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A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.

The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determine unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont. We evaluated nodE function by examining CLOS structures made by wild-type R. leguminosarum bv. trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843.1 H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species. We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H. P., Bloemberg, G. V., van Brussel, A. A. N., Lugtenberg, B. J. J., van der Drift, K. M. G. M., Haverkamp, J., and Thomas-Oates, J. E. (1995) Mol. Plant-Microbe Interact. 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species. No evidence for nodE-dependent CLOSs was found for these strains. These results indicate a need to revise the current model to explain how nodE determines host range in the R. leguminosarumlegume symbiosis.Rhizobium, Bradyrhizobium, and Azorhizobium are bacterial genera that form N 2 -fixing nodules on legume roots. In this symbiosis, the plant produces flavonoids that activate bacterial expression of nod genes necessary for production of "Nod factors" involved in infection and nodulation of the corresponding host plant (1-4). These Nod factors are chitolipooligosaccharides (CLOSs) 1 consisting of ␤-1,4-linked oligomers of N-acetylglucosamine bearing an amide-linked fatty acyl moiety at the nonreducing end and may contain other substituents (e.g. Oacetyl, sulfate, etc.) that make their biological activity hostspecific (5). The current model for nod functions is that the common nod genes encode enzymes that synthesize the common backbone of CLOSs, and the host-specific nod genes encode enzymes that introduce these modifications in CLOS structures making them host-specific (6, 7). Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) is the bacterial symbiont of the legume host, clover (Trifolium spp.). In the most thoroughly studied wild-type strain (ANU843), the ability to nodulate white clover is controlled by regulatory (nodD), common (nodABCIJ), and hostspecific (nodFERL, nodMN) nod genes residing within a 14-kb region on its resident symbiotic plasmid (pSym) (8, 9). Elegant studies have shown that NodE is the main determinant of nodulation host range for R. trifolii and its closest relative, the pea symbiont, R. leguminosarum bv. viciae (10, 11). Tn5 disruption of nodE (but not genes downstream of nodE) in ANU843 results in a unique dual phenotype, which is defective in nodula...

We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv. trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection. CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs. At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself. Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted. Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites. The application of CLOS also enabled a NodC ؊ mutant of R. leguminosarum bv. trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls. However, CLOSs did not rescue the ability of the NodC ؊ mutant to induce marked curlings or infection threads within root hairs. These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.