Foliar Application of Rhizobium leguminosarum bv. viciae Strain 33504-Borg201 Promotes Faba Bean Growth and Enhances Systemic Resistance Against Bean Yellow Mosaic Virus Infection.

The bean yellow mosaic virus (BYMV) is one of the most serious economic diseases affecting faba bean crop production. Rhizobium spp., well known for its high nitrogen fixation capacity in legumes, has received little study as a possible biocontrol agent and antiviral. Under greenhouse conditions, foliar application of molecularly characterized Rhizobium leguminosarum bv. viciae strain 33504-Borg201 to the faba bean leaves 24 h before they were infected with BYMV made them much more resistant to the disease while also lowering its severity and accumulation. Furthermore, the treatment promoted plant growth and health, as evidenced by the increased total chlorophyll (32.75 mg/g f.wt.) and protein content (14.39 mg/g f.wt.), as well as the improved fresh and dry weights of the plants. The protective effects of 33504-Borg201 greatly lowered the levels of hydrogen peroxide (H2O2) (4.92 µmol/g f.wt.) and malondialdehyde (MDA) (173.72 µmol/g f.wt.). The antioxidant enzymes peroxidase (1.58 µM/g f.wt.) and polyphenol oxidase (0.57 µM/g f.wt.) inhibited the development of BYMV in plants treated with 33504-Borg201. Gene expression analysis showed that faba bean plants treated with 33504-Borg201 had higher amounts of pathogenesis-related protein-1 (PR-1) (3.28-fold) and hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (4.13-fold) than control plants. These findings demonstrate the potential of 33,504-Borg201 as a cost-effective and eco-friendly method to protect faba bean plants against BYMV. Implementing this approach could help develop a simple and sustainable strategy for protecting faba bean crops from the devastating effects of BYMV.

Role of a receptor-like kinase K1 in pea Rhizobium symbiosis development.

MAIN CONCLUSION: The LysM receptor-like kinase K1 is involved in regulation of pea-rhizobial symbiosis development. The ability of the crop legume Pisum sativum L. to perceive the Nod factor rhizobial signals may depend on several receptors that differ in ligand structure specificity. Identification of pea mutants defective in two types of LysM receptor-like kinases (LysM-RLKs), SYM10 and SYM37, featuring different phenotypic manifestations and impaired at various stages of symbiosis development, corresponds well to this assumption. There is evidence that one of the receptor proteins involved in symbiosis initiation, SYM10, has an inactive kinase domain. This implies the presence of an additional component in the receptor complex, together with SYM10, that remains unknown. Here, we describe a new LysM-RLK, K1, which may serve as an additional component of the receptor complex in pea. To verify the function of K1 in symbiosis, several P. sativum non-nodulating mutants in the k1 gene were identified using the TILLING approach. Phenotyping revealed the blocking of symbiosis development at an appropriately early stage, strongly suggesting the importance of LysM-RLK K1 for symbiosis initiation. Moreover, the analysis of pea mutants with weaker phenotypes provides evidence for the additional role of K1 in infection thread distribution in the cortex and rhizobia penetration. The interaction between K1 and SYM10 was detected using transient leaf expression in Nicotiana benthamiana and in the yeast two-hybrid system. Since the possibility of SYM10/SYM37 complex formation was also shown, we tested whether the SYM37 and K1 receptors are functionally interchangeable using a complementation test. The interaction between K1 and other receptors is discussed.

Bacteria-zinc co-localization implicates enhanced synthesis of cysteine-rich peptides in zinc detoxification when Brassica juncea is inoculated with Rhizobium leguminosarum.

Some plant growth promoting bacteria (PGPB) are enigmatic in enhancing plant growth in the face of increased metal accumulation in plants. Since most PGPB colonize the plant root epidermis, we hypothesized that PGPB confer tolerance to metals through changes in speciation at the root epidermis. We employed a novel combination of fluorophore-based confocal laser scanning microscopic imaging and synchrotron based microscopic X-ray fluorescence mapping with X-ray absorption spectroscopy to characterize bacterial localization, zinc (Zn) distribution and speciation in the roots of Brassica juncea grown in Zn contaminated media (400 mg kg(-1) Zn) with the endophytic Pseudomonas brassicacearum and rhizospheric Rhizobium leguminosarum. PGPB enhanced epidermal Zn sequestration relative to PGBP-free controls while the extent of endophytic accumulation depended on the colonization mode of each PGBP. Increased root accumulation of Zn and increased tolerance to Zn was associated predominantly with R. leguminosarum and was likely due to the coordination of Zn with cysteine-rich peptides in the root endodermis, suggesting enhanced synthesis of phytochelatins or glutathione. Our mechanistic model of enhanced Zn accumulation and detoxification in plants inoculated with R. leguminosarum has particular relevance to PGPB enhanced phytoremediation of soils contaminated through mining and oxidation of sulphur-bearing Zn minerals or engineered nanomaterials such as ZnS.

[Effect of N-phenyl-2-naphthylamine on activity of adenylate cyclase signal system components and virulence of bacterial phytopathogens and mutualists].

The effect of N-phenyl-2-naphthylamine, negative allelochemical isolated from the exudates of roots of pea (Pisum sativum L.), on the growth and activity of the adenylate cyclase signal system and virulence factors of the bacteria Rhizobium leguminosarum bv. viciae and Pseudomonas siringae pv. pisi was studied. It was demonstrated that N-phenyl-2-naphthylamine at a physiological concentration nonspecifically inhibited the growth of these bacteria in both planktonic cultures and biofilms. One of the reasons for this phenomenon is the reduction of intra- and extracellular concentrations of cAMP due to greater activation of phosphodiesterase, which disrupts cAMP, in comparison to soluble adenylyl cyclase, which synthesizes it. At the same time, N-phenyl-2-naphthylamine did not affect activity of either membrane-bound adenylyl cyclase or bacterial virulence factors.

Mechanisms behind bacteria induced plant growth promotion and Zn accumulation in Brassica juncea.

The growth and metal-extraction efficiency of plants exposed to toxic metals has been reported to be enhanced by inoculating plants with certain bacteria but the mechanisms behind this process remain unclear. We report results from glasshouse experiments on Brassica juncea plants exposed to 400mgZnkg(-1) that investigated the abilities of Pseudomonas brassicacearum and Rhizobium leguminosarum to promote growth, coupled with synchrotron based µXANES analysis to probe Zn speciation in the plant roots. P. brassicacearum exhibited the poorest plant growth promoting ability, while R. leguminosarum alone and in combination with P. brassicacearum enhanced plant growth and Zn phytoextraction. Reduced growth in un-inoculated plants was attributed to accumulation of Zn oxalate and Zn sulfate in roots. In plants inoculated with P. brassicacearum the high concentration of Zn polygalacturonic acid in the root may be responsible for the stunted growth and reduced Zn phytoextraction. The improved growth and increased metal accumulation observed in plants inoculated with R. leguminosarum and in combination with P. brassicacearum was attributed to the storage of Zn in the form of Zn phytate and Zn cysteine in the root. When combined with the observation that both bacteria do not statistically improve B. juncea growth in the absence of Zn, this work suggests that bacteria-induced metal chelation is the key mechanism of plant growth promoting bacteria in toxicity attenuation and microbial-assisted phytoremediation.

Characterization of a gene family of outer membrane proteins (ropB) in Rhizobium leguminosarum bv. viciae VF39SM and the role of the sensor kinase ChvG in their regulation.

The outer membrane of Gram-negative bacteria represents the interface between the bacterium and its external environment. It has a critical role as a protective barrier against harmful substances and is also important in host-bacteria interactions representing the initial physical point of contact between the host cell and bacterial cell. RopB is a previously identified outer membrane protein from Rhizobium leguminosarum bv. viciae that is present in free-living cells but absent in bacteroids (H. P. Roest, I. H. Mulders, C. A. Wijffelman, and B. J. Lugtenberg, Mol. Plant Microbe Interact. 8:576-583, 1995). The functions of RopB and the molecular mechanisms of ropB gene regulation have remained unknown. We identified and cloned ropB and two homologs (ropB2 and ropB3) from the R. leguminosarum VF39SM genome. Reporter gene fusions indicated that the expression of ropB was 8-fold higher when cells were grown in complex media than when they were grown in minimal media, while ropB3 expression was constitutively expressed at low levels in both complex and minimal media. Expression of ropB2 was negligible under all conditions tested. The use of minimal media supplemented with various sources of peptides resulted in a 5-fold increase in ropB expression. An increase in ropB expression in the presence of peptides was not observed in a chvG mutant background, indicating a role for the sensor kinase in regulating ropB expression. Each member of the ropB gene family was mutated using insertional mutagenesis, and the mutants were assayed for susceptibility to antimicrobial agents and symbiotic phenotypes. All mutants formed effective nodules on pea plants, and gene expression for each rop gene in bacteroids was negligible. The functions of ropB2 and ropB3 remain cryptic, while the ropB mutant had an increased sensitivity to detergents, hydrophobic antibiotics, and weak organic acids, suggesting a role for RopB in outer membrane stability.

Rhizobium leguminosarum hupE encodes a nickel transporter required for hydrogenase activity.

Synthesis of the hydrogen uptake (Hup) system in Rhizobium leguminosarum bv. viciae requires the function of an 18-gene cluster (hupSLCDEFGHIJK-hypABFCDEX). Among them, the hupE gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of hupE2, a hydrogenase-unlinked gene identified in the R. leguminosarum genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the Escherichia coli nikABCDE mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX(5)DH and FHGX[AV]HGXE) that are required for HupE functionality. An R. leguminosarum double mutant, SPF22A (hupE hupE2), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (Lens culinaris L.) root nodules but not in pea (Pisum sativum L.) bacteroids. Moreover, heterologous expression of the R. leguminosarum hup system in bacteroid cells of Rhizobium tropici and Mesorhizobium loti displayed reduced levels of hydrogen uptake in the absence of hupE. These data support the role of R. leguminosarum HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions.

[Chemotaxis of Rhizobium leguminosarum bv. viciae to organic substances].

The chemotaxis of nodule bacteria cultures of pea Rhizobium leguminosarum bv. vicia RRL1, RRL3, RRL6 i RRL7 isolated from the plants of Ivano-Frankivsk district to different organic substances was investigated. Saccharose, pyruvate and tartaric acid were the most active attractants among carbohydrates and organic acids studied for all used nodule bacteria strains. Serine, alanine, glycine, tyrosine and cysteine were the most active attractants of amino acids. Dependence of pea nodule bacteria chemotaxic activity on time of growth and attractant concentrations was revealed. The isolate specificity towards organic substances for different strains of Rhizobium leguminosarum bv. vicia was found.

The cin and rai quorum-sensing regulatory systems in Rhizobium leguminosarum are coordinated by ExpR and CinS, a small regulatory protein coexpressed with CinI.

To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.

[A study of the competitive properties of the aluminum-tolerant strain Rhizobium leguminosarum bv. trifolii 9-4A by the antibiotic resistance method].

The virulence, competitive ability, and symbiotic efficiency of 2 Rhizobium leguminosarum bv. trifolii strains--the wild aluminum tolerant strain 9-4A and the commercial strain 348a-were compared when introducing their variants marked with antibiotic resistance into the rhizosphere of red clover (Trifolium pratense L.) plants. High virulence and competitive ability of the strain tolerant to aluminum was demonstrated by a concurrent inoculation of the seeds with these two strains. The resistance acquisition by the commercial strain was accompanied by a decrease in its symbiotic efficiency. Presumably, the resistant variant of aluminum-tolerant isolate retains its symbiotic properties due to its adaptation to acidity factors at the level of membrane function.

Mutagenesis of the carboxy terminal protease CtpA decreases desiccation tolerance in Rhizobium leguminosarum.

To better understand the role of proteases in Rhizobium leguminosarum biovar viciae, a gene with homology to the carboxy-terminal protease (CtpA), which belongs to a novel group of serine proteases, was studied. The ctpA gene was cloned and mutated using allelic exchange and a gusA reporter gene was used to study ctpA expression. Mutational analysis shows that ctpA is critical for the viability of R. leguminosarum when cells are grown on complex semi-solid media but is dispensable when cells are grown in complex liquid media and that this is likely due to an increase in susceptibility to desiccation on semi-solid media. The ctpA mutant also displayed an increased sensitivity to detergents, indicating an alteration in the permeability of the cell envelope. This is the first characterization of a ctpA gene within the Rhizobiaceae and the first report of a ctpA mutant that exhibits an increased sensitivity to desiccation.

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants.

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.

Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae.

Twenty-seven new Rhizobium isolates were obtained from root nodules of wild and crop legumes belonging to the genera Vicia, Lathyrus and Pisum from different agroecological areas in central and southern Italy. A polyphasic approach including phenotypic and genotypic techniques was used to study their diversity and their relationships with other biovars and species of rhizobia. Analysis of symbiotic properties and stress tolerance tests revealed that wild isolates showed a wide spectrum of nodulation and a marked variation in stress tolerance compared with reference strains tested in this study. All rhizobial isolates (except for the isolate CG4 from Galega officinalis) were presumptively identified as Rhizobium leguminosarum biovar viciae both by their symbiotic properties and the specific amplification of the nodC gene. In particular, we found that the nodC gene could be used as a diagnostic molecular marker for strains belonging to the bv. viciae. RFLP-PCR 16S rDNA analysis confirms these results, with the exception of two strains that showed different RFLP-genotypes from those of the reference strains of R. leguminosarum bv. viciae. Analysis of intraspecies relationship among strains by using the RAPD-PCR technique showed a high level of genetic polymorphism, grouping our isolates and reference strains into six different major clusters with a similarity level of 20%. Data from seven parameters of phenotypic and genotypic analyses were evaluated by using principal component analysis which indicated the differences among strains and allowed them to be divided into seven different groups.

Utilization of heterologous siderophores enhances levels of iron available to Pseudomonas putida in the rhizosphere.

Pseudomonas spp. have the capacity to utilize siderophores produced by diverse species of bacteria and fungi, and the present study was initiated to determine if siderophores produced by rhizosphere microorganisms enhance the levels of iron available to a strain of Pseudomonas putida in this natural habitat. We used a previously described transcriptional fusion (pvd-inaZ) between an iron-regulated promoter (pvd) and the ice nucleation reporter gene (inaZ) to detect alterations in iron availability to P. putida. Ice nucleation activity (INA) expressed from the pvd-inaZ fusion by P. putida N1R or N1R Pvd(-), a derivative deficient in the production of a pyoverdine siderophore, was inversely related to the concentration of ferric citrate in a culture medium. In culture, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of the ferric complex of pseudobactin-358, a pyoverdine siderophore produced by P. putida WCS358 that can be utilized as a source of iron by N1R Pvd(-). In the rhizosphere of cucumbers grown in sterilized soil, N1R Pvd(-) (pvd-inaZ) expressed INA, indicating that iron availability was sufficiently low in that habitat to allow transcription of the iron-regulated pvd promoter. Coinoculation with WCS358 or N1R significantly decreased INA expressed by N1R Pvd(-) (pvd-inaZ) in the rhizosphere, whereas coinoculation with a pyoverdine-deficient mutant of WCS358 did not reduce INA expressed by N1R Pvd(-) (pvd-inaZ). These results indicate that iron availability to N1R Pvd(-) (pvd-inaZ) in the rhizosphere was enhanced by the presence of another strain of P. putida that produces a pyoverdine that N1R Pvd(-) (pvd-inaZ) was able to utilize as a source of iron. In culture, strain N1R Pvd(-) also utilized ferric complexes of the siderophores enterobactin and aerobactin as sources of iron. In the rhizosphere of cucumbers grown in sterilized soil, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of strains of Enterobacter cloacae that produced enterobactin, aerobactin, or both siderophores, but INA expressed by N1R Pvd(-) (pvd-inaZ) was not altered in the presence of a mutant of E. cloacae deficient in both enterobactin and aerobactin production. Therefore, the iron status of P. putida was altered by siderophores produced by an unrelated bacterium coinhabiting the rhizosphere. Finally, we demonstrated that INA expressed by N1R containing pvd-inaZ in the rhizosphere differed between plants grown in sterilized versus nonsterilized field soil. The results of this study demonstrate that (i) P. putida expresses genes for pyoverdine production and uptake in the rhizosphere, but the level of gene expression is influenced by other bacteria that coexist with P. putida in this habitat, and (ii) diverse groups of microorganisms can alter the availability of chemical resources in microbial habitats on root surfaces.

Feasibility of using prokaryote biosensors to assess acute toxicity of polycyclic aromatic hydrocarbons.

The aim of this study was to assess the acute toxicity of polycyclic aromatic hydrocarbons using lux-marked bacterial biosensors. Standard solutions of phenanthrene, pyrene and benzo[a]pyrene were produced using 50 mM hydroxpropyl-beta-cyclodextrin solution which contained each respective polycyclic aromatic hydrocarbon at 6.25 times the aqueous solubility limit of the compound. The polycyclic aromatic hydrocarbon solutions were incubated with each of the biosensors for 280 min and the bioluminescence monitored every 20 min. Over the incubation time period, there was no significant decrease in bioluminescence in any of the biosensors tested with the exception of Rhizobium leguminosarum biovar trifolii TA1 luxAB. In this series of incubations, there was a dramatic increase in bioluminescence in the presence of phenanthrene (2.5 times) and benzo[a]pyrene (3 times) above that of the background control (biosensor without polycyclic aromatic hydrocarbon) after 20 min. Over the next 3 h, bioluminescence decreased to that of the control. An ATP assay was carried out on the biosensors to assess if uncoupling of the oxidative phosphorylation mechanisms in the respiratory chain of the cells had occurred. However, it was found that the polycyclic aromatic hydrocarbons had no effect on the organisms indicating that there was no uncoupling. Additionally, mineralisation studies using 14C-labelled polycyclic aromatic hydrocarbons showed that the biosensors could not mineralise the compounds. This study has shown that the three polycyclic aromatic hydrocarbons tested are not acutely toxic to the prokaryotic biosensors tested, although acute toxicity has been shown in other bioassays. These results question the rationale for using prokaryote biosensors to assess the toxicity of hydrophobic chemicals, such as polycyclic aromatic hydrocarbons.

Effectes of high temperature on survival, symbiotic performance and genomic modifications of bean nodulating Rhizobium strains

High temperatures can affect the survival, establishment and symbiotic properties of "Rhizobium" strains. Bean nodulating "Rhizobium" strains are considered particularly sensitive because on this strains genetic recombinations and/or deletions occur frequently, thus compromising the use of these bacteria as inoculants. In this study "R. tropici" and "R. leguminosarum" bv. "phaseoli" strains isolated from Cerrado soils were exposed to thermal stress and the strains' growth, survival and symbiotic relationships as well as alterations in their genotypic and phenotypic were analysed. After successive thermal shocks at 45ºC for four hours, survival capacity appeared to be strain-specifc, independent of thermo-tolerance and was more apparent in "R. tropici" strains (with the exception of FJ2.21) were more stable than "R. leguminosarum" bv. "phaseoli" strains because no significant phenotypic alterations were observed following thermal treatments and they maintained their original genotypic pattern after innoculation in plants.

Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction.

Nod factors secreted by Rhizobium leguminosarum bv. viciae induce root hair deformation, involving a reinitiation of tip growth, and the formation of nodule primordia in Vicia sativa (vetch). Ethylene is a potent inhibitor of cortical cell division, an effect that can be counteracted by applying silver ions (Ag+) or aminoethoxy-vinylglycine (AVG). In contrast to the inhibitory effect on cortical cell division, ethylene promotes the formation of root hairs (which involves tip growth) in the root epidermis of Arabidopsis. We investigate the possible paradox concerning the action of ethylene, putatively promoting Nod factor induced tip growth whilst, at the same time, inhibiting cortical cell division. We show, by using the ethylene inhibitors AVG and Ag+, that ethylene has no role in the reinitiation of root hair tip growth induced by Nod factors (root hair deformation) in vetch. However, root hair formation is controlled, at least in part, by ethylene. Furthermore, we show that ACC oxidase, which catalizes the last step in ethylene biosynthesis, is expressed in the cell layers opposite the phloem in that part of the root where nodule primordia are induced upon inoculation with Rhizobium. Therefore, we test whether endogenously produced ethylene provides positional information controlling the site where nodule primordia are formed by determining the position of nodules formed on pea roots grown in the presence of AVG or Ag+.

Expression of cysteine protease genes in pea nodule development and senescence.

Coding sequences for two cysteine proteases were amplified from cDNA derived from pea nodule mRNA using primers based on conserved regions of plant cysteine proteases. One of the amplified cDNA sequences corresponded to a previously described cysteine protease gene, Cyp15a, expressed in pea shoots in response to dehydration (J.T. Jones and J.E. Mullet, Plant Mol. Biol. 28:1055-1065, 1995). Inside the pea root nodule, in situ hybridization revealed that this gene is expressed strongly in the apical region and more weakly in the uninfected cortex and in the central infected tissue where nitrogen fixation takes place. The complete sequence of the cDNA corresponding to the other gene, PsCyp1, was obtained. Expression of this gene, which was studied both on RNA blots and in situ, showed good correlation with the onset of nodule senescence. In situ hybridization studies revealed that PsCyp1 was expressed in senescent infected tissue at the base of the nodule. This signal was just detectable in normal symbiotically wild-type nodules but was much stronger in the early senescing nodules formed by a symbiotically defective mutant of Rhizobium leguminosarum.

Rhizobium leguminosarum as a plant growth-promoting rhizobacterium: direct growth promotion of canola and lettuce.

Early seedling root growth of the nonlegumes canola (Brassica campestris cv. Tobin, Brassica napus cv. Westar) and lettuce (Lactuca sativa cv. Grand Rapids) was significantly promoted by inoculation of seeds with certain strains of Rhizobium leguminosarum, including nitrogen- and nonnitrogen-fixing derivatives under gnotobiotic conditions. The growth-promotive effect appears to be direct, with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin. Auxotrophic Rhizobium mutants requiring tryptophan or adenosine (precursors for indole-3-acetic acid and demonstrate a new facet of the Rhizobium-plant relationship and that Rhizobium leguminosarum can be considered a plant growth-promoting rhizobacterium (PGPR).

Nod factors produced by Rhizobium leguminosarum biovar viciae induce ethylene-related changes in root cortical cells of Vicia sativa ssp. nigra.

Vicia sativa ssp. nigra plants develop the "Thick short root" (Tsr) phenotype when both (i) the roots are inoculated with the root nodule inducing bacterium Rhizobium leguminosarum biovar viciae, and (ii) the plants, including the roots, are grown in the light. Tsr roots have a reduced length, are locally twice as thick as normal roots and have an increased number of root hairs. Development of the Tsr phenotype is correlated with the presence of nod (nodulation) genes in the rhizobia. Nod factors (lipochitin oligosaccharides), products of these nod genes, can induce the Tsr phenotype in the absence of rhizobia. The Tsr phenotype can be mimicked by addition of the ethylene-releasing compound ethephon. Using several microscopical techniques, we compared roots showing the Tsr phenotype (Tsr roots) with normal roots and roots grown in the presence of the ethylene inhibitor aminoethoxyvinylglycine (AVG). The thickening of Tsr roots appeared to be caused by a swelling of the cortical cells, which corresponded with (i) a reorientation of the interphase cortical microtubules from a transverse to a longitudinal direction, (ii) general cell wall modifications, (iii) frequent absence of middle lamellae, and (iv) local maceration. The same changes could be induced by ethephon and were inhibited by AVG. This strongly suggests that the Tsr phenotype is caused by excessive ethylene production. The ethylene-related changes mentioned above are also seen during infection thread formation, but only very locally. Apparently, Vicia roots when grown in the light overrespond to Nod factors leading to overproduction of ethylene and to a non-local "ripening" process. These phenomena inhibit nodulation of the main root by preventing formation of pre-infected threads and by reducing formation of root nodule primordia. Local controlled production of ethylene, as induced by Nod factors, may, however, be an essential element of the nodulation process.