A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China.

We have analysed 198 fast-growing soybean-nodulating rhizobial strains from four different regions of China for the following characteristics: generation time; number of plasmids; lipopolysaccharide (LPS), nodulation factors (LCOs) and PCR profiles; acidification of growth medium; capacity to grow at acid, neutral, and alkaline pH; growth on LC medium; growth at 28 and 37 degrees C; melanin production capacity; Congo red absorption and symbiotic characteristics. These unbiased analyses of a total subset of strains isolated from specific soybean-cropping areas (an approach which could be called "strainomics") can be used to answer various biological questions. We illustrate this by a comparison of the molecular characteristics of five strains with interesting symbiotic properties. From this comparison we conclude, for instance, that differences in the efficiency of nitrogen fixation or competitiveness for nodulation of these strains are not apparently related to differences in Nod factor structure.

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations.

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

Mutants in the nodFEL promoter of Rhizobium leguminosarum bv. viciae reveal a role of individual nucleotides in transcriptional activation and protein binding.

The highly conserved nod box sequence in the promoters of the inducible nodulation genes of rhizobia is required for transcription activation together with NodD, a LysR-type transcriptional regulator, and a flavonoid as a coinducer. DNA fragments containing nod box sequences form two binding complexes when crude preparations of Rhizobium leguminosarum bv. viciae are used: a NodD-dependent and an additional, NodD-independent complex. The role of individual nucleotides in the conserved nod box sequence in complex formation and in nodulation gene expression was investigated by introducing 13 individual base-pair substitutions in the nodF nod box of R. leguminosarum bv. viciae and studying their effect on promoter activity and protein-DNA complex formation. Two mutants showed decreased NodD binding and decreased promoter activity. Five mutants showed a NodD-dependent complex as with the wild-type nodF nod box, whereas their promoter activity was severely reduced after induction. This result is in agreement with earlier observations that NodD DNA binding also occurs in the absence of inducer. Four mutants were impaired in the formation of the NodD-independent retardation complex. Three of them showed no alterations in promoter activity, meaning that no specific role for the protein forming the NodD-independent complex could be established. The two mutants in the highly conserved LysR motif of the nod box were unable to direct coinducer-dependent promoter activity but, unexpectedly, their retardation patterns were not altered. The remaining two mutants showed constitutive promoter activity. The results are discussed in terms of the relevance of conserved nucleotides and motifs identified in the nod box.

Proteins involved in the production and perception of oligosaccharides in relation to plant and animal development.

Chitin oligosaccharides and their derivatives are involved in developmental and defence-related signalling pathways. Major advances include the structural identification of lectins involved in development that bind chitin oligosaccharides and the links between chitin oligosaccharide and hyaluronan synthesis. Also, recent advances in the understanding of the biological role of oligosaccharides are summarised in a model for multistep glycan recognition.

Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants.

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides.

The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids. The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot-inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot-inoculation, but only after delivery by microtargeting. Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.

Chitin oligosaccharides can induce cortical cell division in roots of Vicia sativa when delivered by ballistic microtargeting.

Rhizobia, bacterial symbionts of leguminous plants, produce lipo-chitin oligosaccharide (LCO) signal molecules that can induce nodule organogenesis in the cortex of legume roots in a host-specific way. The multi-unsaturated fatty acyl and the O-acetyl moieties of the LCOs of Rhizobium leguminosarum biovar viciae were shown to be essential for obtaining root nodule induction in Vicia sativa plants. We have used ballistic microtargeting as a novel approach to deliver derivatives of the nodulation signal molecules inside the roots of V. sativa. This method offers the unique ability to introduce soluble compounds into the tissue at a small area. The mitogenic effect of microtargeting of chitin oligosaccharides, including an analysis of the influence of the chain length and modifications, was tested in a qualitative assay. The role of a cell division factor from the root stele, uridine, has also been examined in these experiments. The results show that O-acetylated chitin oligosaccharides can induce root cortical cell divisions when delivered by microtargeting. For this effect it is essential that uridine is co-targeted. The foci of cortical cell division were often similar to root nodule primordia. Anatomical examination also revealed chimeric structures that share characteristics with lateral root and nodule primordia. Our data favour a model in which the oligosaccharide moiety of the rhizobial LCO induces cortical cell division and the fatty acyl moiety plays a role in transport of the LCO into the plant tissue.

Nucleotide sequence corrections of the uidA open reading frame encoding beta-glucuronidase.

Part of the open reading frame of uidA, encoding beta-glucuronidase, was sequenced and two differences were found with the previously reported nucleotide sequence [Jefferson et al., Proc. Natl. Acad. Sci. USA 83 (1986) 8447-8451]. One is a silent mutation, the other results in the Glu279-->Gln substitution.

The NodD protein does not bind to the promoters of inducible nodulation genes in extracts of bacteroids of Rhizobium leguminosarum biovar viciae.

In a previous study, we showed that in bacteroids, transcription of the inducible nod genes does not occur and expression of nodD is decreased by 65% (H. R. M. Schlaman, B. Horvath, E. Vijgenboom, R.J.H. Okker, and B. J. J. Lugtenberg, J. Bacteriol. 173:4277-4287, 1991). In the present study, we show, using gel retardation, that in crude extracts of bacteroids of Rhizobium leguminosarum biovar (bv.) viciae, NodD protein does not bind to the nodF, nodM, and nodO box and that it binds only weakly to the nodA box. Binding of NodD from bacteroids to nod box DNA could be restored by mild proteinase K treatment, indicating that NodD is present in bacteroids in an altered form or complex which prevents its binding to nod box DNA. In addition, a novel nodA box DNA-protein complex was found which is specific for the nodA promoter region. This novel complex was formed neither with material from cultured bacterial cells nor with an extract from uninfected roots, and it did not contain NodD but another protein. These results are consistent with the hypothesis that the protein present in the novel retardation complex acts as a transcriptional repressor causing the decreased nodD expression in bacteroids. Such a repressor also explains the lack of nodABCIJ transcription despite the weak NodD binding to the nodA box.

Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae.

The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteriods compared with cultured cells, whereas NodD protein was reduced only 3-fold. Northern (RNA) blot hybridization, RNase protection assays, and in situ RNA hybridization together showed that, except for the nodD transcript, none of the other nod gene transcripts were present in bacteroids. The amount of nodD transcript in bacteroids was reduced only two- to threefold compared with that in cultured cells. Identical results were found with a Rhizobium strain harboring multicopies of nodD and with a strain containing a NodD protein (NodD604) which is activated independently of flavonoids. Furthermore, it was found that mature pea nodules contain inhibitors of induced nod gene transcription but that NodD604 was insensitive to these compounds. In situ RNA hybridization on sections from P. sativum and Vicia hirsuta nodules showed that transcription of inducible nod genes is switched off before the bacteria differentiate into bacteroids. This is unlikely to be due to limiting amounts of NodD, the absence of inducing compounds, or the presence of anti-inducers. The observed switch off of transcription during the development of symbiosis is a general phenomenon and is apparently caused by a yet unknown, negative regulation mechanism.

Subcellular localization of the Rhizobium leguminosarum nodI gene product.

By the use of antibodies raised against a fusion protein of lacZ'-nodI (produced in Escherichia coli) which specifically react with NodI protein, it was shown that in wild-type Rhizobium leguminosarum biovar viciae NodI protein (i) is recovered with the cytoplasmic membrane fraction and (ii) is translated as part of the nodABCIJ operon. In addition, it was found that the bacterial chromosomal background strongly influences the expression of several nod genes.

Subcellular localization of the nodD gene product in Rhizobium leguminosarum.

In Rhizobium strains the transcription of symbiosis plasmid-localized nod genes, except nodD, is induced by plant flavonoids and requires the nodD gene product. In order to localize NodD protein in R. leguminosarum, a NodD protein-specific antiserum was raised against a lacZ'-'nodD gene fusion product. Using these antibodies, we determined that the NodD protein is located exclusively in the cytoplasmic membrane of wild-type R. leguminosarum biovar viciae cells. This localization is independent of the presence of inducers. In a Rhizobium strain that overproduced the NodD protein, the protein was present both in the cytoplasmic membrane and the cytosol, indicating an influence of the protein abundance on its ultimate subcellular localization. It was estimated that 20 to 80 molecules of NodD protein were present per wild-type Rhizobium cell. A model which combines the localization and the DNA-binding properties of the NodD protein as well as the observed association of flavonoids with the cytoplasmic membrane is discussed.