The Bradyrhizobium Sp. LmicA16 Type VI Secretion System is Required for Efficient Nodulation of Lupinus Spp.

Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and L. angustifolius . A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild type strain (wt) and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26 kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidy and motility than the wt strain in free living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

Towards Understanding Afghanistan Pea Symbiotic Phenotype Through the Molecular Modeling of the Interaction Between LykX-Sym10 Receptor Heterodimer and Nod Factors

The difference in symbiotic specificity between peas of Afghanistan and European phenotypes was investigated using molecular modeling. Considering segregating amino acid polymorphism, we examined interactions of pea LykX-Sym10 receptor heterodimers with four forms of Nodulation factor (NF) that varied in natural decorations (acetylation and length of the glucosamine chain). First, we showed the stability of the LykX-Sym10 dimer during molecular dynamics (MD) in solvent and in the presence of a membrane. Then, four NFs were separately docked to one European and two Afghanistan dimers, and the results of these interactions were in line with corresponding pea symbiotic phenotypes. The European variant of the LykX-Sym10 dimer effectively interacts with both acetylated and non-acetylated forms of NF, while the Afghanistan variants successfully interact with the acetylated form only. We additionally demonstrated that the length of the NF glucosamine chain contributes to controlling the effectiveness of the symbiotic interaction. The obtained results support a recent hypothesis that the LykX gene is a suitable candidate for the unidentified Sym2 allele, the determinant of pea specificity toward Rhizobium leguminosarum bv. viciae strains producing NFs with or without an acetylation decoration. The developed modeling methodology demonstrated its power in multiple searches for genetic determinants, when experimental detection of such determinants has proven extremely difficult.

Towards understanding Afghanistan pea symbiotic phenotype through the molecular modeling of the interaction between LykX-Sym10 receptor heterodimer and Nod factors

The difference in symbiotic specificity between peas of Afghanistan and European phenotypes was interrogated using molecular modeling. Considering segregating amino acid polymorphism, we examined interactions of pea LykX-Sym10 receptor heterodimers with four forms of Nod factor (NF) that varied in natural decorations (acetylation and length of the glucosamine chain). First, we showed the stability of the LykX-Sym10 dimer during molecular dynamics (MD) in solvent and in the presence of a membrane. Then, four NFs were separately docked to one European and two Afghanistan dimers, and the results of these interactions were in line with corresponding pea symbiotic phenotypes. The European variant of the LykX-Sym10 dimer effectively interacts with both acetylated and non-acetylated forms of NF, while the Afghanistan variants successfully interact with the acetylated form only. We additionally demonstrated that the length of the NF glucosamine chain contributes to controlling the effectiveness of the symbiotic interaction. The obtained results support a recent hypothesis that the LykX gene is a suitable candidate for the unidentified Sym2 allele, the determinant of pea specificity towards Rhizobium leguminosarum bv. viciae strains producing NFs with or without an acetylation decoration. The developed modeling methodology demonstrated its power in multiple searches for genetic determinants, when experimental detection of such determinants has proven extremely difficult.

The macroevolutionary dynamics of symbiotic and phenotypic diversification in lichens

Symbioses are evolutionarily pervasive and play fundamental roles in structuring ecosystems, yet our understanding of their macroevolutionary origins, persistence, and consequences is incomplete. We traced the macroevolutionary history of symbiotic and phenotypic diversification in an iconic symbiosis, lichens. By inferring the most comprehensive time-scaled phylogeny of lichen-forming fungi (LFF) to date (over 3,300 species), we identified shifts among symbiont classes that broadly coincided with the convergent evolution of phylogenetically or functionally similar associations in diverse lineages (plants, fungi, bacteria). While a relatively recent loss of lichenization in Lecanoromycetes was previously identified, our work instead suggests lichenization was abandoned far earlier, interrupting what had previously been considered a direct switch between trebouxiophycean and trentepohlialean algal symbionts. Consequently, some of the most diverse clades of LFF are instead derived from nonlichenized ancestors and re-evolved lichenization with Trentepohliales algae, a clade that also facilitated lichenization in unrelated lineages of LFF. Furthermore, while symbiont identity and symbiotic phenotype influence the ecology and physiology of lichens, they are not correlated with rates of lineage birth and death, suggesting more complex dynamics underly lichen diversification. Finally, diversification patterns of LFF differed from those of wood-rotting and ectomycorrhizal taxa, likely reflecting contrasts in their fundamental biological properties. Together, our work provides a timeline for the ecological contributions of lichens, and reshapes our understanding of symbiotic persistence in a classic model of symbiosis.

Metagenomic data reveal diverse fungal and algal communities associated with the lichen symbiosis

Lichens have traditionally been considered the symbiotic phenotype from the interactions of a single fungal partner and one or few photosynthetic partners. However, the lichen symbiosis has been shown to be far more complex and may include a wide range of other interacting organisms, including non-photosynthetic bacteria, accessory fungi, and algae. In this study, we analyzed metagenomic shotgun sequences to better characterize lichen mycobiomes. Specifically, we inferred the range of fungi associated within lichen thalli from five groups of lichens – horsehair lichens (mycobiont=Bryoria spp.), shadow lichens (taxa in Physciaceae), rock posies (Rhizoplaca spp.), rock tripes (Umbilicaria spp.), and green rock shields (Xanthoparmelia spp.). Metagenomic reads from the multi-copy nuclear ribosomal internal transcribed spacer region, the standard DNA barcode region for fungi, were extracted, clustered, and used to infer taxonomic assignments. Our data revealed diverse lichen-associated mycobiomes, and closely related mycobionts tended to have more similar mycobiomes. Many of the members of the lichen-associated mycobiomes identified here have not previously been found in association with lichens. We found little evidence supporting the ubiquitous presence of Cystobasidiales yeasts in macrolichens, although reads representing this putative symbiotic partner were found in samples of horsehair lichens, albeit in low abundance. Our study further highlights the ecosystem-like features of lichens, with partners and interactions far from being completely understood. Future research is needed to more fully and accurately characterize lichen mycobiomes and how these fungi interact with the major lichen components – the photo- and mycobionts.

Classical Soybean (Glycine max (L.) Merr) Symbionts, Sinorhizobium fredii USDA191 and Bradyrhizobium diazoefficiens USDA110, Reveal Contrasting Symbiotic Phenotype on Pigeon Pea (Cajanus cajan (L.) Millsp)

Pigeon pea (Cajanus cajan (L.) Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including Bradyrhizobium and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts—S. fredii USDA191 and B. diazoefficiens USDA110—and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both S. fredii USDA191 and a T3SS mutant S. fredii RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with B. diazoefficiens USDA110 and B. diazoefficiens Δ136 (a T3SS mutant) resulted in the formation of Fix− and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by B. diazoefficiens USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by B. diazoefficiens Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in B. diazoefficiens USDA110 nodules, while it was abundantly present in B. diazoefficiens Δ136 nodules. Results of competitive nodulation assays indicated that B. diazoefficiens Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of B. diazoefficiens, due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.

Creation and multi-omics characterization of a genomically hybrid strain in the nitrogen-fixing symbiotic bacteriumSinorhizobium meliloti

Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbour a multipartite genome composed by more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacteriumSinorhizobium meliloti. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donorS. melilotistrain to an acceptor strain. Thecis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extra-cellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modelling were employed forin silicoprediction of metabolic flux reorganization. Phenotypes of thecis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with thecis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.