were monitored for more than 7 days. Our results showed that vascular bundles are independent of each other within the stem, and that it takes a long time for R. solanacearum cells to migrate from one vascular bundle to another. For real-time monitoring of bacteria in planta, tomato seedlings were grown on agar medium and bacterial suspension was applied to the root apex. The bacterial invasion process was monitored by fluorescent microscopy. Bacteria invaded taproots within 6 h, and movement of the bacteria was observed until 144 h after inoculation. In susceptible tomato cultivars, strong GFP fluorescence was observed in hypocotyls and lateral roots as well as the taproot. In resistant cultivars, however, GFP fluorescence was rarely observed on lateral roots. Our results show that this monitoring system can be used to assess bacterial pathogenicity efficiently.
我们监测了携带质粒 pRSS12 的青枯雷尔氏菌在番茄幼苗中的生长和运动。该质粒含有绿色荧光蛋白（GFP）基因，在没有选择压力的情况下可以在青枯雷尔氏菌细胞中稳定维持。携带质粒的细菌可以通过可视化 GFP 荧光在植物体内进行追踪。将携带 pRSS12 的质粒转化的青枯雷尔氏菌细胞感染幼苗的茎部，监测细菌的生长和运动，特别是在维管束周围，持续了超过 7 天。我们的结果表明，茎内的维管束彼此独立，青枯雷尔氏菌细胞从一个维管束迁移到另一个维管束需要很长时间。为了实时监测植物体内的细菌，我们将番茄幼苗种植在琼脂培养基上，并将细菌悬浮液应用于根尖。通过荧光显微镜监测细菌的入侵过程。细菌在 6 小时内入侵主根，并且可以观察到细菌的运动，直到接种后 144 小时。在易感番茄品种中，在子叶和侧根以及主根中观察到强烈的 GFP 荧光。然而，在抗性品种中，很少在侧根上观察到 GFP 荧光。我们的结果表明，该监测系统可用于有效地评估细菌的致病性。
