plant surface microbiology.pdf

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Frank B. Dazzo
biological activities on white clover roots (Philip-Hollingsworth et al. 1997).
This approach is far superior to conjugation of CLOSs with certain alternative
fluorochromes, e.g., biodipi, whose relatively large and hydrophobic molecular
structure could significantly perturb the physiological bioactivity of the
CLOS molecules. This NBD-CLOS molecular probe was applied to axenic
seedling roots under microbiologically controlled conditions. At various time
points thereafter, the specimens were rinsed free of unbound conjugate and
examined in vivo by laser scanning confocal microscopy, with results
acquired in real time at subcellular resolution (Philip-Hollingsworth et al.
1997). Figure 8A–H illustrates the key in vivo results of these studies, providing
direct microscopical evidence that the NBD-CLOSs made by wild-type R.
leguminosarum bv. trifolii interact rapidly with clover root hairs, traverse
their cell walls, absorb to their cell membrane, and within minutes are then
internalized within these living cells, where they migrate to the base of the
root hairs and translocate to underlying cortical cells in a discrete region of
the root. Quantitative fluorescence microscopy indicated that NBD-CLOSs
from wild-type R. leguminosarum bv. trifolii were internalized by a significantly
higher proportion of root hairs from the host legume white clover than
from the nonhost legume alfalfa (Philip-Hollingsworth et al. 1997). As predicted,
the structural requirements for internalization of NBD-CLOS analogs
in living root hairs matched those required for Had and Noi bioactivities of
CLOSs in white clover and alfalfa as described above. In contrast, the fluorescent
analog NBD-chitotriose (without a linked lipid) was not taken up by living
clover root hairs or cortical cells, indicating that in vivo internalization of
Fig. 8. Laser scanning confocal microscopy of the direct, dynamic interaction of chitolipooligosaccharides
(CLOSs) from wild-type R. leguminosarum bv. trifolii ANU843
with living cells of white clover roots. Purified CLOSs were conjugated with the fluorochrome
NBD to produce a fluorescent molecular probe with minimal molecular perturbation
that retained Had and Noi bioactivities on white clover roots. A When applied
to roots, these labeled Nod factors rapidly adsorbed to the root hairs. Closer examination
of a time-series sequence of images showed that the NBD-tagged CLOSs adsorbed
to the root hair cell membrane, and then within minutes were internalized within these
epidermal cells (B–F), some migrating to the base of the root hair cell (B–D) and others
remaining on the cell membrane or inside the root hair nucleus (E, F). Within 30 min,
some NBD-CLOSs were translocated to a discrete region of the underlying root cortex
and internalized within selected cortical cells (G, H). Arrowheads in the paired micrographs
of (E, F) point to the root hair nucleus that internalized some labeled CLOSs. The
NBD-CLOSs of ANU843 were internalized by a significantly higher proportion of the
root hairs on white clover than alfalfa roots. Further studies using synthetic CLOS
analogs and axenic seedling bioassays evaluated by these microscopy techniques established
the minimal structural features of these Nod factor molecules that are required
and sufficient for uptake and Had/Noi-inducing activities on both white clover and
alfalfa roots. Scale bar A 50 mm, B–D 15 mm, E, F 10 mm, H 100 mm. Reprinted with permission
from Lipid Research, Inc.