Conditional Requirement for Exopolysaccharide in the Mesorhizobium–Lotus Symbiosis

Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide (EPS) in the formation of determinate nodules, we isolated Mesorhizobium loti mutants affected in various steps of EPS biosynthesis and characterized their symbiotic phenotypes on two Lotus spp. The wild-type M. loti R7A produced both high molecular weight EPS and lower molecular weight (LMW) polysaccharide fractions whereas most mutant strains produced only LMW fractions. Mutants affected in predicted early biosynthetic steps (e.g., exoB) formed nitrogen-fixing nodules on Lotus corniculatus and L. japonicus ‘Gifu’, whereas mutants affected in mid or late biosynthetic steps (e.g., exoU) induced uninfected nodule primordia and, occasionally, a few infected nodules following a lengthy delay. These mutants were disrupted at the stage of infection thread (IT) development. Symbiotically defective EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority of full-length IT observed harbored only the EPS mutant strain and did not show bacterial release, whereas the nitrogen-fixing nodules contained both mutants. Examination of the symbiotic proficiency of the exoU mutant on various L. japonicus ecotypes revealed that both host and environmental factors were linked to the requirement for EPS. These results reveal a complex function for M. loti EPS in determinate nodule formation and suggest that EPS plays a signaling role at the stages of both IT initiation and bacterial release.

Sinorhizobium fredii HH103 rkp-3 Genes Are Required for K-Antigen Polysaccharide Biosynthesis, Affect Lipopolysaccharide Structure and Are Essential for Infection of Legumes Forming Determinate Nodules

The Sinorhizobium fredii HH103 rkp-3 region has been isolated and sequenced. Based on the similarities between the S. fredii HH103 rkpL, rkpM, rkpN, rkpO, rkpP, and rkpQ genes and their corresponding orthologues in Helicobacter pylori, we propose a possible pathway for the biosynthesis of the S. fredii HH103 K-antigen polysaccharide (KPS) repeating unit. Three rkp-3 genes (rkpM, rkpP, and rkpQ) involved in the biosynthesis of the HH103 KPS repeating unit (a derivative of the pseudaminic acid) have been mutated and analyzed. All the rkp-3 mutants failed to produce KPS and their lipopolysaccharide (LPS) profiles were altered. These mutants showed reduced motility and auto-agglutinated when early-stationary cultures were further incubated under static conditions. Glycine max, Vigna unguiculata (determinate nodule–forming legumes), and Cajanus cajan (indeterminate nodules) plants inoculated with mutants in rkpM, rkpQ, or rkpP only formed pseudonodules that did not fix nitrogen and were devoid of bacteria. In contrast, another indeterminate nodule–forming legume, Glycyrrhiza uralensis, was still able to form some nitrogen-fixing nodules with the three S. fredii HH103 rifampicin-resistant rkp-3 mutants tested. Our results suggest that the severe symbiotic impairment of the S. fredii rkp-3 mutants with soybean, V. unguiculata, and C. cajan is mainly due to the LPS alterations rather than to the incapacity to produce KPS.

Anatomy of the Legume Nodule Cortex: Species Survey of Suberisation and Intercellular Glycoprotein

Nodules of 29 species from 23 legume genera were examined for suberisation and glycoprotein deposits. Extensive suberisation of the nodule outer cortex to form a peridem was considered a primitive feature, common to non-legume and caesalpinioid nodules. The periderm was less extensive in nodules of Mimosoideae and Papilionoideae. Vascular bundles within the nodule were always surrounded by a vascular endodermis, defined by the presence of suberin on radial walls. Suberisation of the tangential walls of this endodermis was considered to be a primitive feature (present in all species examined of Caesalpinioideae and Mimosoideae, and in 10 out of 21 Papilionoideae) which may limit solute import to and export from the nodule. Glycoprotein was observed in the apoplast of the cortex in the three papilionoid species examined, but was absent in the caesalpinioid species examined. The common endodermis was recognised as an advanced feature, present only in certain species of the subfamily Papilionoideae (5 of 7, and 11 of 15 species of indeterminate and determinate nodule growth respectively). A membrane impermeant dye (lucifer yellow-CH), supplied in the rhizosphere under a mild vacuum, was observed to infiltrate through the cortex and into the infected zone in caesalpinioid nodules, and as far as the inner cortex in mimosoid and papilionoid nodules. Thus the common endodermis does not serve as an apoplastic barrier, and is unlikely to serve as a significant oxygen 'diffusion barrier'.

The Impairment of the Nodulation Process, Induced by a Bradyrhizobium japonicum Exopolysaccharide Mutant is Determined by the Genotype of the Host Plant

The deletion mutant Bradyrhizobium japonicum ΔP22 produces a structurally altered exopolysaccharide. The nodulation of two cultivars each of Glycine max and Glycine soja, and cultivars of Macroptilium atropurpureum and Vigna radiata, infected with this mutant was examined in order to analyze the role of the exopolysaccharide in the infection process of plants with a determinate nodule type. All host plants analyzed exhibited delayed nodulation and formed fewer nodules per plant. The extent of the impairments depended on the genotype of the host plant. Morphological studies confirmed these results. In V radiata later steps in nodule development proceeded without further disturbances, whereas with G. soja PI 407287 minor changes were detected. In contrast, the inoculation of G. soja PI 468397 and M. atropurpureum lead to the formation of nodules most of which were not infected by Bradyrhizobium japonicum ΔP22 (Inf-). However, on M. atropurpureum at least some effective nitrogen-fixing nodules developed. Such nodules did not emerge from G. soja PI 468397. Inf- nodules were arrested in an early stage of nodule development, and symptoms of plant defense responses were observed.