Dept of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA, USA.
Biotechnol Prog. 2012 Sep-Oct;28(5):1321-8. doi: 10.1002/btpr.1608. Epub 2012 Aug 28.
Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging-based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum-infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post-infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration.
植物病原菌农杆菌附着在宿主植物细胞上是植物转化和农杆菌浸润过程的早期和必要步骤。然而，在复杂的植物组织中，细菌附着行为还没有得到很好的理解。在这里，我们开发了一种基于成像的方法，用于观察和量化原位叶组织中附着的农杆菌。研究了用核酸、蛋白质和活体染料对细菌进行荧光标记，作为生成表达荧光蛋白的重组菌株的快速替代方法。发现 Syto 16 绿色荧光核酸染色在不影响活力或感染力的情况下，可使染色细菌产生最大的信号强度。染色后的细菌保留了染色，并可在 72 小时内检测到。为了证明附着细菌的原位检测，使用共焦荧光显微镜对真空渗透标记细菌后的生菜叶组织切片进行成像，以观察农杆菌。细菌信号与植物细胞表面相关，表明检测到附着在植物细胞上的细菌。细菌对特定叶片组织的附着与农杆菌转化的已知叶片组织能力一致。随着时间的推移，对附着在叶细胞上的细菌进行了定量分析。在渗透后 24 小时内，染色细菌的信号保持稳定，但随着细菌在植物体内繁殖，信号强度降低。用核酸染色农杆菌，然后用感染的叶片组织进行共焦显微镜检查，为评估农杆菌与植物表达宿主的附着提供了一种快速的原位方法，也是通过农杆菌浸润来管理瞬时表达过程的一种工具。
