Broad Host-range Rhizobium Sp Research Articles

Structural Characterization of a K-antigen Capsular Polysaccharide Essential for Normal Symbiotic Infection in Rhizobium sp. NGR234: DELETION OF THE rkpMNO LOCUS PREVENTS SYNTHESIS OF 5,7-DIACETAMIDO-3,5,7,9-TETRADEOXY-NON-2-ULOSONIC ACID

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.

Flavonoids induce temporal shifts in gene-expression of nod-box controlled loci in Rhizobium sp. NGR234.

Rhizobia, soil bacteria of the Rhizobiales, enter the roots of homologous legumes, where they induce the formation of nitrogen-fixing nodules. Signals emanating from both symbiotic partners control nodule development. Efficient nodulation requires precise, temporal regulation of symbiotic genes. Roots continuously release flavonoids that interact with transcriptional activators of the LysR family. NodD proteins, which are members of this family, act both as sensors of the environment and modulate the expression of genes preceded by conserved promoter sequences called nod-boxes. The symbiotic plasmid of the broad host-range Rhizobium sp. NGR234 caries 19 nod-boxes (NB1 to NB19), all of which were cloned upstream of a lacZ-reporter gene. A flavonoid, daidzein was able to induce 18 of the 19 nod-boxes in a NodD1-dependent manner. Interestingly, induction of four nod-boxes (NB6, NB15, NB16 and NB17) is highly dependent on NodD2 and was delayed in comparison with the others. In turn, NodD2 is involved in the repression of the NB8 nodABCIJnolOnoeI operon. Activation of transcription of nodD2 is also dependent on flavonoids despite the absence of a nod-box like sequence in the upstream promoter region. Mutational analysis showed that syrM 2 (another member of the LysR family), which is controlled by NB19, is also necessary for expression of nodD 2. Thus, NodD1, NodD2 and SyrM2 co-modulate a flavonoid-inducible regulatory cascade that coordinates the expression of symbiotic genes with nodule development.

Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp. NGR234.

In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no "model system" to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.

Symbiosis-stimulated chitinase isoenzymes of soybean (Glycine max (L.) Merr.)

and to be colonized by arbuscular mycorrhiza (AM ) fungi (Harrison, 1997). Often, nodule formation by a Isoforms of endochitinase in soybean were studied in given bacterial strain is restricted to only a few host plant relation to root symbiosis. Five selected cultivars dif- genera, and varieties of a given legume species may diVer fering in their nodulation potential were inoculated in nodule formation when inoculated with a given strain. with two strains of Bradyrhizobium japonicum, the Specifically in soybean, allelic genes have been described broad host-range Rhizobium sp. NGR234, and with the which restrict nodulation of certain genotypes by certain mycorrhizal fungus Glomus mosseae. Total chitinase Bradyrhizobium strains ( Vest, 1970; Qian et al., 1996). activity in nodules was up to 7-fold higher than in Other, ‘broad host-range’ rhizobia exist: Rhizobium sp. uninoculated roots and in mycorrhizal roots. The chi- NGR234 is such an example, and it is able to induce tinase activity in nodules varied depending on the nodules on more than 110 genera of legumes as well

Lotus japonicus Nodulates and Fixes Nitrogen with the Broad Host Range Rhizobium sp. NGR234

Lotus japonicus possesses major advantages as a model legume for the study of plant-microbe interactions. The relative absence of genetic information on its normal microbial partner (i.e., Mesorhizobium loti) could limit its utility in research. Here we show for the first time that the broad host range Rhizobium strain NGR234 nodulates and fixes nitrogen in symbiosis with Lotus japonicus ecotypes Gifu and Funakura. We demonstrate that bacterial mutants deficient in nodulation or nitrogen fixation possess the expected phenotype with L. japonicus. Nodulation of L. japonicus was sensitive to nitrate. Vermiculite was an efficient synthetic growth substrate, allowing axenic growth in Magenta jars. The genetic analysis of the Lotus japonicus-Mesorhizobium interaction should be accelerated through the use of this well-defined microsymbiont.

The nodulation of micro-propagated plants ofParasponia andersoniiby tropical legume rhizobia

A simple clonal micro-propagation system for Parasponia andersonii was employed to study the nodulation response of this non-legume to inoculation by the broad host range Rhizobium sp. NGR234, isolated from Lablab purpureus, and also to tropical legume rhizobia isolated from Aeschynomene species. Partially effective nodules, assayed by acetylene reduction and 15 N dilution procedures, were induced with strain NGR234 and its spontaneous streptomycin-resistant mutant ANU240. Effective nodules were produced by one of the Aeschynomene strains (ORS302) tested, with rates of acetylene reduction comparable to those of root nodules produced by Bradyrhizobium strain CP279, originally isolated from P. andersonii. Light and transmission electron microscopy showed that there was a correlation between the nitrogen fixing capability of the symbiosis between NGR234 and Parasponia and the number of persistent infection (fixation) threads within the nodule cells.

Proteins Associated with Root-Hair Deformation and Nodule Initiation inVigna unguiculata

Developmental changes in the synthesis of root-hair proteins were followed in the symbiosis between the promiscuous legume Vigna unguiculata and the broad host-range Rhizobium sp. NGR234. Comparison of the two-dimensional electrophoresis patterns of proteins isolated from root hairs inoculated with wildtype or hair-deformation minus mutants revealed 12 symbiosis-specific proteins. Synthesis of three of these proteins was repressed 4 days after inoculation with Rhizobium. The remaining nine proteins were induced by Rhizobium 1, 2, or 4 days after inoculation. As three of these (15-, 31-, and 44-kDa hadulins) were specifically and transiently expressed in root hairs during the deformation process, we have named them hadulins (hair-deformation specific proteins). Five proteins (including 15- and 31-kDa hadulins) were first observed 24 hr after inoculation. Of these, only the 15-kDa hadulin was not induced by R. fredii USDA257Sl Nod⁺, Fix⁺ on V. unguiculata. Two days after inoculation, three additional proteins became apparent, while another (44-kDa hadulin) appeared on day 4. All 12 proteins seem to be associated with root-hair deformation and nodule development.

Biology and genetics of the broad host range Rhizobium sp. NGR234

Biology and genetics of the broad host range <i>Rhizobium</i> sp. NGR234

Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata

Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to "nod-box"-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.