Abstract
Chemical imaging of plant-bacteria co-cultures makes it possible to characterize bacterial populations
and behaviors and their interactions with proximal organisms, under conditions closest to the environment
in the rhizosphere. Here Raman micro-spectroscopy and confocal Raman imaging are used as
minimally invasive probes to study the rhizosphere bacterial isolate, Pantoea sp. YR343, and its co-culture
with model plant Arabidopsis thaliana by combining enhanced Raman spectroscopies with electron
microscopy and principal component analysis (PCA). The presence of carotenoid pigments in the wild
type Pantoea sp. YR343 was characterized using resonance Raman scattering, which was also used to
confirm successful disruption of the crtB gene in an engineered carotenoid mutant strain. Other components
of the Pantoea sp. YR343 cells were imaged in the presence of resonantly enhanced pigments
using a combination of surface enhanced Raman imaging and PCA. Pantoea sp. YR343 cells decorated
with Ag colloid synthesized ex situ gave spectra dominated by carotenoid scattering, whereas colloids
synthesized in situ produced spectral signatures characteristic of flavins in the cell membrane. Scanning
electron microscopy (SEM) of whole cells and transmission electron microscopy (TEM) images of thinly
sliced cross-sections were used to assess structural integrity of the coated cells and to establish the origin
of spectral signatures based on the position of Ag nanoparticles in the cells. Raman imaging was also
used to characterize senescent green Arabidopsis thaliana plant roots inoculated with Pantoea sp. YR343,
and PCA was used to distinguish spectral contributions from plant and bacterial cells, thereby establishing
the potential of Raman imaging to visualize the distribution of rhizobacteria on plant roots.
