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.

Specialization in a nitrogen-fixing symbiosis: proteome differences between Sinorhizobium medicae bacteria and bacteroids

Using tandem mass spectrometry (MS/MS), we analyzed the proteome of Sinorhizobium medicae WSM419 growing as free-living cells and in symbiosis with Medicago truncatula. 3215 proteins were identified, over half of the ORFs predicted from the genomic sequence. The abundance of 1361 proteins displayed strong lifestyle bias. 1131 proteins had similar levels in bacteroids and free-living cells, and the low levels of 723 proteins prevented statistically significant assignments. Nitrogenase subunits comprised ~12% of quantified bacteroid proteins. Other major bacteroid proteins included symbiosis-specific cytochromes and FixABCX, which transfer electrons to nitrogenase. Bacteroids had normal levels of proteins involved in amino acid biosynthesis, glycolysis/gluconeogenesis and the pentose phosphate pathway, but several amino acid degradation pathways were repressed. This suggests bacteroids maintain a relatively independent anabolic metabolism. TCA cycle proteins were highly expressed in bacteroids and no other catabolic pathway emerged as an obvious candidate to supply energy and reductant to nitrogen fixation. Bacterial stress response proteins were induced in bacteroids. Many WSM419 proteins that are not encoded in Sinorhizobium meliloti Rm1021 were detected and understanding the functions of these proteins might clarify why S. medicae WSM419 forms a more effective symbiosis with M. truncatula than S. meliloti Rm1021.

Sinorhizobium medicae WSM419 genes that improve symbiosis between Sinorhizobium meliloti Rm1021 and Medicago truncatula Jemalong A17 and in other symbiotic systems

Some soil bacteria called rhizobia can interact symbiotically with legumes in which they form nodules on the plant roots where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobia/plant combinations can differ in how successful this symbiosis is—Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17 but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness) had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant induced resistance to rhizobial infection, suggesting an interaction with the plant’s regulation of nodule formation. IMPORTANCE The legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle—symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach may identify new genes that may more generally contribute to symbiotic productivity.

Medicago-Sinorhizobium-Ralstonia a model system to investigate pathogen triggered inhibition of nodulation

How plants deal with beneficial and pathogenic microorganisms and how they can tolerate beneficial ones and face to pathogens in the same time are questions that remain puzzling to plant biologists. Legume plants are good models to explore those issues as their interactions with nitrogen-fixing bacteria, rhizobia, results in a drastic and easy to follow phenotype of nodulation. Intriguingly, despite massive and chronic infection, legumes defense reactions are essentially suppressed during the whole symbiotic process rising the question about a potential negative effect of plant immune responses on the establishment of nodulation. In the present study, we used the model legume, Medicago truncatula, co-inoculated with a mutualistic and with a phytopathogenic bacteria, Sinorhizobium medicae and Ralstonia solanacerarum. We show that the presence of R. solanacearum drastically inhibits the nodulation process. The type three secretion system (TTSE) of R. solanacearum, that is important for the inhibition of PAMP (Pathogen Associated Molecular Patterns) triggered immunity (PTI), strongly contributes to inhibit nodulation. Thus, our results question the negative effect of PTI on nodulation. By including a pathogenic bacterium in the interaction system, our study provides a new angle to address the influence of the biotic environment on the nodulation process.

Co-inoculation of lucerne (Medicago sativa) with an AM fungus and a rhizobium reduces occurrence of spring black stem and leaf spot caused by Phoma medicaginis

Spring black stem and leaf spot of lucerne (alfalfa, Medicago sativa L.), caused by Phoma medicaginis, is an important disease in temperate regions of the world. It is now a serious disease threatening global lucerne production. This experiment was designed to test the combined effects of the arbuscular mycorrhizal (AM) fungus Funneliformis mosseae and the rhizobium Sinorhizobium medicae on growth, nutrient uptake and disease severity in lucerne. The results showed that F. mosseae increased plant phosphorus and nitrogen uptake and plant dry weight, and this beneficial effect was enhanced when in association with S. medicae. Rhizobial and AM fungal effects were mutually promoting; inoculation with AM fungus significantly increased the formation of root nodules, and inoculation with rhizobium increased the percentage of root length colonised by AM fungus (P < 0.05). After infection with P. medicaginis, typical leaf spot symptoms with the lowest disease incidence and disease index occurred on plants that were host to both F. mosseae and S. medicae. Plants with both symbiotic microorganisms had higher activities (concentrations) of phenylalanine ammonia-lyase, chitinase, β-1,3-glucanase, lignin, hydroxyproline-rich glycoprotein and jasmonic acid. Therefore, the tested AM fungus (F. mosseae) and rhizobium (S. medicae) have the potential to reduce damage and yield loss in lucerne from spring black stem and leaf spot caused by P. medicaginis.