A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae.

Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism.

Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid.

We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp. Strains and in Agrobacterium tumefaciens. Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nif region (Kondorosi, A., E. Kondorosi, C.E. Pankhurst, W. J. Broughton, and Z. Banfalvi, 1982, Mol. Gen. Genet., 188:433-439). In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa. These structures were largely composed of meristematic cells but they were not invaded by bacteria. Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures. The donor strain of R. meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M. sativa that did not fix nitrogen (Fix-). In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably. Any bacteroids formed degenerated prematurely. In some cases, however, reversion to a Fix+ phenotype occurred after 4 to 6 wk. Bacteria released into newly infected cells in these nodules showed normal development into bacteriods.

Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes.

Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc., into two Nod- Rhizobium mutants as well as into Agrobacterium tumefaciens. Subsequently, cosmid clones of pMPIK3030a were mobilized into Nod+ Rhizobium that cannot nodulate the chosen hosts. Nodule development was monitored by examining the ultrastructure of nodules formed by the transconjugants. pMPIK3030a could complement Nod- and Nif- deletions in R. leguminosarum and R. meliloti as well as enable A. tumefaciens to nodulate. Three non-overlapping sets of cosmids were found that conferred upon a slow-growing Rhizobium species, as well as on R. loti and R. meliloti, the ability to nodulate Psophocarpus and Vigna, thus pointing to the existence of three sets of host-specificity genes. Recipients harboring these hsn regions had truly broadened host-range since they could nodulate both their original hosts as well as MPIK3030 hosts.

Molecular basis of symbiotic promiscuity.

Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the "keys" to a succession of legume "doors". Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys.

Early legume responses to inoculation with Rhizobium sp. NGR234.

Interactions between legumes and rhizobia are controlled by the sequential exchange of symbiotic signals. Two different techniques, 2D-PAGE electrophoresis and differential display were used to study the effects of rhizobial signals on legume development. Application of variously substituted lipo-oligo-saccharidic Nod-factors to roots of Vigna unguiculata resulted in changes in the phosphorylation patterns of microsomal proteins. Reliable amino-acid sequences were obtained for one Nod-factor enhanced protein which was highly homologous to the 57-kDa subunit from Arabidopsis thaliana vacuolar membrane H(+)-ATPase. Immuno-blotting techniques demonstrated that Nod-factors cause rapid and massive increases of this enzyme in treated roots, suggesting that H(+)-ATPases play symbiotic roles. Concomitantly, we used differential display (DD) techniques on mRNA isolated from root-hairs to analyse early root responses to NGR234. Significant matches of several DD clones to known sequences were found. Clone D2.62 was homologous to a multitude of receptor kinases including S receptor-like kinases of A. thaliana and clone D4.1 showed similarities to Lotus japonicus phosphatidylinositol transfer-like protein III and late nodulin 16. Independent confirmatory analyses of these differentially expressed clones indicated expression at very low levels.

Genealogy of legume-Rhizobium symbioses.

Accumulating evidence suggests that lateral transfer of nodulation capacity is an important driving force in symbiotic evolution. As a consequence, many distantly related soil bacteria have acquired the capacity to invade plants and fix nitrogen within them. In addition to these proteins required for bacteroid development and nitrogen fixation, core symbiotic competence seems to require flavonoids, NodD proteins, lipochitooligosaccharidic Nod-factors, extra-cellular polysaccharides, as well as various exported proteins. Plants respond to different levels and combinations of these substances in species specific ways. After contact has been initiated by flavonoids and NodD proteins, constant signal exchange fine-tunes these symbiotic demands, especially to overcome defence reactions.

Rhizobium type III secretion systems: legume charmers or alarmers?

Mutagenesis and sequence analyses of rhizobial genomes have revealed the presence of genes encoding type III secretion systems. Considered as a machine used by plant and animal pathogens to deliver virulence factors into their hosts, this secretion apparatus has recently been proven to play a role in symbiotic bacteria-leguminous plant interactions.

Amino acid composition and relationships of lupin and serradella leghaemoglobins.

The amino acid compositions of two lupin and two serradella leghaemoglobins were determined by conventional techniques. They are as follows: Lupin component I: Ala20, Arg1, (Asp + Asn)17, (Glu + Gln)17, Gly9, His4, Ile9, Leu15, Lys17, Met1, Phe8, Pro5, Ser12, Thr9, Trp3, Tyr2 and Val17. Lupin component II: Ala26, Arg1, (Asp + Asn)18, (Glu + Gln)20, Gly9, His5, Ile10, Leu16, Lys18, Met1, Phe8, Pro6, Ser12, Thr10, Trp3, Tyr2 and Val18. Serradella component I: Ala32, Arg1, (Asp + Asn)10, (Glu + Gln)18, Gly8, His3, Ile5, Leu5, Lys13, Phe8, Pro4, Ser14, Thr7, Try2, Tyr3 and Val12. Serradella component II: Ala37, Arg1, (Asp + Asn)11, (Glu + Gln)20, Gly9, His3, Ile6, Leu17, Lys15, Phe9, Pro5, Ser16, Thr8, Try2, Tyr3 and Val13. Notable features of these are (a) the absence of cyst(e)ine, which suggests that each is a single polypeptide chain (b) the presence of a methionine residue in the lupin leghaemoglobins, and (c) the high contents of alanine, aspartic acid plus asparagine, glutamic acid plus glutamine, leucine, lysine, serine and valine. The relationships of leghaemoglobins to each other and to other groups of haem-containing proteins have been investigated using a statistical procedure based on amino acid composition.

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans.

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.

Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges.

Genetically, Rhizobium sp. strain NGR234 and R. fredii USDA257 are closely related. Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257. What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains. The two bacteria nodulated P. andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested. Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae. Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae. On Vigna spp. (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the "cowpea" (V. unguiculata) miscellany. USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera). If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234. The only exceptions were with Apios americana, Glycine max, and G. soja. Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes. Relationships between the ability to nodulate and the origin of the host were not apparent. As both P. andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward.

Induction of chalcone synthase expression by rhizobia and nod factors in root hairs and roots.

Chalcone synthase (CHS) of Vigna unguiculata is encoded by a gene family that is abundantly transcribed in leaves and nodules. Inoculation with Rhizobium sp. NGR234, which nodulates V. unguiculata, or with NGR delta nodABC, a mutant deficient in Nod factor production, induced rapid accumulation of CHS mRNAs in roots and root hairs. As both Nod+ and Nod- bacteria provoke responses, induction of CHS gene expression may involve symbiotic or defense responses. Four days after inoculation with the wild-type Rhizobium sp., the transcript levels increased in roots but decreased in root hairs. Use of a region unique to the 5' end of a specific CHS gene (VuCHS1) showed that increases of transcript levels in root hairs 24 h after inoculation with both rhizobia were specific to this gene. Transcripts of this gene in roots were only detectable 4 days after treatment with NGR234. It is possible therefore that accumulation of VuCHS1 follows the infection pathway of rhizobia entering legume roots. Purified Nod factors induced accumulation of transcripts, showing that they might be part of the signal transduction pathway leading to CHS expression.

Rhizobia modulate root-hair-specific expression of extensin genes.

Three cDNAs (ext3, ext127, and ext26), originally isolated by differential screening from a root-hair cDNA library of Vigna unguiculata, were found to encode extensin-like cell wall proteins. Transcripts homologous to these cDNAs were only detected in root hairs where mRNA levels decreased 1 day after inoculation with rhizobia. This coincided with the onset of root-hair deformation, the first morphological step in the Rhizobium-legume interaction. Decreases in transcript levels following inoculation with wild-type Rhizobium sp. NGR234 were more pronounced than with NGR delta nodABC, a mutant deficient in Nod-factor production. Inoculation with a rhizobial strain carrying a mutation in a gene encoding a transcriptional activator for nod genes (NGR delta nodD1) did not repress mRNA levels, indicating that a second nodulation signal may be present that is nodD dependent. Application of purified NodNGR factors only affected transcript levels of ext3. The genomic locus of the gene homologous to ext26 (Ext26G) was cloned. In the 5' flanking region, several potential TATA boxes and CAP signals were identified. Part of the promoter region shares homology with the Pisum sativum seed lectin promoter and the Nicotiana tabacum nitrate reductase promoter region. Nonetheless, the function of these homologous regions in gene regulation is unknown.

nodSU, two new nod genes of the broad host range Rhizobium strain NGR234 encode host-specific nodulation of the tropical tree Leucaena leucocephala.

Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii. Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a. Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata. In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V. unguiculata. Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene. Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M. Göttfert, S. Hitz, and H. Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990). We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS.

In vitro sulfotransferase activity of NoeE, a nodulation protein of Rhizobium sp. NGR234.

Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium liberate morphogenetic lipochitin-oligosaccharides (Nod factors) into legume rhizospheres. Nod factors, which are synthesized by the products of rhizobial nodulation (nod) genes, vary in core length as well as in the number and type of substitutions. In Rhizobium sp. NGR234, the N-acylated pentamers of N-acetyl-D-glucosamine carry an O-methylfucose group on the reducing terminus that is substituted, on a mutually exclusive basis, with either an acetyl or a sulfuryl group. A sulfotransferase encoded by noeE is required for adjunction of activated sulfate donated by 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Here we show that when expressed in NGR234 cured of its symbiotic plasmid (= ANU265) or when purified as a fusion protein (MBP-NoeE), NoeE transfers sulfate from PAPS to fucosylated lipochitin-oligosaccharides. Enzyme assays showed that sulfotransferase activity is dependent on the presence of an acyl group (stearic and vaccenic acids were tested) since no activity was detected when fucosylated oligochitins (oligomers of two to six N-acetyl-D-glucosamine units) were used as substrates. Thus, NoeE is unique in that it is the only characterized sulfotransferase that is specific for fucosylated Nod factors. It probably acts after NodA, which acylates the amino-sugar backbone.

Biological activity of Rhizobium sp. NGR234 Nod-factors on Macroptilium atropurpureum.

The broad host range of Rhizobium sp. NGR234 is based mainly on its ability to secrete a family of lipooligosaccharide Nod factors. To monitor Nod-factor purification, we used the small seeded legume Macroptilium atropurpureum, which responds evenly and consistently to Nod factors. At concentrations between approximately equal to 10(-11) M and 10(-9) M, this response takes the form of deformation of the root hairs. Higher concentrations (approximately equal to 10(-9) to 10(-7) M), provoked profound "shepherd's crook" type curling of the root hairs. Similar concentrations of Nod factors of Bradyrhizobium japonicum, Rhizobium leguminosarum, and R. meliloti also provoked marked curling of the root hairs, but the latter two species are unable to nodulate Macroptilium. On the other hand, plant hormones, hormone-like substances, inhibitors of hormone action, as well as substituents of Nod factors were without effect in this bioassay. We thus conclude that only Nod factors are capable of inducing shepherd's crook type curling of Macroptilium root hairs. Perturbations in the auxin-cytokinin balance induced "pseudo" nodulation on M. atropurpureum, as did NodNGR factors at concentrations between 10(-7) and 10(-6) M. Concomitant inoculation of Macroptilium with a NodABC- mutant of NGR234 and sulfated NodNGR factors (NodNGR[S]) gave rise to plants that slowly greened, showing that the NodNGR factors permitted entry of the Nod- mutant into the roots.

Accumulation of transcripts encoding a lipid transfer-like protein during deformation of nodulation-competent Vigna unguiculata root hairs.

A cDNA library was constructed from RNA of Vigna unguiculata root hairs harvested 1 day and 4 days after inoculation with Rhizobium sp. NGR234. A heterologous probe was used to identify a cDNA clone, the predicted 99-amino-acid sequence of which shares homology with a nonspecific lipid transfer protein (LTP) of Hordeum vulgare. Other characteristics, including an estimated molecular weight of 10.4 kD, an isoelectric point of 8.6, and a signal peptide with a hydrophobic region at the amino-terminal end, are shared by most LTPs. A transcript of 630 nt was found in all tissues tested, except nodules. Levels of mRNA increased in root hairs 24 hr after treatment with Rhizobium sp. NGR234, with different hormones, or with Nod factors. Amounts of transcripts were dependent on the concentration of Nod factors. Accumulation of transcripts during nodule development correlated with root hair deformation, the first visible step in the Rhizobium-legume symbiosis.