Page 2 Plant-Bacteria Interactions Edited by Iqbal Ahmad, John ...

174j 9 Rice–Rhizobia Association: Evolution of an Alternate Niche
Strains of A. caulinodans, Bradyrhizobium and Rhizobium were genetically modified
with the lacZ reporter gene to study their mode of invasion and extent of
colonization in rice roots [40]. To study the timing and route of entry into rice
tissues further, rhizobia have also been tagged with DNA sequences encoding the
green fluorescent protein (Gfp) that imparts a green autofluorescence in the bacteria.
This gene expression enables a nondestructive assay to be used to locate the
bacteria, quantify their local abundance and follow their association with the root of
young rice seedlings at single-cell resolution using epifluorescence microscopy
[41,42]. After inoculation with R. leguminosarum bv. trifolii strain ANU843, the
bacterial cells were observed along the root grooves and in microcolonies on the
root surfaces of the primary main root. Gradually, this colonization progressed into
lateral root cracks, the epidermis and finally deeper within intercellular spaces of the
root cortex. The bacteria form long lines of fluorescent microcolonies inside lateral
roots. Furthermore, no specific morphological changes of roots such as root hair
curling, formation of infection threads or root nodule primordial were observed on
the rice roots inoculated with rhizobial strains [42].
Quantitative microscopy is being used to evaluate spatial patterns of rhizobial
colonization on rice roots to better understand how the rhizobia–rice association
develops. This work involves scanning electron microscopy of rice roots inoculated
with a reference biofertilizer strain of endophytic R. leguminosarum bv. trifolii analyzed
at single-cell resolution using CMEIAS (Center for Microbial Ecology Image
Analysis System) image analysis software developed for these studies in computerassisted
microscopy. New measurement features have been developed in CMEIAS,
for example the Cluster Index [50], to extract information from digital images of
microbial cells on the root surface needed to compute plotless, plot-based and
geostatistical analyses that describe and mathematically model spatial patterns of
their root surface colonization. This includes geostatistically defendable interpolation
of their distribution and dispersion, even within areas of the root that are not
sampled [39,51–53]. Typical scanning electron micrographs depicting various morphological
features of the colonization of the Sakha 102 variety of rice roots by the
rhizobial strain E11 used in these studies are illustrated in Figure 9.4a–e. These
images reveal that these bacteria (1) attach in both supine and polar orientations,
preferentially to the nonroot hair epidermis, in contrast to their preferential attachment
in polar orientation to root hairs of their host legume, (2) commonly colonize
small crevices at junctions between epidermal cells (white arrows in Figure 9.4a–c),
suggesting this route to be a portal of entry into the root and (3) produce eroded pits
on the rice root epidermis (Figure 9.4d and e). Similarly eroded plant structures are
produced in the Rhizobium–white clover symbiosis by plant cell wall degrading
enzymes bound to the bacterial cell surface, and these pits represent incomplete
attempts of bacterial penetration that had only progressed through isotropic, noncrystalline
outer layers of the plant cell wall [54]. Consistent with these results,
activity gel electrophoresis indicated that the rice-adapted rhizobia produced a
cell-bound CMcellulase [7]. This enzyme likely participates in the invasion and
dissemination of the rhizobial endophyte within host roots.