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Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii's capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga's fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide-a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Soil potassium (K) supplement depends intensively on the application of chemical fertilizers, which have substantial harmful environmental effects. However, some bacteria can act as inoculants by converting unavailable and insoluble K forms into plant-accessible forms. Such bacteria are an eco-friendly approach for enhancing plant K absorption and consequently reducing utilization of chemical fertilization. Therefore, the present research was undertaken to isolate, screen, and characterize the K solubilizing bacteria (KSB) from the rhizosphere soils of northern India. Overall, 110 strains were isolated, but only 13 isolates showed significant K solubilizing ability by forming a halo zone on solid media. They were further screened for K solubilizing activity at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C for 5, 10, and 20 days. All the bacterial isolates showed mineral K solubilization activity at these different temperatures. However, the content of K solubilization increased with the upsurge in temperature and period of incubation. The isolate KSB (Grz) showed the highest K solubilization index of 462.28% after 48 h of incubation at 20 °C. The maximum of 23.38 µg K/mL broth was solubilized by the isolate KSB (Grz) at 20 °C after 20 days of incubation. Based on morphological, biochemical, and molecular characterization (through the 16S rDNA approach), the isolate KSB (Grz) was identified as Mesorhizobium sp. The majority of the strains produced HCN and ammonia. The maximum indole acetic acid (IAA) (31.54 µM/mL) and cellulase (390 µM/mL) were produced by the isolate KSB (Grz). In contrast, the highest protease (525.12 µM/mL) and chitinase (5.20 µM/mL) activities were shown by standard strain Bacillus mucilaginosus and KSB (Gmr) isolate, respectively.

Objective: The effects of monosaccharide constituents of lignocellulosic materials on exopolysaccharide (EPS) production by Mesorhizobium sp. Semia 816 were studied., Results: According to the results, by using sugars commonly found in lignocellulosic biomass as carbon sources (glucose, arabinose and xylose), no significant differences were observed in the production of EPS, reaching 3.39 g/L, 3.33 g/L and 3.27 g/L, respectively. Differences were observed in monosaccharide composition, mainly in relation to rhamnose and glucuronic acid contents (1.8 times higher when arabinose was compared with xylose). However, the biopolymers showed no differences in relation to rheological properties, with EPS aqueous-based suspensions (1.0% w/v) presenting pseudoplastic behavior, and a slight difference in degradation temperatures. Using soybean hulls hydrolysate as carbon source, slightly higher values were obtained (3.93 g/L)., Conclusion: The results indicate the potential of the use of lignocellulosic hydrolysates containing these sugars as a source of carbon in the cultivation of Mesorhizobium sp. Semia 816 for the production of EPS with potential industrial applications.

Peroxiredoxins (Prxs) play an essential role in the antioxidant activity and symbiotic capacity of Mesorhizobium huakuii. A mutation in the M. huakuii prxA gene (encoding a Prx5-like peroxiredoxin) was generated by homologous recombination. The mutation of prxA did not affect M. huakuii growth, but the strain displayed decreased antioxidative capacity under organic cumene hydroperoxide (CUOOH) conditions. The higher resistance of the prxA mutant strain compared with the wild-type strain to more than 1 mmol/L H 2 O 2 was associated with a significantly higher level of glutathione reductase activity and a significantly lower level of intracellular hydrogen peroxide content. Real-time quantitative PCR showed that under 1 mmol/L H 2 O 2 conditions, expression of the stress-responsive genes katG and katE was significantly upregulated in the prxA mutant. Although the prxA mutant can form nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 53.25% reduction in nitrogen fixation capacity. This phenotype was linked to an absence of bacteroid differentiation and deregulation of the transcription of the symbiotic genes nifH, nifD, and fdxN. Expression of the prxA gene was induced during symbiosis. Thus, the PrxA protein is essential for antioxidant capacity and symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and cellular antioxidative systems., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Evidences for an involvement of the bacterial type IV secretion system (T4SS) in the symbiotic relationship between rhizobia and legumes have been pointed out by several recent studies. However, information regarding this secretion system in Mesorhizobium is still very scarce. The aim of the present study was to investigate the phylogeny and expression of the traG gene, which encodes a substrate receptor of the T4SS. In addition, the occurrence and genomic context of this and other T4SS genes, namely, genes from tra/trb and virB/virD4 complexes, were also analyzed in order to unveil the structural and functional organization of T4SS in mesorhizobia. The location of the T4SS genes in the symbiotic region of the analyzed rhizobial genomes, along with the traG phylogeny, suggests that T4SS genes could be horizontally transferred together with the symbiosis genes. Regarding the T4SS structural organization in Mesorhizobium , the virB/virD4 genes were absent in all chickpea ( Cicer arietinum L.) microsymbionts and in the Lotus symbiont Mesorhizobium japonicum MAFF303099 T . Interestingly, the presence of genes belonging to another secretion system (T3SS) was restricted to these strains lacking the virB/virD4 genes. The traG gene expression was detected in M. mediterraneum Ca36 T and M. ciceri LMS-1 strains when exposed to chickpea root exudates and also in the early nodules formed by M. mediterraneum Ca36 T , but not in older nodules. This study contributes to a better understanding of the importance of T4SS in mutualistic symbiotic bacteria.

In the present study, a new extracellular polysaccharide (EPS-M816) was obtained during the growth of Mesorhizobium loti Semia 816 on a crude glycerol-based medium. EPS-M816 precipitate mainly consisted of carbohydrates (82.54%) and proteins (11.31%), and the weight average molecular weight was estimated at 1.646 × 10 6  Da. The biopolymer was characterized by FT-IR and NMR spectroscopy, and was found to have typical functional groups of other rhizobial polysaccharides. Furthermore, the rheological and emulsifying properties were investigated. The EPS-M816 solution (1.0% w/v) showed typical pseudoplastic non-Newtonian fluid behavior, and the addition of sodium and potassium chloride (1 mol L -1 ) increased the apparent viscosity. Regarding its emulsification activity, EPS-M816 formed emulsions with different food-grade vegetable oils (soybean, rice, canola, sunflower and corn oils), showing emulsification index values over 65% in 24 h, indicative of strong emulsion-stabilizing capacity. The biopolymer was able to form gels with texture parameters similar to those reported for xanthan gum and low syneresis. Overall, these results suggest that EPS-M816 is a good candidate for application in the food, cosmetics and pharmaceutical industries as a thickening, gelling, stabilizing and emulsifying agent., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A total of 219 endophytic actinobacteria, isolated from roots, stems and leaves of chickpea, were characterized for antagonistic potential against Botrytis cinerea, causal organism of Botrytis grey mold (BGM) disease, in chickpea. Among them, three most potential endophytes, AUR2, AUR4 and ARR4 were further characterized for their plant growth-promoting (PGP) and nodulating potentials and host-plant resistance against B. cinerea, in chickpea. The sequences of 16 S rDNA gene of the three endophytes were matched with Streptomyces but different species. In planta, the isolate AUR4 alone was able to significantly enhance PGP traits including seed numbers (11.8 vs. 9.8/Plant), seed weight (8 vs. 6.8 g/Plant), pod numbers (13.6 vs. 11.5/Plant), pod weight (9.3 vs. 7.5 g/Plant) and biomass (10.9 vs. 8 g/Plant) over the un-inoculated control in chickpea genotype JG11. Interestingly, consortium of the selected endophytes, AUR2, AUR4 and ARR4 were found less effective than single inoculation. Co-inoculation of the selected endophytes with Mesorhizobium ciceri significantly enhanced nodulation and nitrogenase activity in five chickpea genotypes including ICCV2, ICCV10, ICC4958, Annigeri and JG11 over the un-inoculated control. The selected endophytes showed antagonistic potential in planta by significant reduction of disease incidence (28─52%) in both single inoculation and consortium treatments over the un-inoculated control across the genotypes ICC4954 (susceptible), ICCV05530 (moderately resistant) and JG11 (unknown resistance). Further, antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, phenylalanine ammonia-lyase and polyphenol oxidase and phenolics were found induced in the leaves of chickpea inoculated with selected endophytes over un-inoculated control. Principal component analysis revealed that, the antioxidant enzymes and phenolics were found in the magnitude of ICC4954 < JG11 < ICCV05530 which correlates with their resistance level. The selected endophytes enhanced the plant growth and also host plant resistance against BGM in chickpea., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biotic elicitation is an important biotechnological strategy for triggering the accumulation of secondary metabolites in adventitious root cultures. These biotic elicitors can be obtained from safe, economically important strains of bacteria found in the rhizosphere and fermented foods. Here, we assayed the effects of filtered cultures of five nitrogen-fixing bacteria and four types of fermentation bacteria on mutant adventitious Panax ginseng root cultures induced in a previous study by colchicine treatment. The biomass, pH, and electrical conductivity (EC) of the culture medium were altered at 5 days after treatment with bacteria. The saponin content was highest in root cultures treated with Mesorhizobium amorphae (GS3037), with a concentration of 105.58 mg g -1 dry weight saponin present in these cultures versus 74.48 mg g -1 dry weight in untreated root cultures. The accumulation of the ginsenosides Rb 2 and Rb 3 dramatically increased (19.4- and 4.4-fold, and 18.8- and 4.8-fold) 5 days after treatment with M. amorphae (GS3037) and Mesorhizobium amorphae (GS336), respectively. Compound K production increased 1.7-fold after treatment with M. amorphae (GS3037) compared with untreated root cultures. These results suggest that treating mutant adventitious root cultures with biotic elicitors represents an effective strategy for increasing ginsenoside production in Panax ginseng.

The unicellular green alga Lobomonas rostrata requires an external supply of vitamin B 12 (cobalamin) for growth, which it can obtain in stable laboratory cultures from the soil bacterium Mesorhizobium loti in exchange for photosynthate. We investigated changes in protein expression in the alga that allow it to engage in this mutualism. We used quantitative isobaric tagging (iTRAQ) proteomics to determine the L. rostrata proteome grown axenically with B 12 supplementation or in coculture with M. loti. Data are available via ProteomeXchange (PXD005046). Using the related Chlamydomonas reinhardtii as a reference genome, 588 algal proteins could be identified. Enzymes of amino acid biosynthesis were higher in coculture than in axenic culture, and this was reflected in increased amounts of total cellular protein and several free amino acids. A number of heat shock proteins were also elevated. Conversely, photosynthetic proteins and those of chloroplast protein synthesis were significantly lower in L. rostrata cells in coculture. These observations were confirmed by measurement of electron transfer rates in cells grown under the two conditions. The results indicate that, despite the stability of the mutualism, L. rostrata experiences stress in coculture with M. loti, and must adjust its metabolism accordingly., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Rhizobial bacteria are known for their capacity to fix nitrogen for legume hosts. However ineffective rhizobial genotypes exist and can trigger the formation of nodules but fix little if any nitrogen for hosts. Legumes must employ mechanisms to minimize exploitation by the ineffective rhizobial genotypes to limit fitness costs and stabilize the symbiosis. Here we address two key questions about these host mechanisms. What stages of the interaction are controlled by the host, and can hosts detect subtle differences in nitrogen fixation? We provide the first explicit evidence for adaptive host control in the interaction between Lotus japonicus and Mesorhizobium loti. In both single inoculation and co-inoculation experiments, less effective rhizobial strains exhibited reduced in planta fitness relative to the wildtype M. loti. We uncovered evidence of host control during nodule formation and during post-infection proliferation of symbionts within nodules. We found a linear relationship between rhizobial fitness and symbiotic effectiveness. Our results suggest that L. japonicus can adaptively modulate the fitness of symbionts as a continuous response to symbiotic nitrogen fixation.

Endophytic microorganisms asymptomatically colonise plant tissues. Exploring the assembly dynamics of bacterial endophytic communities is essential to understand the functioning of the plant holobiont and to optimise their possible use as biopesticides or plant biostimulants. The variation in endophytic communities in above and below-ground organs in Vitis vinifera in the field were studied. To understand the specific effect of temperature on endophytic communities, a separate experiment was set up where grapevine cuttings were grown under controlled conditions at three different temperatures. The findings revealed the succession of endophytic communities over the year. Endophytic communities of roots and stems differ in terms of composition and dynamic response to temperature. Noticeably, compositional differences during the seasons affected bacterial taxa more in stems than in roots, suggesting that roots offer a more stable and less easily perturbed environment. Correlation abundance networks showed that the presence of several taxa (including Bradyrhizobium, Burkholderia, Dyella, Mesorhizobium, Propionibacterium and Ralstonia) is linked in both the field and the greenhouse., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Objective: We studied the functions and characteristics of hfq gene in Mesorhizobium huakuii 7653R in adverse environment and symbiotic with its host plant., Methods: The hfq mutant of 7653R was constructed via homologous recombination with small cloned fragments on suicide plasmids pK19mob to insert target gene. We applied 7653RΔhfq to characterize stress tolerance and symbiosis with host plant, in comparison with the complementary strains 7653R △hfq-C and the wild type., Results: Mutant 7653RΔhfq presented lower growth rate, and higher mortality after heat shock-pretreated than that of the wild type, as well as the decreasing adaptability under the stress of 4.5% ethanol and 50 mmol H2O2. The defection of hfq affected the expression of some sRNAs in 7653R. Moreover, the mutant displayed significant reduced nodulation ability and nitrogenase activity compared with the wild type., Conclusion: As a crucial post transcriptional regulatory factor, hfq plays an important role in Mesorhizobium Huakuii 7653R on both processes of stress resistance and symbiosis with the host plant Astragalus sinicus L.

Integrative and conjugative elements (ICEs) are ubiquitous mobile genetic elements present as "genomic islands" within bacterial chromosomes. Symbiosis islands are ICEs that convert nonsymbiotic mesorhizobia into symbionts of legumes. Here we report the discovery of symbiosis ICEs that exist as three separate chromosomal regions when integrated in their hosts, but through recombination assemble as a single circular ICE for conjugative transfer. Whole-genome comparisons revealed exconjugants derived from nonsymbiotic mesorhizobia received three separate chromosomal regions from the donor Mesorhizobium ciceri WSM1271. The three regions were each bordered by two nonhomologous integrase attachment (att) sites, which together comprised three homologous pairs of attL and attR sites. Sequential recombination between each attL and attR pair produced corresponding attP and attB sites and joined the three fragments to produce a single circular ICE, ICEMcSym 1271 A plasmid carrying the three attP sites was used to recreate the process of tripartite ICE integration and to confirm the role of integrase genes intS, intM, and intG in this process. Nine additional tripartite ICEs were identified in diverse mesorhizobia and transfer was demonstrated for three of them. The transfer of tripartite ICEs to nonsymbiotic mesorhizobia explains the evolution of competitive but suboptimal N 2 -fixing strains found in Western Australian soils. The unheralded existence of tripartite ICEs raises the possibility that multipartite elements reside in other organisms, but have been overlooked because of their unusual biology. These discoveries reveal mechanisms by which integrases dramatically manipulate bacterial genomes to allow cotransfer of disparate chromosomal regions., Competing Interests: The authors declare no conflict of interest.

A bacterial strain ZY1 capable of utilizing PCBs as its carbon source was isolated from the root nodules of Chinese milk vetch (Astragalus sinicus L.). The strain was identified as Mesorhizobium sp. according to its physiological-biochemical properties and the analysis of its 16S rRNA gene sequence. When the initial OD600 was 0.15, 62.7% of 15 mg L(-1) 3,3',4,4'-TCB in a liquid culture was degraded by Mesorhizobium sp. ZY1 within 10 days. Mesorhizobium sp. ZY1 also greatly increased the biotransformation of soil PCBs. Pot experiments indicated that the soil PCB concentrations of a single incubation of strain ZY1 (R) and a single planting of A. sinicus (P) decreased by 20.5% and 23.0%, respectively, and the concentration of PCBs in soil treated with A. sinicus and strain ZY1 decreased by 53.1%. We also observed that A. sinicus-Mesorhizobium sp. ZY1 treatment (PR) improved plant biomass and the concentration of PCBs in plants compared with a single A. sinicus planting treatment (P). The results suggest that the synergistic association between A. sinicus and PCBs-degrading Mesorhizobium sp. ZY1 can stimulate the phytoextraction of PCBs and the rhizosphere microflora to degrade PCBs, and might be a promising bioremediation strategy for PCB-contaminated soil.

Legumes interact with rhizobial bacteria to form nitrogen-fixing root nodules. Host signalling following mutual recognition ensures a specific response, but is only partially understood. Focusing on the stage of epidermal infection with Mesorhizobium loti, we analysed endogenous small RNAs (sRNAs) of the model legume Lotus japonicus to investigate their involvement in host response regulation. We used Illumina sequencing to annotate the L. japonicus sRNA-ome and isolate infection-responsive sRNAs, followed by candidate-based functional characterization. Sequences from four libraries revealed 219 novel L. japonicus micro RNAs (miRNAs) from 114 newly assigned families, and 76 infection-responsive sRNAs. Unlike infection-associated coding genes such as NODULE INCEPTION (NIN), a micro RNA 172 (miR172) isoform showed strong accumulation in dependency of both Nodulation (Nod) factor and compatible rhizobia. The genetics of miR172 induction support the existence of distinct epidermal and cortical signalling events. MIR172a promoter activity followed a previously unseen pattern preceding infection thread progression in epidermal and cortical cells. Nodule-associated miR172a expression was infection-independent, representing the second of two genetically separable activity waves. The combined data provide a valuable resource for further study, and identify miR172 as an sRNA marking successful epidermal infection. We show that miR172 acts upstream of several APETALA2-type (AP2) transcription factors, and suggest that it has a role in fine-tuning AP2 levels during bacterial symbiosis., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

The importance of protozoa as environmental reservoirs of pathogens is well recognized, while their impact on survival and symbiotic properties of rhizobia has not been explored. The possible survival of free-living rhizobia inside amoebae could influence bacterial abundance in the rhizosphere of legume plants and the nodulation competitiveness of microsymbionts. Two well-characterized strains of Mesorhizobium: Mesorhizobium loti NZP2213 and Mesorhizobium huakuii symbiovar loti MAFF303099 were assayed for their growth ability within the Neff strain of Acanthamoeba castellanii. Although the association ability and the initial uptake rate of both strains were similar, recovery of viable M. huakuii MAFF303099 after 4 h postinfection decreased markedly and that of M. loti NZP2213 increased. The latter strain was also able to survive prolonged co-incubation within amoebae and to self-release from the amoeba cell. The temperature 28 °C and PBS were established as optimal for the uptake of Mesorhizobium by amoebae. The internalization of mesorhizobia was mediated by the mannose-dependent receptor. M. loti NZP2213 bacteria released from amoebae developed 1.5 times more nodules on Lotus corniculatus than bacteria cultivated in an amoebae-free medium.

Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium ciceri are chickpea (Cicer arietinum L.) rhizobia that share a high similarity of the symbiotic genes nodC and nifH, but they have different geographic distributions. M. muleiense has been isolated and found only in alkaline soils of Xinjiang, China, whereas the other two strains have been found in the Mediterranean and India. To investigate the species stability of M. muleiense during natural evolution and its capability of competitive nodulation against the other two exotic species, re-sampling of nodules in the field and competition experiments between the three species were conducted. The results showed that the predominant microsymbiont associated with chickpea grown in Xinjiang was still M. muleiense, but the predominant genotypes of M. muleiense had changed significantly during the four years since a previous survey. The data also showed that M. mediterraneum and M. ciceri were more competitive than the residential strain of M. muleiense CCBAU 83963(T) in sterilized vermiculite or soils from Xinjiang. However, in non-sterilized soils, M. muleiense was the predominant nodule occupier. These results indicated that natural or adapting evolution of M. muleiense was occurring in fields subjected to changing environmental factors. In addition, the biogeography and symbiotic associations of rhizobia with their host legumes were also influenced by biological factors in the soil, such as indigenous rhizobia and other organisms., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.

The present study was carried out with the aim of evaluating the effectiveness and potentiality of three compatible rhizosphere microbes, viz., fluorescent Pseudomonas aeruginosa (PHU094), Trichoderma harzianum (THU0816) and Mesorhizobium sp. (RL091), in promoting plant growth and mobilizing phenolic acid biosynthesis in chickpea under challenge of Sclerotium rolfsii. The microbes were applied as seed coating in different combinations in two experimental sets and the pathogen was inoculated after 25 days of sowing in one set. Results revealed that microbe application led to higher growth in chickpea particularly in the triple microbe combination compared to their individual treatments and control. Similarly, pathogen challenged plants accumulated higher amount of phenolic compounds both at the site of attack of the pathogen i.e. collar region as well as leaves compared to unchallenged plants. All the bioagents were found to trigger the level of phenolic compounds at collar region in varying degrees as compared to the healthy control (A). However, the most effective treatment was D7 (combined application of PHU094, THU0816 and RL091 with pathogen challenge) among all the treatments. Shikimic acid was maximally induced amongst all the phenolic compounds. In leaves also, the most effective treatment was D7 where shikimic acid, t-chlorogenic acid, ferulic acid, myricetin, quercetin and syringic acid were produced in higher amounts as compared to treatment B where the plants were challenged only with the pathogen., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Competitiveness for nodulation is a desirable trait in rhizobia strains used as inoculant. In Sinorhizobium meliloti 1021 mutation in either of the trehalose utilization genes thuA or thuB influences its competitiveness for root colonization and nodule occupancy depending on the interacting host. We have therefore investigated whether mutation in the thuA ortholog in Mesorhizobium loti MAFF303099 also leads to a similar competitive phenotype on its hosts. The results show that M. loti thuA mutant Ml7023 was symbiotically effective and was as competitive as the wild type in colonization and nodule occupancy on Lotus corniculatus and Lotus japonicus. The thuA gene in M. loti was not induced during root colonization or in the infection threads unlike in S. meliloti, despite its induction by trehalose and high osmolarity in in vitro assays.

Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide (EPS) in the formation of determinate nodules, we isolated Mesorhizobium loti mutants affected in various steps of EPS biosynthesis and characterized their symbiotic phenotypes on two Lotus spp. The wild-type M. loti R7A produced both high molecular weight EPS and lower molecular weight (LMW) polysaccharide fractions whereas most mutant strains produced only LMW fractions. Mutants affected in predicted early biosynthetic steps (e.g., exoB) formed nitrogen-fixing nodules on Lotus corniculatus and L. japonicus 'Gifu', whereas mutants affected in mid or late biosynthetic steps (e.g., exoU) induced uninfected nodule primordia and, occasionally, a few infected nodules following a lengthy delay. These mutants were disrupted at the stage of infection thread (IT) development. Symbiotically defective EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority of full-length IT observed harbored only the EPS mutant strain and did not show bacterial release, whereas the nitrogen-fixing nodules contained both mutants. Examination of the symbiotic proficiency of the exoU mutant on various L. japonicus ecotypes revealed that both host and environmental factors were linked to the requirement for EPS. These results reveal a complex function for M. loti EPS in determinate nodule formation and suggest that EPS plays a signaling role at the stages of both IT initiation and bacterial release.

Nodulation in legumes requires the recognition of rhizobially made Nod factors. Genetic studies have revealed that the perception of Nod factors involves LysM domain receptor-like kinases, while biochemical approaches have identified LECTIN NUCLEOTIDE PHOSPHOHYDROLASE (LNP) as a Nod factor-binding protein. Here, we show that antisense inhibition of LNP blocks nodulation in Lotus japonicus. This absence of nodulation was due to a defect in Nod factor signaling based on the observations that the early nodulation gene NODULE INCEPTION was not induced and that both Nod factor-induced perinuclear calcium spiking and calcium influx at the root hair tip were blocked. However, Nod factor did induce root hair deformation in the LNP antisense lines. LNP is also required for infection by the mycorrhizal fungus Glomus intraradices, suggesting that LNP plays a role in the common signaling pathway shared by the rhizobial and mycorrhizal symbioses. Taken together, these observations indicate that LNP acts at a novel position in the early stages of symbiosis signaling. We propose that LNP functions at the earliest stage of the common nodulation and mycorrhization symbiosis signaling pathway downstream of the Nod factor receptors; it may act either by influencing signaling via changes in external nucleotides or in conjunction with the LysM receptor-like kinases for recognition of Nod factor.

Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein-tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.

One of the earliest responses of legumes to symbiotic signalling is oscillation of the calcium concentration in the nucleoplasm of root epidermal cells. Integration and decoding of the calcium-spiking signal involve a calcium- and calmodulin-dependent protein kinase (CCaMK) and its phosphorylation substrates, such as CYCLOPS. Here we describe the Lotus japonicus ccamk-14 mutant that originated from a har1-1 suppressor screen. The ccamk-14 mutation causes a serine to asparagine substitution at position 337 located within the calmodulin binding site, which we determined to be an in vitro phosphorylation site in CCaMK. We show that ccamk-14 exerts cell-specific effects on symbiosis. The mutant is characterized by an increased frequency of epidermal infections and significantly compromised cortical infections by Mesorhizobium loti and also the arbuscular mycorrhiza fungus Rhizophagus irregularis. The S337 residue is conserved across angiosperm CCaMKs, and testing discrete substitutions at this site showed that it participates in a negative regulation of CCaMK activity, which is required for the cell-type-specific integration of symbiotic signalling., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Remodeling of the plant cell cytoskeleton precedes symbiotic entry of nitrogen-fixing bacteria within the host plant roots. Here we identify a Lotus japonicus gene encoding a predicted ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) as essential for rhizobial infection but not for arbuscular mycorrhiza symbiosis. In other organisms ARPC1 constitutes a subunit of the ARP2/3 complex, the major nucleator of Y-branched actin filaments. The L. japonicus arpc1 mutant showed a distorted trichome phenotype and was defective in epidermal infection thread formation, producing mostly empty nodules. A few partially colonized nodules that did form in arpc1 contained abnormal infections. Together with previously described L. japonicus Nck-associated protein1 and 121F-specific p53 inducible RNA mutants, which are also impaired in the accommodation of rhizobia, our data indicate that ARPC1 and, by inference a suppressor of cAMP receptor/WASP-family verpolin homologous protein-ARP2/3 pathway, must have been coopted during evolution of nitrogen-fixing symbiosis to specifically mediate bacterial entry.

Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor (SNARE) proteins are crucial for signal transduction and development in plants. Here, we investigate a Lotus japonicus symbiotic mutant defective in one of the SNARE proteins. When in symbiosis with rhizobia, the growth of the mutant was retarded compared with that of the wild-type plant. Although the mutant formed nodules, these exhibited lower nitrogen fixation activity than the wild type. The rhizobia were able to invade nodule cells, but enlarged symbiosomes were observed in the infected cells. The causal gene, designated LjSYP71 (for L. japonicus syntaxin of plants71), was identified by map-based cloning and shown to encode a Qc-SNARE protein homologous to Arabidopsis (Arabidopsis thaliana) SYP71. LjSYP71 was expressed ubiquitously in shoot, roots, and nodules, and transcripts were detected in the vascular tissues. In the mutant, no other visible defects in plant morphology were observed. Furthermore, in the presence of combined nitrogen, the mutant plant grew almost as well as the wild type. These results suggest that the vascular tissues expressing LjSYP71 play a pivotal role in symbiotic nitrogen fixation in L. japonicus nodules.

The symbiosis receptor kinase (SymRK) is required for morphological changes of legume root hairs triggered by rhizobial infection. How protein turnover of SymRK is regulated and how the nodulation factor signals are transduced downstream of SymRK are not known. In this report, a SymRK-interacting E3 ubiquitin ligase (SIE3) was shown to bind and ubiquitinate SymRK. The SIE3-SymRK interaction and the ubiquitination of SymRK were shown to occur in vitro and in planta. SIE3 represents a new class of plant-specific E3 ligases that contain a unique pattern of the conserved CTLH (for C-terminal to LisH), CRA (for CT11-RanBPM), and RING (for Really Interesting New Gene) domains. Expression of SIE3 was detected in all tested tissues of Lotus japonicus plants, and its transcript level in roots was enhanced by rhizobial infection. The SIE3 protein was localized to multiple subcellular locations including the nuclei and plasma membrane, where the SIE3-SymRK interaction took place. Overexpression of SIE3 promoted nodulation in transgenic hairy roots, whereas downregulation of SIE3 transcripts by RNA interference inhibited infection thread development and nodule organogenesis. These results suggest that SIE3 represents a new class of E3 ubiquitin ligase, acts as a regulator of SymRK, and is involved in rhizobial infection and nodulation in L. japonicus.