Paenibacillus polymyxa has widely been studied as a model of plant-growth promoting rhizobacteria (PGPR). Here, the genome sequences of 9 P. polymyxa strains, together with 26 other sequenced Paenibacillus spp., were comparatively studied. Phylogenetic analysis of the concatenated 244 single-copy core genes suggests that the 9 P. polymyxa strains and 5 other Paenibacillus spp., isolated from diverse geographic regions and ecological niches, formed a closely related clade (here it is called Poly-clade). Analysis of single nucleotide polymorphisms (SNPs) reveals local diversification of the 14 Poly-clade genomes. SNPs were not evenly distributed throughout the 14 genomes and the regions with high SNP density contain the genes related to secondary metabolism, including genes coding for polyketide. Recombination played an important role in the genetic diversity of this clade, although the rate of recombination was clearly lower than mutation. Some genes relevant to plant-growth promoting traits, i.e. phosphate solubilization and IAA production, are well conserved, while some genes relevant to nitrogen fixation and antibiotics synthesis are evolved with diversity in this Poly-clade. This study reveals that both P. polymyxa and its closely related species have plant growth promoting traits and they have great potential uses in agriculture and horticulture as PGPR.

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation. We sequenced the genomes of 11 N₂-fixing Paenibacillus strains and demonstrated the genomic diversity of the genus Paenibacillus by comparing these strains to each other and to 20 other strains (4 N₂-fixing and 16 non-N₂-fixing strains) that were sequenced previously. Phylogenetic analysis of the concatenated 275 single-copy core genes suggests that ancestral Paenibacillus did not fix nitrogen and the N₂-fixing strains fall into two sub-groups, which were likely originated from a N₂-fixing common ancestor. A minimal and compact nif cluster comprising nine nif genes encoding Mo-nitrogenase is highly conserved in the 15 N₂-fixing strains. Variations in the nif cluster and in the chromosomal regions surrounding the nif cluster between two sub-groups imply at least two independent acquisitions with insertion of distinct nif cluster variants in different genomic sites of Paenibacillus in early evolutionary history. Phylogeny of the concatenated NifHDK sequences suggests that Paenibacillus and Frankia are sister groups. The nif cluster, a functional unit for nitrogen fixation, was lost, producing some non-N₂-fixing strains. There were recent events of acquisition of vnf and anf genes, causing further diversification of some strains. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes.

Plant growth behavior is a function of genetic network architecture. The importance of root microbiome variation driving plant functional traits is increasingly recognized, but the genetic mechanisms governing this variation are less studied. Here, we collect roots and rhizosphere soils from nine Populus species belonging to four sections (Leuce, Aigeiros, Tacamahaca, and Turanga), generate metabolite and transcription data for roots and microbiota data for rhizospheres, and conduct comprehensive multi-omics analyses. We demonstrate that the roots of vigorous Leuce poplar enrich more Pseudomonas, compared with the poorly performing poplar. Moreover, we confirm that Pseudomonas is strongly associated with tricin and apigenin biosynthesis and identify that gene GLABRA3 (GL3) is critical for tricin secretion. The elevated tricin secretion via constitutive transcription of PopGL3 and Chalcone synthase (PopCHS4) can drive Pseudomonas colonization in the rhizosphere and further enhance poplar growth, nitrogen acquisition, and secondary root development in nitrogen-poor soil. This study reveals that plant-metabolite-microbe regulation patterns contribute to the poplar fitness and thoroughly decodes the key regulatory mechanisms of tricin, and provides insights into the interactions of the plant’s key metabolites with its transcriptome and rhizosphere microbes.

Plants in natural surroundings frequently encounter diverse forms of stress, and microbes are known to play a crucial role in assisting plants to withstand these challenges. However, the mining and utilization of plant-associated stress-resistant microbial sub-communities from the complex microbiome remains largely elusive. This study was based on the microbial communities over 13 weeks under four treatments (control, drought, salt, and disease) to define the shared core microbiota and stress-specific microbiota. Through co-occurrence network analysis, the dynamic change networks of microbial communities under the four treatments were constructed, revealing distinct change trajectories corresponding to different treatments. Moreover, by simulating species extinction, the impact of the selective removal of microbes on network robustness was quantitatively assessed. It was found that under varying environmental conditions, core microbiota made significant potential contributions to the maintenance of network stability. Our assessment utilizing null and neutral models indicated that the assembly of stress-specific microbiota was predominantly driven by deterministic processes, whereas the assembly of core microbiota was governed by stochastic processes. We also identified the microbiome features from functional perspectives: the shared microbiota tended to enhance the ability of organisms to withstand multiple types of environmental stresses and stress-specific microbial communities were associated with the diverse mechanisms of mitigating specific stresses. Using a culturomic approach, 781 bacterial strains were isolated, and nine strains were selected to construct different SynComs. These experiments confirmed that communities containing stress-specific microbes effectively assist plants in coping with environmental stresses. Collectively, we not only systematically revealed the dynamics variation patterns of rhizosphere microbiome under various stresses, but also sought constancy from the changes, identified the potential contributions of core microbiota and stress-specific microbiota to plant stress tolerance, and ultimately aimed at the beneficial microbial inoculation strategies for plants. Our research provides novel insights into understanding the microbe-mediated stress resistance process in plants.

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Pseudogenes (Ψs), nonfunctional relatives of functional genes, form by duplication or retrotransposition, and loss of gene function by disabling mutations. Evolutionary analysis provides clues to Ψ origins and effects on gene regulation. However, few systematic studies of plant Ψs have been conducted, hampering comparative analyses. Here, we examined the origin, evolution, and expression patterns of Ψs and their relationships with noncoding sequences in seven angiosperm plants. We identified ∼250,000 Ψs, most of which are more lineage specific than protein-coding genes. The distribution of Ψs on the chromosome indicates that genome recombination may contribute to Ψ elimination. Most Ψs evolve rapidly in terms of sequence and expression levels, showing tissue- or stage-specific expression patterns. We found that a surprisingly large fraction of nontransposable element regulatory noncoding RNAs (microRNAs and long noncoding RNAs) originate from transcription of Ψ proximal upstream regions. We also found that transcription factor binding sites preferentially occur in putative Ψ proximal upstream regions compared with random intergenic regions, suggesting that Ψs have conditioned genome evolution by providing transcription factor binding sites that serve as promoters and enhancers. We therefore propose that rapid rewiring of Ψ transcriptional regulatory regions is a major mechanism driving the origin of novel regulatory modules.