DNA Methylation Patterns Differ between Free-Living Rhizobium leguminosarum RCAM1026 and Bacteroids Formed in Symbiosis with Pea (Pisum sativum L.)

Rhizobium leguminosarum (Rl) is a common name for several genospecies of rhizobia able to form nitrogen-fixing nodules on the roots of pea (Pisum sativum L.) while undergoing terminal differentiation into a symbiotic form called bacteroids. In this work, we used Oxford Nanopore sequencing to analyze the genome methylation states of the free-living and differentiated forms of the Rl strain RCAM1026. The complete genome was assembled; no significant genome rearrangements between the cell forms were observed, but the relative abundances of replicons were different. GANTC, GGCGCC, and GATC methylated motifs were found in the genome, along with genes encoding methyltransferases with matching predicted target motifs. The GGCGCC motif was completely methylated in both states, with two restriction–modification clusters on different replicons enforcing this specific pattern of methylation. Methylation patterns for the GANTC and GATC motifs differed significantly depending on the cell state, which indicates their possible connection to the regulation of symbiotic differentiation. Further investigation into the differences of methylation patterns in the bacterial genomes coupled with gene expression analysis is needed to elucidate the function of bacterial epigenetic regulation in nitrogen-fixing symbiosis

Transcriptome Analysis of Alternative Splicing Events Induced by Arbuscular Mycorrhizal Fungi (Rhizophagus irregularis) in Pea (Pisum sativum L.) Roots

Alternative splicing (AS), a process that enables formation of different mRNA isoforms due to alternative ways of pre-mRNA processing, is one of the mechanisms for fine-tuning gene expression. Currently, the role of AS in symbioses formed by plants with soil microorganisms is not fully understood. In this work, a comprehensive analysis of the transcriptome of garden pea (Pisum sativum L.) roots in symbiosis with arbuscular mycorrhiza was performed using RNAseq and following bioinformatic analysis. AS profiles of mycorrhizal and control roots were highly similar, intron retention accounting for a large proportion of the observed AS types (67%). Using three different tools (SUPPA2, DRIMSeq and IsoformSwitchAnalyzeR), eight genes with AS events specific for mycorrhizal roots of pea were identified, among which four were annotated as encoding an apoptosis inhibitor protein, a serine/threonine-protein kinase, a dehydrodolichyl diphosphate synthase, and a pre-mRNA-splicing factor ATP-dependent RNA helicase DEAH1. In pea mycorrhizal roots, the isoforms of these four genes with preliminary stop codons leading to a truncated ORFs were up-regulated. Interestingly, two of these four genes demonstrating mycorrhiza-specific AS are related to the process of splicing, thus forming parts of the feedback loops involved in fine-tuning of gene expression during mycorrhization

The Succession of the Cellulolytic Microbial Community from the Soil during Oat Straw Decomposition

The process of straw decomposition is dynamic and is accompanied by the succession of the microbial decomposing community, which is driven by poorly understood interactions between microorganisms. Soil is a complex ecological niche, and the soil microbiome can serve as a source of potentially active cellulolytic microorganisms. Here, we performed an experiment on the de novo colonization of oat straw by the soil microbial community by placing nylon bags with sterilized oat straw in the pots filled with chernozem soil and incubating them for 6 months. The aim was to investigate the changes in decomposer microbiota during this process using conventional sequencing techniques. The bacterial succession during straw decomposition occurred in three phases: the early phase (first month) was characterized by high microbial activity and low diversity, the middle phase (second to third month) was characterized by low activity and low diversity, and the late phase (fourth to sixth months) was characterized by low activity and high diversity. Analysis of amplicon sequencing data revealed three groups of co-changing phylotypes corresponding to these phases. The early active phase was abundant in the cellulolytic members from Pseudomonadota, Bacteroidota, Bacillota, and Actinobacteriota for bacteria and Ascomycota for fungi, and most of the primary phylotypes were gone by the end of the phase. The second intermediate phase was marked by the set of phylotypes from the same phyla persisting in the community. In the mature community of the late phase, apart from the core phylotypes, non-cellulolytic members from Bdellovibrionota, Myxococcota, Chloroflexota, and Thermoproteota appeared. Full metagenome sequencing of the microbial community from the end of the middle phase confirmed that major bacterial and fungal members of this consortium had genes of glycoside hydrolases (GH) connected to cellulose and chitin degradation. The real-time analysis of the selection of these genes showed that their representation varied between phases, and this occurred under the influence of the host, and not the GH family factor. Our findings demonstrate that soil microbial community may act as an efficient source of cellulolytic microorganisms and that colonization of the cellulolytic substrate occurs in several phases, each characterized by its own taxonomic and functional profile