The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.     

Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis

In the biosynthesis of lipochitin oligosaccharides (LCOs) theRhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence ofnodA in thenodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which thenodA, nodB andnodC genes are separately present on different plasmids. Efficient nodulation was only obtained ifnodC was present on a low-copy-number vector. Our results confirm the notion thatnodA ofRhizobium leguminosarum biovarviciae is essential for nodulation onVicia. Surprisingly, replacement ofR. l. bv.viciae nodA by that ofBradyrhizobium sp. ANU289 results in a nodulation-minus phenotype onVicia. Further analysis revealed that theBradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of theR. l. bv.viciae nodF E-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion thatnodA is a commonnod gene should be revised.     

Rhizobium NodI and NodJ proteins play a role in the efficiency of secretion of lipochitin oligosaccharides.

Thin-layer chromatographic analysis of extracts of D-[1-14C]glucosamine-labelled rhizobia was used to analyze the effects of nodI, nodJ, and nodT on secretion of lipochitin oligosaccharide (LCO) signal molecules. Secretion was analyzed by comparing quantities of radiolabelled LCOs present in the cellular and spent growth medium fractions. A second rapid and sensitive method was introduced to estimate the secreted LCO fractions by using D-[1-14C]glucosamine-labelled cells grown in medium supplemented with chitinase. At various times after induction of LCO synthesis, the quantity of degradation products of LCOs was compared with the amount of nondegraded LCOs. In wild-type strains of Rhizobium leguminosarum biovars viciae and trifolii the nodI and nodJ genes (but not the nodT gene) strongly enhance the secretion of LCOs during the first 5 h after the induction of LCO synthesis. In LCO-overproducing strains the enhancement of secretion was observed only during the first 3 h after induction. At times later than 5 h after induction, a significant influence of the presence of the nodI and nodJ genes on LCO secretion was detectable neither in the wild type nor in LCO-overproducing strains. By using plasmids in which the nodI and nodJ genes are cloned separately under control of a flavonoid-inducible promoter, it was shown that both genes are needed for a wild-type level of LCO secretion. Therefore, these results demonstrate that nodI and nodJ play a role in determining the efficiency of LCO secretion.     

Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.     

Structural motifs in the RNA encoded by the early nodulation gene enod40 of soybean

The plant gene enod40 is highly conserved among legumes and also present in various non-legume species. It is presumed to play a central regulatory role in the Rhizobium–legume interaction, being expressed well before the initiation of cortical cell divisions resulting in nodule formation. Two small peptides encoded by enod40 mRNA as well as its secondary structure have been shown to be key elements in the signalling processes underlying nodule organogenesis. Here results concerning the secondary structure of mRNA of enod40 in soybean are presented. This study combined a theoretical approach, involving structure prediction and comparison, as well as structure probing. Our study indicates five conserved domains in enod40 mRNA among numerous leguminous species. Structure comparison suggests that some domains are also conserved in non-leguminous species and that an additional domain exists that was found only in leguminous species developing indeterminate nodules. Enzymatic and chemical probing data support the structure for three of the domains, and partially for the remaining two. The rest of the molecule appears to be less structured. Some of the domains include motifs, such as U-containing internal loops and bulges, which seem to be conserved. Therefore, they might be involved in the regulatory role of enod40 RNA.     

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.