GFP-aided confocal laser scanning microscopy can monitor Agrobacterium tumefaciens cell morphology and gene expression associated with infection

We tagged Agrobacterium tumefaciens cells with a mini-Tn5 transposon contai ning a promoterless gene encoding a green fluorescent protein (GFP). Some o f the GFP-tagged individual bacterial cells exhibited strong green fluoresc ence, which reflected the expression levels of the GFP-tagged genes. Those cells could be readily detected with a confocal laser scanning microscope ( CLSM), We observed that the fluorescence and morphology of A. tumefaciens c ells grown in plant tissues resembled those grown in a minimal medium of lo w pH, which is required for expression of the virulence genes responsible f or tumorigenesis. This suggests that GFP-aided CLSM can be used to determin e which growth medium is more representative of the nutritional conditions that a pathogen encounters in plant tissues. We also observed that the fluo rescence and morphology of A. tumefaciens cells changed dramatically during the course of infection. Our data suggested that A. tumefaciens cells were probably better fed upon successful colonization. We believe that GFP-aide d CLSM can help study the fate of A. tumefaciens cells inside plant tissues by monitoring cell morphology and gene expression associated with the infe ction process in situ. (C) 1999 Federation of European Microbiological Soci eties. Published by Elsevier Science B.V. All rights reserved.