Helmi R. M. Schlaman

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.

Flavonoids Synthesized in Cortical Cells During Nodule Initiation Are Early Developmental Markers in White Clover

We examined the site-specific induction of the flavonoid pathway before and during nodule initiation in white clover with transgenic plants, fluorescence microscopy, and microspectrofluorometry to test if flavonoids play a role in nodule organogenesis. A chalcone synthase regulated βglucuronidase (GUS) transgene (CHS3:gusA) was upregulated from 3 h post inoculation (p.i.) until cell division (around 40 h p.i.) in inner cortex cells underlying the inoculation site. Intracellular fluorescence occurred in vacuoles of those inner cortex cells from 13 h p.i. until the fluorescent cells divided. Fluorescence emission spectra of contents of individual fluorescing cortex cells were measured in situ and compared with emission spectra of compounds purified from root extracts. The fluorescing compound located in cells of the inner cortex after Rhizobium leguminosarum bv. trifolii infection was identified as a water-soluble derivative of 7,4′-dihydroxyflavone. Nodule primordium cells contained a different fluorescent compound, identified as the isoflavonoid formononetin. CHS3:gusA expression and flavonoid accumulation were only induced in inner cortex cells by a nodulating Rhizobium strain and by clover-specific lipo-chitinoligosaccharides, but not by non-nodulating rhizobia. Fluorescence was also induced by compatible rhizobia in other legumes such as alfalfa, pea, and siratro in the cells that participate in nodule initiation. Our results show that fluorescent flavonoids are useful markers in nodule organogenesis in clover and may have direct roles in nodule formation.

Genetic Organization and Transcriptional Regulation of Rhizobial Nodulation Genes

The ability of Rhizobium spp., Mesorhizobium spp., Sinorhizobium spp., Bradyrhizobium spp. and Azorhizobium spp., collectively called rhizobia, to nodulate plants is determined by both plant and bacterial genes. Nodulation genes are defined as those rhizobial genes which play a role in nodulation or which are coordinately regulated with such genes. In chronological order of discovery they were designated as nod, nol and noe genes followed by a subsequent letter of the alphabet. Thus, nodA, nodB and nodC were the first nodulation genes described (Rossen et al., 1984; Torok et al., 1984), and noeL in Rhizobium sp. NGR234 was the one most recently identified (Freiberg et al., 1997). Homologous nodulation genes in various rhizobia have identical names. Protein products of nodulation genes commonly are represented with a capitalized abbreviation (e.g. NodA is the protein encoded by the nodA gene). The nodD-homologue syrM was identified as a symbiotic regulator involved both in enhanced exopolysaccharide synthesis and in nodulation gene regulation (Mulligan and Long, 1989) and is therefore also discussed in this chapter. Initial studies on nodulation genes were carried out mainly in Sinorhizobium meliloti and in Rhizobium leguminosarum biovars viciae and trifolii. The identification of nodulation genes was advanced enormously by the discovery that in these rhizobia many of the nodulation genes are localized extra chromosomal on a large indigenous plasmid (Johnston and Beringer, 1977). These plasmids encoding symbiotic functions are the so-called Sym plasmids whose size can be even as large as one third of the chromosome in the case of S. meliloti (Banfalvi et al., 1981). When these rhizobial strains are cured of their Sym plasmid, they are unable to nodulate, whereas re-introduction of the homologous or a heterologous Sym plasmid restored nodulation (Banfalvi et al., 1981; Beynon et al., 1980; Djordjevic et al., 1983; Hooykaas et al., 1981; Johnston et al., 1978; Kondorosi et al., 1982; van Brussel et al., 1982). Transferring the rhizobial Sym plasmid into Agrobacterium tumefaciens and Philobacterium myrsinacearum also results in a capacity to induce root nodule formation (Hooykaas et al., 1981, 1982; Rodriquez-Quinones et al., 1989; van Veen et al., 1988). The development of genetic tools, such as the availability of broad-host range cloning and cosmid vectors (Ditta et al., 1980; Friedman et al., 1982), construction of cosmid libraries, complementation and mapping of nodulation-deficient mutants, introduction of random and directed transposon mutagenesis techniques coupled to marker exchange (Beringer et al., 1978; Buchanan-Wollaston, 1979; Meade et al., 1982) or used in combination with phage transduction methods (Pees et al., 1986; Wijffelman et al., 1985), led to the identification of regions involved in nodulation and, subsequently the first nodulation genes (Djordjevic et al., 1985; Downie et al., 1983, 1985; Fisher et al., 1985; Kondorosi et al., 1984; Rossen et al., 1984; Schofield et al., 1984; Torok et al., 1984). DNA sequence analysis, as well as complementation studies, revealed that some nodulation genes are conserved in all rhizobia, whereas others are restricted to a few or a single species or strain. Thus, in early literature nodulation genes were designated as “common” or “host specific” (hsn) respectively (Horvath et al., 1986; Kondorosi et al., 1984; Putnoky and Kondorosi, 1986). The DNA sequence revealed also that promoters of many nodulation genes contain a conserved DNA sequence called the nod box that allows coordinated regulation of nod genes (Rostas et al., 1986).

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 β-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.

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.

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.

Analysis of promoter activity of the early nodulin Enod40 in Lotus japonicus.

Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-benzylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L. japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.

Induction of hairy roots for symbiotic gene expression studies

The model legume Lotus japonicus can be transformed and regenerated efficiently with Agrobacterium tumefaciens or A. rhizogenes. However, it takes between 8 to 12 months to obtain seeds of transgenic plants. We therefore developed a rapid and efficient transformation protocol using A. rhizogenes to induce transgenic hairy roots that can be inoculated with Mesorhizobium loti 2 weeks after transformation. The first nodules emerge 8 to 10 days after inoculation, as on the roots of wild type Lotus plants and expression of plant genes involved in any step of nodulation can be completed within two months after the start of a transformation-nodulation experiment. A large number of seedlings can be tranformed in one experiment, allowing addressing of a number of variables in one single tranformationnodulation experiment.

Nucleotide sequence corrections of the uidA open reading frame encoding β-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 production of species-specific highly unsaturated fatty acyl-containing LCOs from Rhizobium leguminosarum bv. trifolii is stringently regulated by nodD and involves the nodRL genes.

A proportion of the Nod factors of some Rhizobium leguminosarum bv. trifolii strains is characterized by the presence of highly unsaturated fatty acyl chains containing trans double bonds in conjugation with the carbonyl group of the glycan oligosaccharide backbone. These fatty acyl chains are C18:3, C20:3, C18:4, or C20:4 and have UV-absorption maxima at 303 and 330 nm. These Nod factors are presumed to be important for host-specific nodulation on clover species. However, in wild-type R. leguminosarum bv. trifolii ANU843, Nod factors with these characteristic acyl chains were not observed using standard growth conditions. They were observed only when nod genes were present in multiple copies or when transcription was artificially increased to higher levels by introduction of extra copies of the transcriptional regulator gene nodD. In a screen for the genetic requirements for production of the Nod factors with these characteristic structures, it was found that the region downstream of nodF and nodE is essential for the presence of highly unsaturated fatty acyl moieties. Mu-lacZ insertion in this region produced a mutant that did not produce detectable levels of the highly unsaturated fatty acyl-bearing Nod factors. The Mu-lacZ insertion was translationally fused to a putative new gene, designated nodR, in the nodE-nodL intergenic region; however, no predicted function for the putative NodR protein has been obtained from database homology searches. In a set of 12 wild-type strains of R. leguminosarum by. trifolii originating from various geographical regions that were analyzed for the presence of a nodR-like gene, it was found that seven strains carry a homologous NodR open reading frame. Taken together, our results suggest a tightly controlled regulation of nod genes, in which we propose that it is the balance of transcriptional levels of nodFE and the nodRL genes that is critical for determining the presence of highly unsaturated fatty acyl moieties in the Nod factors produced by R. leguminosarum bv. trifolii.