Nodulation Host Range Research Articles

One door closes, another opens: when nodulation impairment with natural hosts extends rhizobial host-range.

The rhizobium-legume symbiosis is the best-understood plant-microbe association. The high degree of specificity observed in this relationship is supported by a complex exchange of signals between the two components of the symbiosis. Findings reported in last years indicate that multiple molecular mechanisms, such as the production of a particular set of nodulation factors at a very specific concentration or a suitable arsenal of effectors secreted through the type III secretion system, have been adjusted during evolution to ensure and optimize the recognition of specific rhizobial strains by its legume host. Qualitative or quantitative changes in the production of these symbiotic molecular determinants are detrimental for nodulation with its natural host but, in some cases, can also result beneficial for the rhizobium since it extends the nodulation host-range to other legumes. Potential repercussion of the extension in the nodulation host-range of rhizobia is discussed.

Symbiosis islands of Loteae-nodulating Mesorhizobium comprise three radiating lineages with concordant nod gene complements and nodulation host-range groupings.

Mesorhizobium is a genus of soil bacteria, some isolates of which form an endosymbiotic relationship with diverse legumes of the Loteae tribe. The symbiotic genes of these mesorhizobia are generally carried on integrative and conjugative elements termed symbiosis islands (ICESyms). Mesorhizobium strains that nodulate Lotus spp. have been divided into host-range groupings. Group I (GI) strains nodulate L. corniculatus and L. japonicus ecotype Gifu, while group II (GII) strains have a broader host range, which includes L. pedunculatus. To identify the basis of this extended host range, and better understand Mesorhizobium and ICESym genomics, the genomes of eight Mesorhizobium strains were completed using hybrid long- and short-read assembly. Bioinformatic comparison with previously sequenced mesorhizobia genomes indicated host range was not predicted by Mesorhizobium genospecies but rather by the evolutionary relationship between ICESym symbiotic regions. Three radiating lineages of Loteae ICESyms were identified on this basis, which correlate with Lotus spp. host-range grouping and have lineage-specific nod gene complements. Pangenomic analysis of the completed GI and GII ICESyms identified 155 core genes (on average 30.1 % of a given ICESym). Individual GI or GII ICESyms carried diverse accessory genes with an average of 34.6 % of genes unique to a given ICESym. Identification and comparative analysis of NodD symbiotic regulatory motifs – nod boxes – identified 21 branches across the NodD regulons. Four of these branches were associated with seven genes unique to the five GII ICESyms. The nod boxes preceding the host-range gene nodZ in GI and GII ICESyms were disparate, suggesting regulation of nodZ may differ between GI and GII ICESyms. The broad host-range determinant(s) of GII ICESyms that confer nodulation of L. pedunculatus are likely present amongst the 53 GII-unique genes identified.

Impact of a G2-EPSPS & GAT Dual Transgenic Glyphosate-Resistant Soybean Line on the Soil Microbial Community under Field Conditions Affected by Glyphosate Application.

In the past thirty years, the biosafety of the aboveground part of crops, including horizontal gene transferal through pollen dispersal and hybridization, has been the focus of research; however, microbial communities in the underground part are attracting increasing attention. In the present study, the soybean root-associated bacterial communities of the G2-EPSPS plus GAT transgenic soybean line Z106, its recipient variety ZH10, and Z106 treated with glyphosate (Z106J) were compared at the seedling, flowering, and seed filling stages by high-throughput sequencing of the V4 hypervariable regions of 16S rRNA gene amplicons using Illumina MiSeq. The results obtained showed no significant differences in the alpha/beta diversities of root-associated bacterial communities at the three stages among ZH10, Z106, and Z106J under field growth conditions; however, the relative abundance of four main nitrogen-fixing bacterial genera significantly differed among ZH10, Z106, and Z106J. Ternary plot results indicated that in the root compartment, the proportional contributions of rhizobial nitrogen-fixing Ensifer fredii and Bradyrhizobium elkanii, which exhibit an extremely broad nodulation host range, markedly differed among the three treatments at the three stages. Thus, the present results indicate that transgenic G2-EPSPS and GAT soybean may induce different changes in functional bacterial species in soil, such as E. fredii and B. elkanii, from ZH10, which were compensated for/enriched at the flowering and seed filling stages, respectively, to some extent through as of yet unknown mechanisms by transgenic soybean treated with glyphosate.

Bradyrhizobium niftali sp. nov., an effective nitrogen-fixing symbiont of partridge pea [Chamaecrista fasciculata (Michx.) Greene], a native caesalpinioid legume broadly distributed in the USA.

Information about the symbionts of legumes of the Caesalpinioideae subfamily is still limited, and we performed a polyphasic approach with three Bradyrhizobium strains-CNPSo 3448T, CNPSo 3394 and CNPSo 3442-isolated from Chamaecrista fasciculata, a native legume broadly distributed in the USA. In the phylogenetic analysis of both the 16S rRNA gene and the intergenic transcribed spacer, the CNPSo strains were clustered within the Bradyrhizobium japonicumsuperclade. Multilocus sequence analysis with six housekeeping genes-glnII, gyrB, recA, rpoB, atpD and dnaK-indicated that Bradyrhizobium diazoefficiens is the closest species, with 83 % of nucleotide identity. In the genome analyses of CNPSo 3448T, average nucleotide identity and digital DNA-DNA hybridization results confirmed higher similarity with B. diazoefficiens, with values estimated of 93.35 and 51.50 %, respectively, both below the threshold of the same species, confirming that the CNPSo strains represent a new lineage. BOX-PCR profiles indicated high intraspecific genetic diversity between the CNPSo strains. In the analyses of the symbiotic genes nodC and nifH the CNPSo strains were clustered with Bradyrhizobium arachidis, Bradyrhizobium forestalis, Bradyrhizobium cajani, Bradyrhizobium kavangense and Bradyrhizobium vignae, indicating a different phylogenetic history compared to the conserved core genes. Other physiological (C utilization, tolerance to antibiotics and abiotic stresses), chemical (fatty acid profile) and symbiotic (nodulation host range) properties were evaluated and are described. The data from our study support the description of the CNPSo strains as the novel species Bradyrhizobiumniftali sp. nov., with CNPSo 3448T (=USDA 10051T=U687T=CL 40T) designated as the type strain.

The Sinorhizobium fredii HH103 MucR1 Global Regulator Is Connected With the nod Regulon and Is Required for Efficient Symbiosis With Lotus burttii and Glycine max cv. Williams.

Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.

The Role of Flavonoids in Nodulation Host-Range Specificity: An Update.

Flavonoids are crucial signaling molecules in the symbiosis between legumes and their nitrogen-fixing symbionts, the rhizobia. The primary function of flavonoids in the interaction is to induce transcription of the genes for biosynthesis of the rhizobial signaling molecules called Nod factors, which are perceived by the plant to allow symbiotic infection of the root. Many legumes produce specific flavonoids that only induce Nod factor production in homologous rhizobia, and therefore act as important determinants of host range. Despite a wealth of evidence on legume flavonoids, relatively few have proven roles in rhizobial infection. Recent studies suggest that production of key “infection” flavonoids is highly localized at infection sites. Furthermore, some of the flavonoids being produced at infection sites are phytoalexins and may have a role in the selection of compatible symbionts during infection. The molecular details of how flavonoid production in plants is regulated during nodulation have not yet been clarified, but nitrogen availability has been shown to play a role.

Bacterial Molecular Signals in the Sinorhizobium fredii-Soybean Symbiosis.

Sinorhizobium (Ensifer) fredii (S. fredii) is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides), and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system.

Genome sequence of Ensifer medicae strain WSM1115; an acid-tolerant Medicago-nodulating microsymbiont from Samothraki, Greece.

Ensifer medicae strain WSM1115 forms effective nitrogen fixing symbioses with a range of annual Medicago species and is used in commercial inoculants in Australia. WSM1115 is an aerobic, motile, Gram-negative, non-spore-forming rod. It was isolated from a nodule recovered from the root of burr medic (Medicago polymorpha) collected on the Greek Island of Samothraki. WSM1115 has a broad host range for nodulation and N2 fixation capacity within the genus Medicago, although this does not extend to all medic species. WSM1115 is considered saprophytically competent in moderately acid soils (pH(CaCl2) 5.0), but it has failed to persist at field sites where soil salinity exceeded 10 ECe (dS/m). Here we describe the features of E. medicae strain WSM1115, together with genome sequence information and its annotation. The 6,861,065 bp high-quality-draft genome is arranged into 7 scaffolds of 28 contigs, contains 6,789 protein-coding genes and 83 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI565.

Rhizobium leguminosarum bv. trifolii SRDI565 (syn. N8-J) is an aerobic, motile, Gram-negative, non-spore-forming rod. SRDI565 was isolated from a nodule recovered from the roots of the annual clover Trifolium subterraneum subsp. subterraneum grown in the greenhouse and inoculated with soil collected from New South Wales, Australia. SRDI565 has a broad host range for nodulation within the clover genus, however N2-fixation is sub-optimal with some Trifolium species and ineffective with others. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI565, together with genome sequence information and annotation. The 6,905,599 bp high-quality-draft genome is arranged into 7 scaffolds of 7 contigs, contains 6,750 protein-coding genes and 86 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Isolation and characterization of alfalfa-nodulating rhizobia present in acidic soils of central argentina and uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809-1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.

NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals.

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2-O-methylfucose residue at the reducting-terminal N-acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum). Here we show that transfer of nodZ to Rhizobium leguminosarum blovar (bv) viciae, which produces LCOs that are not modified at the reducing-terminal N-acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N-acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum, Glycine soja, Vigna unguiculate and Leucaena leucocephala. Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.

Diversity of Rhizobia Nodulating Phaseolus vulgaris L. in Two Kenyan Soils with Contrasting pHs

Rhizobia were isolated from two Kenyan soils with pHs of 4.5 and 6.8 and characterized on the basis of their host ranges for nodulation and nitrogen fixation, colony morphologies, restriction fragment fingerprints, and hybridization with a nifH probe. The populations of rhizobia nodulating Phaseolus vulgaris in the two soils were similar in numbers and in effectiveness of N(inf2) fixation but were markedly different in composition. The population in the Naivasha soil (pH 6.8) was dominated by isolates specific in host range for nodulation to P. vulgaris; these all had multiple copies, in most cases four, of the structural nitrogenase gene nifH. Only one of the isolates from this soil formed effective nodules on Leucaena leucocephala, and this isolate had only a single copy of nifH. By contrast, the population in the acid Daka-ini soil (pH 4.5) was composed largely of broad-host-range isolates which had single copies of nifH. The isolates from the Daka-ini soil which were specific to P. vulgaris generally had three copies of nifH, although one isolate had only two copies. These rhizobial isolates are indigenous to Kenyan soils and yet have marked similarities to previously described Rhizobium species from other continents.

A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functional analysis of nodE genes was constructed. By testing chimeric nodE genes constructed by the exchange of polymerase chain reaction (PCR)-generated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues). Mass spectrometric analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R. leguminosarum bv. viciae. The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C18:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively. Therefore, the main difference between the nodE-determined LCOs of biovar viciae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly developed spot application assay, we show that the C18:2- and C18:3-containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results, we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the polyunsaturated fatty acyl moieties of their LCOs, which is mediated by the host-specific central domain of the NodE protein.

Host range, RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10 R. fredii strains with Rhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. The R. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated by R. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and common nod genes, nodDABC; the soybean cultivar specificity gene, nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence from R. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes, nolBTUVWX, was monomorphic. Using antisera against whole cells of three R. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a single R. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences among R. fredii strains were as great as those between NGR234 and R. fredii, our results confirm that NGR234 has a distinctly wider host range than R. fredii.

Lipo-oligosaccharide nodulation factors: A new class of signaling molecules mediating recognition and morphogenesis

Jean Dbnari6 and Julie Cullimore Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes CNRS-INRA, 31326 Castanet-Tolosan Cedex France The legume-rhizobia symbiosis is estimated to fix per an- num as much nitrogen as the fertilizer industry and is of great agronomic and ecological importance. This efficient nitrogen-fixing association occurs as a result of the forma- tion of a new specialized organ, the root nodule, which is induced by the prokaryotic symbiont on its specific plant partner (see Nap and Bisseling, 1990). The rhizobial genes that determine the host recognition and nodulation have recently been found to specify the synthesis of excreted lipo-oligosaccharide signals (Figure 1) which are capable of eliciting, at extremely low concentrations, many of the plant responses characteristic of the bacteria themselves. These responses include the initiation of cell division, in- duction of specific changes in cell morphology, and trig- gering of a plant organogenic program leading to the for- mation of the nodule. This review focuses on the role of this new class of signaling molecules in host-partner rec- ognition and nodule development in the legume-rhizobia symbiosis. Rhizobia, Prokaryotes that Elicit a Specific Plant Organogenesis Rhizobia (now classified into three genera, Rhizobium, Sradyrhizobium and Azorhizobium) are soil bacteria that can elicit the formation of nitrogen-fixing nodules on the roots of selected species of the legume family. Some of these bacteria exhibit a narrow host range of nodulation; for example three of the species discussed in this review, R. meliloti, R. leguminosarum biovar viciae and B. japoni- cum nodulate respectively alfalfa, pea/vetch and soybean and not each other’s hosts. In contrast R. sp. NGR234 has a broad host range, nodulating over 70 legume genera and even a non-legume, Parasponia (see Denarie et al., ‘I 992). Symbiotic nodule formation is based on two processes, plant infection and induction of a novel organ (Nap and Bisseling, 1990). To penetrate into their hosts, rhizobia elicit stimulation and reorientations of plant cell wall growth, presumably due to a restructuring of the cytoskele- ton. These changes result in both the entrapment of the bacteria within root hair curls and also the initiation and development of infection threads, tubular structures ,through which bacteria pass on their way down the root hair to the underlying cortical cell layers (Figure 2A). Ahead of the advancing threads a major developmental switch is elicited in the root cortex. Cortical cells are induced to dedifferentiate and divide, and a meristem is then formed, whose functioning generates the nodule. Nodules have a particular ontogeny and anatomy; they are not mere tu- mors but fully differentiated organs divided into a number of cell-types and tissues. Alfalfa nodules for example (Fig- ure 28) contain a distal meristem, an infection zone, a zone where the cells are filled with nitrogen-fixing bacteria, and vascular bundles that ensure metabolic exchanges with the rest of plant. Genetic manipulation of the prokaryotic partner has led to the identification of a set of rhizobial genes (the nod genes) required for infection, nodulation and the control of host specificity (Figure 1). The expression these genes isdependent upon plant signals, usuallyflavonoids, excreted in root exudates. In the presence of appropriate plant inducers, rhizobial NodD regulatory proteins activate the transcription of thestructural nodgenes. Among these, and present in all rhizobia, are common nodA% genes, in which mutations lead to a complete abolition of infection and nodulation. In addition, there are nod genes that are species/strain specific, such as noV De- narie et al., 1992; Fisher and Long, 1992). Nodulation Genes Specify the Production of Lipo-Oligosaccharide Nod Factors What could be the biochemical function of the rhizobial nod gene products? A link between the genetic determi-

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar‐specific nodulation of soybean by Rhizobium fredii USDA257

Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolW and nolX are of the opposite polarity. The initiation codon for nolW lies 155 bp upstream from that of nolB, and its is separated from nolX by 281 bp. The predicted NolT and NolW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudII1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU and nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with nolB-, nolU-, or nolX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU-ORF4-nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Leaf curl syndrome of pigeonpea ( Cajanus cajan (L.) Millsp.) is a systemic response to effective nodulation by the Rhizobium strain IC3342

Rhizobium strain IC3342 is an unusual bacterium that causes a leaf curl syndrome in pigeonpea ( Cajanus cajan Millsp.) Growth characteristics, plasmid profile, conserved nif and nod gene sequences, and nodulation host range of this strain resemble that of the fast-growing Rhizobium strain ANU240 (NGR234). Leaf curl induction occurred only in hosts effectively nodulated by this strain. A plasmid-cured, non-nodulating derivative failed to induce leaf curl symptoms. The strain IC3342 competed poorly with fast- and slow-growing root-nodule bacteria, but the observed nodule occupancy of 10% was enough to produce leaf curl symptoms. Suppression of nodule development by added inorganic nitrogen also prevented symptom expression. Approach grafting of a healthy pigeonpea plant and a plant with leaf curl symptoms resulted in the development of leaf curl symptoms on the growing shoots of the healthy plant within 8 days of graft union. Further symptom expression ceased after graft separation. Feeding xylem sap from the leaf curled plant to a healthy plant induced the initial symptom of the syndrome, bending of the growing leaf tip. We conclude that the leaf curl induction is a systemic response for which effective nodulation is an apparent prerequisite.

Novel organization of the common nodulation genes in Rhizobium leguminosarum bv. phaseoli strains

Nodulation by Rhizobium, Bradyrhizobium, and Azorhizobium species in the roots of legumes and nonlegumes requires the proper expression of plant genes and of both common and specific bacterial nodulation genes. The common nodABC genes form an operon or are physically mapped together in all species studied thus far. Rhizobium leguminosarum bv. phaseoli strains are classified in two groups. The type I group has reiterated nifHDK genes and a narrow host range of nodulation. The type II group has a single copy of the nifHDK genes and a wide host range of nodulation. We have found by genetic and nucleotide sequence analysis that in type I strain CE-3, the functional common nodA gene is separated from the nodBC genes by 20 kb and thus is transcriptionally separated from the latter genes. This novel organization could be the result of a complex rearrangement, as we found zones of identity between the two separated nodA and nodBC regions. Moreover, this novel organization of the common nodABC genes seems to be a general characteristic of R. leguminosarum bv. phaseoli type I strains. Despite the separation, the coordination of the expression of these genes seems not to be altered.

Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein.

The nucleotide sequence of the nodE gene of Rhizobium trifolii strain ANU843 was determined. Like the nodE gene of R. leguminosarum strain 248 it encodes a protein with a predicted mol. wt of 42.0 kd. The predicted NodE proteins of R.trifolii and R.leguminosarum have a homology of 78%. Using antibodies raised against the NodE protein of R.trifolii it was shown that the NodE products of R.leguminosarum and R.trifolii are localized in the cytoplasmic membrane. Furthermore, these NodE proteins are predicted to contain at least two non-polar transbilayer alpha-helices. The nodE genes of R.trifolii and R.leguminosarum were separated from the nodF genes that precede them in the respective nodFE operons. Using the resulting clones, in which NodE was produced under control of the flavonoid-inducible nodABCIJ promoter of R.leguminosarum, it was shown that the NodE product is the main factor that distinguishes the host range of nodulation of R.trifolii and R.leguminosarum. Hybrid nodE genes, which consist of a 5' part of the R.leguminosarum nodE gene and a 3' part of the R.trifolii gene, were constructed. From the properties of these hybrid genes it was concluded that a central region of 185 amino acids at the most, containing only 44 non-identical amino acids, determines the difference in the host-specific characteristics of the two NodE proteins.

Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix phenotype to the Agrobacterium transconjugants. A Fix phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris.