The nolL gene from Rhizobium etli determines nodulation efficiency by mediating the acetylation of the fucosyl residue in the nodulation factor

The nodulation factors (Nod factors) of Rhizobium etli and R. loti carry a 4-O-acetyl-L-fucosyl group at the reducing end. It has been claimed, based on sequence analysis, that NolL from R. loti participates in the 4-O-acetylation of the fucosyl residue of the Nod factors, as an acetyl-transferase (D. B. Scott, C. A. Young, J. M. Collins-Emerson, E. A. Terzaghi, E. S. Rockman, P. A. Lewis, and C. E. Pankhurst. Mol. Plant-Microbe Interact. 9:187-197, 1996). Further support for this hypothesis was obtained by studying the production of Nod factors in an R. etli nolL::Km mutant. Chromatographic and mass spectrometry analysis of the Nod factors produced by this strain showed that they lack the acetyl-fucosyl substituent, having a fucosyl group instead. Acetyl-fucosylation was restored upon complementation with a wild-type nolL gene. These results indicate that the nolL gene determines 4-O-acetylation of the fucosyl residue in Nod factors. Analysis of the predicted NolL polypeptide suggests a transmembranal location and that it belongs to the family of integral membrane transacylases (J. M. Slauch, A. A. Lee, M. J. Mahan, and J. J. Mekalanos. J. Bacteriol. 178:5904-5909, 1996). NolL from R. loti was also proposed to function as a transporter; our results show that NolL does not determine a differential secretion of Nod factors from the cell. We also performed plant assays that indicate that acetylation of the fucose conditions efficient nodulation by R. etli of some Phaseolus vulgaris cultivars, as well as of an alternate host (Vigna umbellata).     

A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum

Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizobium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-alpha-(2-aminoethoxyvinyl) glycine (AVG).     

Rhizobial NodL O-acetyl transferase and NodS N-methyl transferase functionally interfere in production of modified Nod factors

The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an O-acetyl group on C-6 of the nonreducing N-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the nodL gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains of Mesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of the N-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference between nodL and nodS, we have cloned the nodS gene of M. loti and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti involves the nodulation factor-dependent induction of root hairs

In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule organogenesis. However, diverse mechanisms of nodulation have been described in a variety of legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, we have isolated and performed a detailed analysis of four independent L. japonicus root hair developmental mutants. We show that although important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule primordia (NP) organogenesis and the colonization of the nodule structures. In the genetic background of the L. japonicus root hairless 1 mutant, the nodulation factor-dependent formation of NP provides the structural basis for alternative modes of invasion by M. loti. Surprisingly, one mode of root colonization involves nodulation factor-dependent induction of NP-associated cortical root hairs and epidermal root hairs, which, in turn, support bacterial invasion. In addition, entry of M. loti through cracks at the cortical surface of the NP is described. These novel mechanisms of nodule colonization by M. loti explain the fully functional, albeit significantly delayed, nodulation phenotype of the L. japonicus ROOT HAIRLESS mutant.     

慢生根瘤菌属结瘤基因的进化及遗传分析

根瘤菌中存在一系列控制固氮结瘤因子(lipo-chito-oligosaccharides)合成的结瘤基因(nodulation genes)。其中, nodA基因是合成结瘤因子所必需的, 该基因负责酰基转移酶的合成, 能将不饱和脂肪酸转移到结瘤因子寡聚糖骨架上; 基因nodZ, nolL和noeI为宿主专一性结瘤基因, 分别转录合成岩藻糖基转移酶, 岩藻糖乙酰化酶和岩藻糖甲基化酶。通过GenBank调取慢生根瘤菌属及其他根瘤菌属的结瘤基因nodA, nodZ, nolL和noeI, 构建系统发育树, 进行进化和遗传分析。结果表明, 慢生根瘤菌属各个菌株的nodA, nodZ, nolL和noeI具有很高的相关性, 但是与根据保守基因16S rDNA和dnaK分类情况不完全相符。这表明慢生根瘤菌属的结瘤基因主要是通过直系遗传的, 同时可能为适应宿主及环境条件, 结瘤基因有少量的平行转移。结果表明, 慢生根瘤菌属各个菌株的nodA, nodZ, nolL和noeI具有很高的同源性, 同时发现基于保守基因16S rDNA和dnaK对慢生根瘤菌的分类情况与慢生根瘤菌属各菌株在nodA, nodZ, nolL和noeI具有较高同源性的事实不完全相符。这表明慢生根瘤菌属的结瘤基因主要是通过直系遗传的, 同时可能为适应宿主及环境条件, 结瘤基因有少量的平行转移。     

Salicylic acid inhibits indeterminate-type nodulation but not determinate-type nodulation

LCOs (lipochitin oligosaccharides, Nod factors) produced by the rhizobial symbiote of Vicia sativa subsp. nigra (vetch, an indeterminate-type nodulating plant) are mitogenic when carrying an 18:4 acyl chain but not when carrying an 18:1 acyl chain. This suggests that the 18:4 acyl chain specifically contributes to signaling in indeterminate-type nodulation. In a working hypothesis, we speculated that the 18:4 acyl chain is involved in oxylipin signaling comparable to, for example, signaling by derivatives of the 18:3 fatty acid linolenic acid (the octadecanoid pathway). Because salicylic acid (SA) is known to interfere with oxylipin signaling, we tested whether nodulation of vetch could be affected by addition of 10(-4) M SA. This concentration completely blocked nodulation of vetch by Rhizobium leguminosarum bv. viciae and inhibited the mitogenic effect of 18:4 LCOs but did not affect LCO-induced root-hair deformation. SA did not act systemically, and only biologically active SA derivatives were capable of inhibiting nodule formation. SA also inhibited R. leguminosarum bv. viciae association with vetch roots. In contrast, addition of SA to Lotus japonicus (a determinate-type nodulating plant responding to 18:1 LCOs) did not inhibit nodulation by Mesorhizobium loti. Other indeterminate-type nodulating plants showed the same inhibiting response toward SA, whereas SA did not inhibit the nodulation of other determinate-type nodulating plants. SA may be a useful tool for studying fundamental differences between signal transduction pathways of indeterminate- and determinate-type nodulating plants.     

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 reducing-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 biovar (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 unguiculata 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.     

Phylogenetic analyses of symbiotic nodulation genes support vertical and lateral gene co-transfer within the Bradyrhizobium genus

Symbiotic nitrogen fixing bacteria-known as rhizobia-harbour a set of nodulation (nod) genes that control the synthesis of modified lipo-chitooligosaccharides, called Nod factors that are required for legume nodulation. The nodA gene, which is essential for symbiosis, is responsible for the attachment of the fatty acid group to the oligosaccharide backbone. The nodZ, nolL, and noeI genes are involved in specific modifications of Nod factors common to bradyrhizobia, i.e., the transfer of a fucosyl group on the Nod factor core, fucose acetylation and fucose methylation, respectively. PCR amplification, sequencing and phylogenetic analysis of nodA gene sequences from a collection of diverse Bradyrhizobium strains revealed the monophyletic character with the possible exception of photosynthetic Bradyrhizobium, despite high sequence diversity. The distribution of the nodZ, nolL, and noeI genes in the studied strains, as assessed by gene amplification, hybridization or sequencing, was found to correlate with the nodA tree topology. Moreover, the nodA, nodZ, and noeI phylogenies were largely congruent, but did not closely follow the taxonomy of the strains shown by the housekeeping 16S rRNA and dnaK genes. Additionally, the distribution of nodZ, noeI, and nolL genes suggested that their presence may be related to the requirements of their legume hosts. These data indicated that the spread and maintenance of nodulation genes within the Bradyrhizobium genus occurred through vertical transmission, although lateral gene transfer also played a significant role.     

Cell biological changes of outer cortical root cells in early determinate nodulation.

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylene-biosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.     

Aberrant nodulation response of Vigna umbellata to a Bradyrhizobium japonicum NodZ mutant and nodulation signals.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.     

Rapid colorimetric quantification of lipo-chitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti

Nod factors are lipids with a chitinlike headgroup produced by gram-negative Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are essential signaling molecules for accomplishing symbiosis between the bacteria and roots of legume plants. Despite their important role in the Rhizobium-legume interaction, no fast and sensitive Nod factor quantification methods exist. Here, we report two different quantification methods. The first is based on the enzymatic hydrolysis of Nod factors to release N-acetylglucosamine (GlcNAc), which can subsequently be quantified. It is shown that the degrading enzyme, glusulase, releases exactly two GlcNAc units per pentameric nodulation factor from Mesorhizobium loti factor, allowing quantification of LCOs from Mesorhizobium loti. The second method is based on a specific type of Nod factors that are sulfated on the reducing GlcNAc, allowing quantification analogous to the quantification of sulfolipids. Here, a two-phase extraction method is used in the presence of methylene blue, which specifically forms an ion pair with sulfated lipids. The blue ion pair partitions into the organic phase, after which the methylene blue signal can be quantified. To enable Nod factor quantification with this method, the organic phase was modified and the partitioning was evaluated using fluorescent and radiolabeled sulfated Nod factors. It is shown that sulfated LCOs can be quantified with this method, using sodium dodecyl sulfate for calibration. Both methods allow Nod factor quantification in parallel enabling a fast and easy detection of nanomole quantities of Nod factors. Accurate Nod factor quantification will be crucial for characterization and cross-comparison of the affinity for Nod factors of newly identified Nod factor binding proteins or putative Nod factor receptors.     

Engineered ACC deaminase-expressing free-living cells of Mesorhizobium loti show increased nodulation efficiency and competitiveness on Lotus spp

Ethylene inhibits the establishment of symbiosis between rhizobia and legumes. Several rhizobia species express the enzyme ACC deaminase, which degrades the ethylene precursor 1-cyclopropane-1-carboxilate (ACC), leading to reductions in the amount of ethylene evolved by the plant. M. loti has a gene encoding ACC deaminase, but this gene is under the activity of the NifA-RpoN-dependent promoter; thus, it is only expressed inside the nodule. The M. loti structural gene ACC deaminase (acdS) was integrated into the M. loti chromosome under a constitutive promoter activity. The resulting strain induced the formation of a higher number of nodules and was more competitive than the wild-type strain on Lotus japonicus and L. tenuis. These results suggest that the introduction of the ACC deaminase activity within M. loti in a constitutive way could be a novel strategy to increase nodulation competitiveness of the bacteria, which could be useful for the forage inoculants industry.     

Transgenic expression of the soybean apyrase in Lotus japonicus enhances nodulation

The soybean apyrase, GS52, was previously characterized as an early nodulin that is expressed in roots and localized to the plasma membrane. Transgenic Lotus japonicus plants were constructed constitutively expressing the GS52 apyrase. Segregation and Southern-blot analysis identified four single-copy sense lines, several double-copy sense lines, and one double-copy antisense line for further analysis. The single- and double-copy sense gs52 L. japonicus lines had enhanced nodulation that correlated with expression of the transgene. The sense transgenic lines were also found to have increased infection thread formation and enhanced infection zone length when infected by Mesorhizobium loti, the natural symbiont of L. japonicus. The data presented show that expression of the GS52 apyrase can enhance nodulation in L. japonicus and points to an important role for this group of enzymes in nodulation.     

Cloning, functional expression and characterization of Mesorhizobium loti arylamine N-acetyltransferases: rhizobial symbiosis supplies leguminous plants with the xenobiotic N-acetylation pathway

Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes involved in the detoxification of numerous aromatic chemicals. The NAT-dependent N-acetylation pathway has not previously been detected in plants. We demonstrate here the occurrence of the NAT-dependent pathway in leguminous plants, due to symbiosis with Mesorhizobium loti. We cloned two NAT enzymes from M. loti and showed that these two recombinant enzymes catalysed the N-acetylation of several known NAT substrates, including aniline-derived pesticide residues. We also demonstrate the existence of a functional NAT-dependent acetylation pathway in the root nodules of Lotus japonicus inoculated with M. loti. M. loti is the first non-eukaryotic organism shown to express two catalytically active NAT isoforms. This work also provides the first evidence for acquisition of a xenobiotic detoxification pathway by a plant through symbiosis with a soil microbe.     

Rhizobium nodulation protein NodC is an important determinant of chitin oligosaccharide chain length in Nod factor biosynthesis.

Synthesis of chitin oligosaccharides by NodC is the first committed step in the biosynthesis of rhizobial lipochitin oligosaccharides (LCOs). The distribution of oligosaccharide chain lengths in LCOs differs between various Rhizobium species. We expressed the cloned nodC genes of Rhizobium meliloti, R. leguminosarum bv. viciae, and R. loti in Escherichia coli. The in vivo activities of the various NodC proteins differed with respect to the length of the major chitin oligosaccharide produced. The clearest difference was observed between strains with R. meliloti and R. loti NodC, producing chitintetraose and chitinpentaose, respectively. In vitro experiments, using UDP-[14C]GlcNAc as a precursor, show that this difference reflects intrinsic properties of these NodC proteins and that it is not influenced by the UDP-GlcNAc concentration. Analysis of oligosaccharide chain lengths in LCOs produced by a R. leguminosarum bv. viciae nodC mutant, expressing the three cloned nodC genes mentioned above, shows that the difference in oligosaccharide chain length in LCOs of R. meliloti and R. leguminosarum bv. viciae is due only to nodC. The exclusive production of LCOs which contain a chitinpentaose backbone by R. loti strains is not due to NodC but to end product selection by Nod proteins involved in further modification of the chitin oligosaccharide. These results indicate that nodC contributes to the host specificity of R. meliloti, a conclusion consistent with the results of several studies which have shown that the lengths of the oligosaccharide backbones of LCOs can strongly influence their activities on host plants.     

Dual effect of Mesorhizobium loti T3SS functionality on the symbiotic process

Mesorhizobium loti MAFF303099 has a functional type III secretory system (T3SS) involved in the nodulation process on Lotus tenuis and Lotus japonicus. Four putative M.?loti T3SS effectors (Mlr6358, Mlr6331, Mlr6361, and Mlr6316) have been previously described, and it has been demonstrated that the N-terminal regions of Mlr6361 and Mlr6358 mediate the secretion via a T3SS. Here, we demonstrate the capacity of Mlr6316 and Mlr6331 N-terminal regions to direct the secretion of a translational fusion to a reporter peptide through T3SS. By using single, double, and triple mutants, we demonstrated the positive and negative participation of some of these proteins in the determination of competitiveness on Lotus spp. Low competitiveness values correlated with low nodulation efficiency for a mutant deficient in three of the putative M.?loti effectors. Our data suggest that the net effect of M.?loti T3SS function on symbiotic process with Lotus results from a balance between positive and negative effects.     

Nitric oxide production induced in roots of Lotus japonicus by lipopolysaccharide from Mesorhizobium loti

Lipopolysaccharide (LPS) is a bacterial molecule that induces nitric oxide (NO) production and triggers defense systems in plant-pathogen interactions. NO production is induced in the roots of Lotus japonicus after inoculation of the roots with its microsymbiont Mesorhizobium loti. However, the rhizobial molecule that induces NO production has not yet been identified. We investigated NO production in the roots of L. japonicus by treatment with LPS of M. loti. LPS was prepared by phenol-hot water extraction and separated into several fractions: polysaccharide, lipooligosaccharide, oligosaccharide and lipid A. In the roots of L. japonicus, NO production was observed with an NO-specific fluorescent dye 4, 10 and 24 h after treatment with each fraction of LPS. NO production was detected 4 h after treatment with all fractions. NO production was also detectable 24 h after treatment, except after treatment with the polysaccharide and oligosaccharide fractions. Expression of a class 1 hemoglobin gene and application of an NO scavenger showed that the treatment with LPS and LOS induced a similar response to inoculation with M. loti. These data suggest that LPS of M. loti induces NO production after inoculation with M. loti.     

Mesorhizobium loti produces nodPQ-dependent sulfated cell surface polysaccharides

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.