Brandl M T, Quiñones B, Lindow S E
Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3454-9. doi: 10.1073/pnas.061014498.
We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC.
我们研究了ipdC基因的表达空间模式，ipdC是一种参与草生欧文氏菌吲哚乙酸生物合成的植物诱导基因，在植物上的单个细胞间进行研究，以便更好地了解该表型在细菌附生生态学中的作用以及参与ipdC调控的因素。将携带ipdC与gfp转录融合体的非致病性草生欧文氏菌菌株299R接种到菜豆植株上，接种48小时后从单片叶子上回收，并用针对菌株299R的16S rRNA寡核苷酸探针进行荧光原位杂交。通过罗丹明滤光片捕获的落射荧光图像用于区分菌株299R的5-羧基四甲基罗丹明标记细胞与其他叶片微生物区系。通过分析数字图像对单个细胞的绿色荧光强度进行定量分析，结果显示从菜豆叶片回收的299R细胞中约65%的细胞ipdC表达高于在培养基中的表达。此外，10%的细胞表现出比中位荧光强度高得多的绿色荧光水平，表明它们在植物上相对于ipdC表达比在培养基中更具异质性。在对叶片样品上的细胞进行荧光原位杂交后，通过共聚焦激光扫描显微镜对叶片表面的299R细胞进行原位检查，结果显示即使是紧邻的细胞也表现出显著不同的绿色荧光强度，因此，它们处于诱导ipdC差异表达的物理或化学微环境中。
