The effectiveness of the use of Sinorhizobium meliloti in the cultivation of variable alfalfa (Medicago varia Mart.)

As a result of three-year studies, we have established a growth in the yield and feed advantages of variable alfalfa in the variants with inoculation of seeds with virulent active strains of rhizobia. The total yield augmentation of green mass in the experimental variants over the three years of alfalfa life were 6.8–13.7 % compared to the control ones. The positive effect of inoculation with virulent active rhizobia strains on the total collection of dry matter was expressed in its increase in experimental conditions by 12.6–21.7 %. The highest yield of green mass, as well as dry matter was obtained in the variant with the inoculation of alfalfa seeds with the main production strain 425. The researched factor has a positive effect on the collection of feed units, digestible protein and feed protein units from 1 ha. The collection of feed units per hectare in the experimental versions increases by 1.1–1.3 times, the collection of digestible protein – by 1.2–1.4 times. The maximum substance of feed units and digestible protein per hectare was observed in the version with seed inoculation with strain 425a. The provision of a feed unit with digestible protein increases by 10.44–18.18 g or by 6.1–10.6 %.

AGROENERGY EFFICIENCY OF IMPROVED TECHNOLOGIES FOR CREATION AND USE OF ALFALFA-CEREAL HAYMAKING IN THE NON-CHERNOZEM ZONE

On the basis of field experience in the creation of alfalfa-grass stands on sod-podzolic soil of the Non-Chernozem zone, a high agroenergetic efficiency of improved technologies has been established, including two zoned varieties of alfalfa changeable (Lugovaya 67 and Pastbishchnaya 88) in combination with agrotechnical methods (change in the composition of leguminous species in the previous period and pre-sowing inoculation of alfalfa seeds with complementary strains of Sinorhizobium meliloti). As a result of the application of the agro-energy method for assessing the production of exchangeable energy and total anthropogenic costs in uniform indicators according to the international SI system (GJ/ha), their payback was established — 8–11 times. This was achieved due to a high share of natural factors (88–91% of total costs), additional nitrogen input into the production process due to increased symbiotic fixation (141–171 kg/ha per year) and long-term use — for 7 years. The productivity of 1 hectare was 62–64 GJ/ha metabolic energy, the collection of protein was 956–1120 kg/ha, the saving of anthropogenic costs due to the exclusion of the use of nitrogen fertilizers was 17–21 GJ/ha.

Membrane Homeoviscous Adaptation in Sinorhizobium Submitted to a Stressful Thermal Cycle Contributes to the Maintenance of the Symbiotic Plant–Bacteria Interaction

Here, we estimate fast changes in the fluidity of Sinorhizobium meliloti membranes submitted to cyclic temperature changes (10°C–40°C–10°C) by monitoring the fluorescence polarization (P) of DPH and TMA-DPH of the whole cell (WC) as well as in its outer (OM) and inner (IM) membranes. Additionally, the long-term response to thermal changes is demonstrated through the dynamics of the phospholipid and fatty acid composition in each membrane. This allowed membrane homeoviscous adaptation by the return to optimal fluidity levels as measured by the PDPH/TMA-DPH in WC, OM, IM, and multilamellar vesicles of lipids extracted from OM and IM. Due to probe-partitioning preferences and membranes’ compositional characteristics, DPH and TMA-DPH exhibit different behaviors in IM and OM. The rapid effect of cyclic temperature changes on the P was the opposite in both membranes with the IM being the one that exhibited the thermal behavior expected for lipid bilayers. Interestingly, only after the incubation at 40°C, cells were unable to recover the membrane preheating P levels when cooled up to 10°C. Solely in this condition, the formation of threads and nodular structures in Medicago sativa infected with S. meliloti were delayed, indicating that the symbiotic interaction was partially altered but not halted.

The ChpR transcriptional regulator of Sinorhizobium meliloti senses 3,5,6-trichloropyridinol, a degradation product of the organophosphate pesticide chlorpyrifos

The global use of organophosphate insecticides (OPPs) and the growing concern of off-target side effects due to OPP exposure has prompted the need for sensitive and economical detection methods. Here we set out to engineer a previously identified OPP responsive transcription factor, ChpR, from Sinorhizobium melilotii to respond to alternative OPPs and generate a repertoire of whole-cell biosensors for OPPs. The ChpR transcription factor and cognate promoter P chpA, have been shown to activate transcription in the presence of the OPP chlorpyrifos (CPF). Utilizing a GFP reporter regulated by ChpR in a whole-cell biosensor we found that the system responds significantly better to 3,5,6-trichloro-2-pyridinol (TCP), the main degradation product of CPF, compared to CPF itself. This biosensor was able to respond to TCP at 390 nM within 4 h compared to 50 µM of CPF in 7 h. The ChpR-P chpA , and the activating ligand TCP, were able to regulate expression of a kanamycin resistance/sucrose sensitivity (kan/sacB) selection/counterselection module suitable for high throughput mutagenesis screening studies. The ability to control both GFP and the kan/sacB module demonstrates the utility of this reporter for the detection of CPF affected areas. The ChpR-P chpA system serves as an additional positive regulator switch to add to the growing repertoire of controllers available within synthetic biology.

The legume-rhizobia symbiosis can be supported on Mars soil simulants

Legumes (soybeans, peas, lentils, etc.) play important roles in agriculture on Earth because of their food value and their ability to form a mutualistic beneficial association with rhizobia bacteria. In this association, the host plant benefits from atmospheric nitrogen fixation by rhizobia. The presence of nitrogen in the Mars atmosphere offers the possibility to take advantage of this important plant-microbe association. While some studies have shown that Mars soil simulants can support plant growth, none have investigated if these soils can support the legume-rhizobia symbiosis. In this study, we investigated the establishment of the legume-rhizobia symbiosis on different Mars soil simulants (different grades of the Mojave Mars Simulant (MMS)-1: Coarse, Fine, Unsorted, Superfine, and the MMS-2 simulant). We used the model legume, Medicago truncatula, and its symbiotic partners, Sinorhizobium meliloti and Sinorhizobium medicae, in these experiments. Our results show that root nodules could develop on M. truncatula roots when grown on these Mars soil simulants and were comparable to those formed on plants that were grown on sand. We also detected nifH (a reporter gene for nitrogen fixation) expression inside these nodules. Our results indicate that the different Mars soil simulants used in this study can support legume-rhizobia symbiosis. While the average number of lateral roots and nodule numbers were comparable on plants grown on the different soil simulants, total plant mass was higher in plants grown on MMS-2 soil than on MMS-1 soil and its variants. Our results imply that the chemical composition of the simulants is more critical than their grain size for plant mass. Based on these results, we recommend that the MMS-2 Superfine soil simulant is a better fit than the MMS-1 soil and it’s variants for future studies. Our findings can serve as an excellent resource for future studies investigating beneficial plant-microbe associations for sustainable agriculture on Mars.

Endophytic fungi facilitate the coexistence of host grasses and neighboring legumes by changing rhizobial abundance

Purpose: Grass-endophyte and legume-rhizobium symbionts coexist in grasslands. However, the effects of endophyte infection on legume-rhizobium symbionts remain poorly understood, especially in natural grasslands. Methods: In this study, Achnatherum sibiricum - Epichloë endophytes and Medicago ruthenica -rhizobia were selected as materials to investigate whether and how endophyte infection affected the growth of legume-rhizobia symbionts. It was hypothesized that endophytes can facilitate the coexistence of grass-legume systems. Results: The results demonstrated that endophyte infection affected the growth of both rhizobia and M. ruthenica -rhizobia symbionts, and the results depended on rhizobial identity. Endophyte infection inhibited the growth of Mesorhizobium ciceri , which significantly promoted the growth of M. ruthenica , and promoted Sinorhizobium meliloti , which had no significant effect on the growth of M. ruthenica . Endophyte infection also changed the interaction between A. sibiricum and M. ruthenica . When inoculated with M. ciceri , endophyte infection weakened the promoting effect of A. sibiricum on M. ruthenica , while when inoculated with S. meliloti , endophyte infection enhanced the promoting effect. Endophyte infection affected the growth of M. ruthenica -rhizobia symbionts by affecting rhizobia abundance in roots and nitrogen content in plant leaves. Conclusion: In conclusion, endophyte infection was beneficial to biomass accumulation and species coexistence in grass-legume mixed planting systems. In this study, it was proposed that endophyte infection may change the growth of legume-rhizobia symbionts by affecting the growth and nitrogen fixation of rhizobia.

A newly-evolved chimeric lysin motif receptor-like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership

Rhizobial lipochitooligosaccharidic Nod factors, specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis. A Sinorhizobium meliloti nodF/nodL mutant produces Nod factors that differ from wild-type ones in lacking an O-acetate, and with a different acyl chain on the terminal non-reducing sugar. This mutant is defective in nodulation with various Medicago hosts. We examined the nodulation ability of M. truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the nodF/nodL mutant. We then applied genetic and functional approaches to study the genetic basis and mechanism of nodulation of R108 by this mutant. We show that the nodF/nodL mutant can nodulate R108 but not A17. Using genomics and reverse genetics, we identified a newly-evolved gene in R108, LYK2bis, which is responsible for the phenotype. Transformation with LYK2bis allows A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in specific NF signalling and interacts with the key receptor protein NFP. Our findings reveal that a newly-evolved gene in R108, LYK2bis, extends nodulation specificity to strains producing non-O-acetylated NFs. Interaction between LYK2bis and NFP provides a means of integrating the nodulation signalling pathways.

Direct regulation of cell cycle regulatory gene expression by NtrX to promote Sinorhizobium meliloti cell division

Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by the CtrA signaling network. The gene expression of regulatory proteins in the network is affected by nutrient signaling. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes from the CtrA signaling network. Three sets of S. meliloti ntrX mutants, including the plasmid insertion strain, the depletion strain and the substitution of the 53rd aspartate (ntrXD53E) from a plasmid in the wild-type strain (Sm1021), showed similar cell division defects, such as slow growth, abnormal morphology of partial cells and delayed DNA synthesis. Transcript quantitative evaluation indicated that the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZ1 was down-regulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by western blotting, which were consistent with the transcription data. The transcriptional regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein. The NtrX protein binds directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG. Our findings reveal that NtrX is a novel transcriptional regulator of the CtrA signaling pathway genes, and positively affects bacterial cell division, associated with nitrogen metabolism.

Plant Peptides (NCRs) and Plant Immunity Play Vital Roles in Determining Specificity Between Alfalfa Cultivars and Sinorhizobium Meliloti Strain

Alfalfa expresses significantly distinct sets of genes in response to infection by different rhizobia strains at the below-species level (i.e., biotype or strain). However, differences in the transcriptomic profiles of two alfalfa cultivars nodulated by a single rhizobium strain have been largely unexamined. In this study, the comparative RNA-seq analysis of two alfalfa cultivars, Medicago sativa cv. Gannong No. 3 and Gannong No. 9 inoculated with one Sinorhizobium meliloti strain LL2, with varying in symbiotic performance, was conducted, followed by a hub gene interaction network construction based on weighted gene co-expression network analysis (WGCNA). The G9-LL2 symbiotic system showed better nodule-formation, nitrogen-fixing, and growth characteristics than the G3-LL2 system. Compared with the uninoculated control (CK), the LL2-inoculated G9 plants (10053) produced more differentially expressed genes (DEGs) than the LL2-inoculated G3 plants (7112). A group of 227 (2623 shared) genes displayed completely distinguished expression in G9 (6.63 < log2(FC) < 15.45) and G3 (‒3.05 < log2(FC) < 12.05), which are primarily involved in encoding nodule-specific cysteine-rich peptides (NCRs), nodulin, and leghemoglobin. Although genes with predicted roles in nitrogen metabolism were primarily upregulated, and almost all of those in ubiquitin-mediated proteolysis and plant-pathogen interaction were suppressed, interestingly, a consistently higher expression level measured by log2(FC) was observed in G9 plants. Hub gene interaction networks showed that the NCRs, late nodulin, and genes related to plant immunity (TIR-NBS-LRR, defensin, thioredoxin, thionine, and polygalacturonase) regulated other genes at the source node positions. After the successful initiation of nodulation in both alfalfa cultivars G3 and G9 by S. meliloti strain LL2, G9 achieved preferable outcomes of rhizobia-alfalfa symbiosis by equilibrating the antagonism and compatibility of plant immunity. It elevated PTI and suppressed defense and ETI, as well as enhancing nitrogen fixation and utilization efficiency by inducing the expression of genes encoding NCRs, nodulin, and leghemoglobin. Hub genes predominantly underlying the highly specific rhizobia-alfalfa symbiosis, positively governed by NCRs and fine-tuned immune antagonism, comprise NCRs, late nodulin, and TIR-NBS-LRR. These findings provide insights into the genetic mechanisms underlying the modification and efficient utilization of semi-compatible and incompatible rhizobia resources.