plant surface microbiology.pdf

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Frank B. Dazzo
microscopy. The bulk of this capsule consists of a large acidic heteropolysaccharide
(Dazzo and Hubbell 1975). Bioassays scored by quantitative phase
contrast microscopy indicate that oligosaccharide fragments produced by
enzymatic depolymerization of this polysaccharide are biologically active in
promoting root hair infectibility in white clover seedlings inoculated with R.
leguminosarum bv. trifolii (Abe et al. 1984; Hollingsworth et al. 1984). The
complete structures of the acidic heteropolysaccharides of several strains of
R. leguminosarum bv. trifolii have been elucidated and shown to consist of
repeated octasaccharide units of 5Glc:2GlcA:1Gal containing a tetrasaccharide
backbone of 2Glc:2GlcA substituted with O-acetate and a tetrasaccharide
sidechain of 3Glc:1Gal bearing pyruvyl substitutions on the terminal Gal and
penultimate Glc, and a O-hydroxybutyrate substitution on the terminal Gal
(Hollingsworth et al. 1988; Philip-Hollingsworth et al. 1989a). Trifoliin A
binds selectively to this acidic heteropolysaccharide, and the symbiont-specificity
in this protein–carbohydrate interaction involves recognition of the
sites of linkage and stoichiometry of noncarbohydrate substitutions in the
octasaccharide repeat unit (Abe et al. 1984; Hollingsworth et al. 1984, 1988;
Philip-Hollingsworth et al. 1989b). Subsequent biochemical studies revealed
host-range related structural features of R. leguminosarum bv. trifolii acidic
heteropolysaccharides that distinguish these cell surface polymers and those
of the closely related pea symbiont, R. leguminosarum bv. viciae, based on
subtle differences in molar stoichiometry and positions of attachment of
these noncarbohydrate substitutions (Philip-Hollingsworth et al. 1989a, b).
Other studies have shown a link between rhizobial genes involved in determining
the acidic heteropolysaccharide structures and the legume host-range
in R. leguminosarum and Rhizobium sp. (Acacia; Philip-Hollingsworth et al.
1989b; Lopez-Lara et al. 1993, 1995). This relationship is expressed in some,
but not all genetic backgrounds of R. leguminosarum (Orgambide et al. 1992).
Recently, we have presented a micrograph of a portion of an isolated molecule
of the R. leguminosarum bv. trifolii acidic polysaccharide acquired using a
field-emission scanning/transmission electron microscope at extremely high
magnification (Dazzo and Wopereis 2000). Image analysis of the branches
projecting perpendicular to the main polymer backbone in that micrograph
indicate that they are within the same size range as the predicted 20±2
angstrom length of the substituted tetrasaccharide side-chain. Molecular
microscopy!
A role of the capsular polysaccharide from R. leguminosarum bv. trifolii in
symbiotic recognition was clearly shown by labeling this polymer with the
fluorochrome FITC and documenting its direct interaction with white clover
roots using epifluorescence microscopy (Dazzo and Brill 1977). Figure 6A
illustrates the result, providing direct evidence for the existence and distribution
of receptor sites on clover root hairs that specifically recognized the capsular
polysaccharide of this rhizobial microsymbiont. Further studies using
fluorescence microscopy indicated that these receptor sites are saturable,