An isoflavone catabolism gene cluster underlying interkingdom interactions in the soybean rhizosphere.

Plant roots secrete various metabolites, including plant specialized metabolites, into the rhizosphere, and shape the rhizosphere microbiome, which is crucial for the plant health and growth. Isoflavones are major plant specialized metabolites found in legume plants, and are involved in interactions with soil microorganisms as initiation signals in rhizobial symbiosis and as modulators of the legume root microbiota. However, it remains largely unknown the molecular basis underlying the isoflavone-mediated interkingdom interactions in the legume rhizosphere. Here, we isolated Variovorax sp. strain V35, a member of the Comamonadaceae that harbors isoflavone-degrading activity, from soybean roots and discovered a gene cluster responsible for isoflavone degradation named ifc. The characterization of ifc mutants and heterologously expressed Ifc enzymes revealed that isoflavones undergo oxidative catabolism, which is different from the reductive metabolic pathways observed in gut microbiota. We further demonstrated that the ifc genes are frequently found in bacterial strains isolated from legume plants, including mutualistic rhizobia, and contribute to the detoxification of the antibacterial activity of isoflavones. Taken together, our findings reveal an isoflavone catabolism gene cluster in the soybean root microbiota, providing molecular insights into isoflavone-mediated legume-microbiota interactions.

Expression of Syo_1.56 SARP Regulator Unveils Potent Elasnin Derivatives with Antibacterial Activity.

Actinomycetes are prolific producers of natural products, particularly antibiotics. However, a significant proportion of its biosynthetic gene clusters (BGCs) remain silent under typical laboratory conditions. This limits the effectiveness of conventional isolation methods for the discovery of novel natural products. Genetic interventions targeting the activation of silent gene clusters are necessary to address this challenge. Streptomyces antibiotic regulatory proteins (SARPs) act as cluster-specific activators and can be used to target silent BGCs for the discovery of new antibiotics. In this study, the expression of a previously uncharacterized SARP protein, Syo_1.56, in Streptomyces sp. RK18-A0406 significantly enhanced the production of known antimycins and led to the discovery of 12 elasnins (1-12), 10 of which were novel. The absolute stereochemistry of elasnin A1 was assigned for the first time to be 6S. Unexpectedly, Syo_1.56 seems to function as a pleiotropic rather than cluster-specific SARP regulator, with the capability of co-regulating two distinct biosynthetic pathways, simultaneously. All isolated elasnins were active against wild-type and methicillin-resistant Staphylococcus aureus with IC50 values of 0.5-20 µg/mL, some of which (elasnins A1, B2, and C1 and proelasnins A1, and C1) demonstrated moderate to strong antimalarial activities against Plasmodium falciparum 3D7. Elasnins A1, B3, and C1 also showed in vitro inhibition of the metallo-ß-lactamase responsible for the development of highly antibiotic-resistant bacterial strains.

Uncovering microbiomes of the rice phyllosphere using long-read metagenomic sequencing.

The plant microbiome is crucial for plant growth, yet many important questions remain, such as the identification of specific bacterial species in plants, their genetic content, and location of these genes on chromosomes or plasmids. To gain insights into the genetic makeup of the rice-phyllosphere, we perform a metagenomic analysis using long-read sequences. Here, 1.8 Gb reads are assembled into 26,067 contigs including 142 circular sequences. Within these contigs, 669 complete 16S rRNA genes are clustered into 166 bacterial species, 121 of which show low identity (<97%) to defined sequences, suggesting novel species. The circular contigs contain novel chromosomes and a megaplasmid, and most of the smaller circular contigs are defined as novel plasmids or bacteriophages. One circular contig represents the complete chromosome of a difficult-to-culture bacterium Candidatus Saccharibacteria. Our findings demonstrate the efficacy of long-read-based metagenomics for profiling microbial communities and discovering novel sequences in plant-microbiome studies.

A Novel Rhizobium sp. Chiba-1 Strain Exhibits a Host Range for Nodule Symbiosis in Lotus Species.

Rhizobia are soil bacteria that induce the formation of nodules in the roots of leguminous plants for mutualistic establishment. Although the symbiotic mechanism between Lotus japonicus and its major symbiotic rhizobia, Mesorhizobium loti, has been extensively characterized, our understanding of symbiotic mechanisms, such as host specificity and host ranges, remains limited. In the present study, we isolated a novel Rhizobium strain capable of forming nodules on L. burttii from agricultural soil at Iwate prefecture in Japan. We conducted genomic and host range ana-lyses of various Lotus species. The results obtained revealed that the novel isolated Rhizobium sp. Chiba-1 was closely related to R. leguminosarum and had a wide host range that induced nodule development, including L. burttii and several L. japonicus wild-type accessions. However, L. japonicus Gifu exhibited an incompatible nodule phenotype. We also identified the formation of an epidermal infection threads that was dependent on the Lotus species and independent of nodule organ development. In conclusion, this newly isolated Rhizobium strain displays a distinct nodulation phenotype from Lotus species, and the results obtained herein provide novel insights into the functional mechanisms underlying host specificity and host ranges.

Draft genome sequence of Cupriavidus sp. strain TKC, isolated from a γ-hexachlorocyclohexane-degrading community.

Cupriavidus sp. strain TKC was isolated from a microbial community enriched with γ-hexachlorocyclohexane (γ-HCH). This strain did not show γ-HCH-degrading activity but was one of the major members of the community. Here, we present the draft genome sequence of the strain TKC with a size of 7 Mb.

Subgenome dominance shapes novel gene evolution in the decaploid pitcher plant Nepenthes gracilis.

Subgenome dominance after whole-genome duplication generates distinction in gene number and expression at the level of chromosome sets, but it remains unclear how this process may be involved in evolutionary novelty. Here we generated a chromosome-scale genome assembly of the Asian pitcher plant Nepenthes gracilis to analyse how its novel traits (dioecy and carnivorous pitcher leaves) are linked to genomic evolution. We found a decaploid karyotype and a clear indication of subgenome dominance. A male-linked and pericentromerically located region on the putative sex chromosome was identified in a recessive subgenome and was found to harbour three transcription factors involved in flower and pollen development, including a likely neofunctionalized LEAFY duplicate. Transcriptomic and syntenic analyses of carnivory-related genes suggested that the paleopolyploidization events seeded genes that subsequently formed tandem clusters in recessive subgenomes with specific expression in the digestive zone of the pitcher, where specialized cells digest prey and absorb derived nutrients. A genome-scale analysis suggested that subgenome dominance likely contributed to evolutionary innovation by permitting recessive subgenomes to diversify functions of novel tissue-specific duplicates. Our results provide insight into how polyploidy can give rise to novel traits in divergent and successful high-ploidy lineages.

Metabolism-linked methylotaxis sensors responsible for plant colonization in Methylobacterium aquaticum strain 22A.

Motile bacteria take a competitive advantage in colonization of plant surfaces to establish beneficial associations that eventually support plant health. Plant exudates serve not only as primary growth substrates for bacteria but also as bacterial chemotaxis attractants. A number of plant-derived compounds and corresponding chemotaxis sensors have been documented, however, the sensors for methanol, one of the major volatile compounds released by plants, have not been identified. Methylobacterium species are ubiquitous plant surface-symbiotic, methylotrophic bacteria. A plant-growth promoting bacterium, M. aquaticum strain 22A exhibits chemotaxis toward methanol (methylotaxis). Its genome encodes 52 methyl-accepting chemotaxis proteins (MCPs), among which we identified three MCPs (methylotaxis proteins, MtpA, MtpB, and MtpC) responsible for methylotaxis. The triple gene mutant of the MCPs exhibited no methylotaxis, slower gathering to plant tissues, and less efficient colonization on plants than the wild type, suggesting that the methylotaxis mediates initiation of plant-Methylobacterium symbiosis and engages in proliferation on plants. To examine how these MCPs are operating methylotaxis, we generated multiple gene knockouts of the MCPs, and Ca2+-dependent MxaFI and lanthanide (Ln3+)-dependent XoxF methanol dehydrogenases (MDHs), whose expression is regulated by the presence of Ln3+. MtpA was found to be a cytosolic sensor that conducts formaldehyde taxis (formtaxis), as well as methylotaxis when MDHs generate formaldehyde. MtpB contained a dCache domain and exhibited differential cellular localization in response to La3+. MtpB expression was induced by La3+, and its activity required XoxF1. MtpC exhibited typical cell pole localization, required MxaFI activity, and was regulated under MxbDM that is also required for MxaF expression. Strain 22A methylotaxis is realized by three independent MCPs, two of which monitor methanol oxidation by Ln3+-regulated MDHs, and one of which monitors the common methanol oxidation product, formaldehyde. We propose that methanol metabolism-linked chemotaxis is the key factor for the efficient colonization of Methylobacterium on plants.

Genome and transcriptome analyses reveal genes involved in the formation of fine ridges on petal epidermal cells in Hibiscus trionum.

Hibiscus trionum, commonly known as the 'Flower of an Hour', is an easily cultivated plant in the Malvaceae family that is widespread in tropical and temperate regions, including drylands. The purple base part of its petal exhibits structural colour due to the fine ridges on the epidermal cell surface, and the molecular mechanism of ridge formation has been actively investigated. We performed genome sequencing of H. trionum using a long-read sequencing technology with transcriptome and pathway analyses to identify candidate genes for fine structure formation. The ortholog of AtSHINE1, which is involved in the biosynthesis of cuticular wax in Arabidopsis thaliana, was significantly overexpressed in the iridescent tissue. In addition, orthologs of AtCUS2 and AtCYP77A, which contribute to cutin synthesis, were also overexpressed. Our results provide important insights into the formation of fine ridges on epidermal cells in plants using H. trionum as a model.

Tomato root-associated Sphingobium harbors genes for catabolizing toxic steroidal glycoalkaloids.

IMPORTANCE: Saponins are a group of plant specialized metabolites with various bioactive properties, both for human health and soil microorganisms. Our previous works demonstrated that Sphingobium is enriched in both soils treated with a steroid-type saponin, such as tomatine, and in the tomato rhizosphere. Despite the importance of saponins in plant-microbe interactions in the rhizosphere, the genes involved in the catabolism of saponins and their aglycones (sapogenins) remain largely unknown. Here we identified several enzymes that catalyzed the degradation of steroid-type saponins in a Sphingobium isolate from tomato roots, RC1. A comparative genomic analysis of Sphingobium revealed the limited distribution of genes for saponin degradation in our saponin-degrading isolates and several other isolates, suggesting the possible involvement of the saponin degradation pathway in the root colonization of Sphingobium spp. The genes that participate in the catabolism of sapogenins could be applied to the development of new industrially valuable sapogenin molecules.

Insights into ecological roles of uncultivated bacteria in Katase hot spring sediment from long-read metagenomics.

Diverse yet-uncultivated bacteria and archaea, i.e., microbial dark matter, are present in terrestrial hot spring environments. Numerous metagenome-assembled genomes (MAGs) of these uncultivated prokaryotes by short-read metagenomics have been reported so far, suggesting their metabolic potential. However, more reliable MAGs, i.e., circularized complete MAGs (cMAGs), have been rarely reported from hot spring environments. Here, we report 61 high-quality (HQ)-MAGs, including 14 cMAGs, of diverse uncultivated bacteria and archaea retrieved from hot spring sediment (52°C, pH 7.2) by highly accurate long-read sequencing using PacBio Sequel II. The HQ MAGs were affiliated with one archaeal and 13 bacterial phyla. Notably, nine of the 14 cMAGs were the first reported cMAGs for the family- to class-level clades that these cMAGs belonged to. The genome information suggests that the bacteria represented by MAGs play a significant role in the biogeochemical cycling of carbon, nitrogen, iron, and sulfur at this site. In particular, the genome analysis of six HQ MAGs including two cMAGs of Armatimonadota, of which members are frequently abundant in hot spring environments, predicts that they are aerobic, moderate thermophilic chemoorganoheterotrophs, and potentially oxidize and/or reduce iron. This prediction is consistent with the environmental conditions where they were detected. Our results expand the knowledge regarding the ecological potential of uncultivated bacteria in moderately-high-temperature environments.

Complete Genome Sequence of Pseudomonas amygdali pv. tabaci Strain 6605, a Causal Agent of Tobacco Wildfire Disease.

Pseudomonas amygdali pv. tabaci strain 6605 is the bacterial pathogen causing tobacco wildfire disease that has been used as a model for elucidating virulence mechanisms. Here, we present the complete genome sequence of P. amygdali pv. tabaci 6605 as a circular chromosome from reads using a PacBio sequencer.

Nitrogen Deficiency-induced Bacterial Community Shifts in Soybean Roots.

Nitrogen deficiency affects soybean growth and physiology, such as symbiosis with rhizobia; however, its effects on the bacterial composition of the soybean root microbiota remain unclear. A bacterial community analysis by 16S rRNA gene amplicon sequencing showed nitrogen deficiency-induced bacterial community shifts in soybean roots with the marked enrichment of Methylobacteriaceae. The abundance of Methylobacteriaceae was low in the roots of field-grown soybean without symptoms of nitrogen deficiency. Although Methylobacteriaceae isolated from soybean roots under nitrogen deficiency did not promote growth or nodulation when inoculated into soybean roots, these results indicate that the enrichment of Methylobacteriaceae in soybean roots is triggered by nitrogen-deficiency stress.

Tobacco Root Endophytic Arthrobacter Harbors Genomic Features Enabling the Catabolism of Host-Specific Plant Specialized Metabolites.

Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.

Telomeres and a repeat-rich chromosome encode effector gene clusters in plant pathogenic Colletotrichum fungi.

Members of the Colletotrichum gloeosporioides species complex are causal agents of anthracnose in many commercially important plants. Closely related strains have different levels of pathogenicity on hosts despite their close phylogenetic relationship. To gain insight into the genetics underlying these differences, we generated and annotated whole-genome assemblies of multiple isolates of C. fructicola (Cf) and C. siamense (Cs), as well as three previously unsequenced species, C. aenigma (Ca), C. tropicale and C. viniferum with different pathogenicity on strawberry. Based on comparative genomics, we identified accessory regions with a high degree of conservation in strawberry-pathogenic Cf, Cs and Ca strains. These regions encode homologs of pathogenicity-related genes known as effectors, organized in syntenic gene clusters, with copy number variations in different strains of Cf, Cs and Ca. Analysis of highly contiguous assemblies of Cf, Cs and Ca revealed the association of related accessory effector gene clusters with telomeres and repeat-rich chromosomes and provided evidence of exchange between these two genomic compartments. In addition, expression analysis indicated that orthologues in syntenic gene clusters showed a tendency for correlated gene expression during infection. These data provide insight into mechanisms by which Colletotrichum genomes evolve, acquire and organize effectors.

Rhizobia use a pathogenic-like effector to hijack leguminous nodulation signalling.

Legume plants form a root-nodule symbiosis with rhizobia. This symbiosis establishment generally relies on rhizobium-produced Nod factors (NFs) and their perception by leguminous receptors (NFRs) that trigger nodulation. However, certain rhizobia hijack leguminous nodulation signalling via their type III secretion system, which functions in pathogenic bacteria to deliver effector proteins into host cells. Here, we report that rhizobia use pathogenic-like effectors to hijack legume nodulation signalling. The rhizobial effector Bel2-5 resembles the XopD effector of the plant pathogen Xanthomonas campestris and could induce nitrogen-fixing nodules on soybean nfr mutant. The soybean root transcriptome revealed that Bel2-5 induces expression of cytokinin-related genes, which are important for nodule organogenesis and represses ethylene- and defense-related genes that are deleterious to nodulation. Remarkably, Bel2-5 introduction into a strain unable to nodulate soybean mutant affected in NF perception conferred nodulation ability. Our findings show that rhizobia employ and have customized pathogenic effectors to promote leguminous nodulation signalling.

Lanthanide-Dependent Methanol and Formaldehyde Oxidation in Methylobacterium aquaticum Strain 22A.

Lanthanides (Ln) are an essential cofactor for XoxF-type methanol dehydrogenases (MDHs) in Gram-negative methylotrophs. The Ln3+ dependency of XoxF has expanded knowledge and raised new questions in methylotrophy, including the differences in characteristics of XoxF-type MDHs, their regulation, and the methylotrophic metabolism including formaldehyde oxidation. In this study, we genetically identified one set of Ln3+- and Ca2+-dependent MDHs (XoxF1 and MxaFI), that are involved in methylotrophy, and an ExaF-type Ln3+-dependent ethanol dehydrogenase, among six MDH-like genes in Methylobacterium aquaticum strain 22A. We also identified the causative mutations in MxbD, a sensor kinase necessary for mxaF expression and xoxF1 repression, for suppressive phenotypes in xoxF1 mutants defective in methanol growth even in the absence of Ln3+. Furthermore, we examined the phenotypes of a series of formaldehyde oxidation-pathway mutants (fae1, fae2, mch in the tetrahydromethanopterin (H4MPT) pathway and hgd in the glutathione-dependent formaldehyde dehydrogenase (GSH) pathway). We found that MxaF produces formaldehyde to a toxic level in the absence of the formaldehyde oxidation pathways and that either XoxF1 or ExaF can oxidize formaldehyde to alleviate formaldehyde toxicity in vivo. Furthermore, the GSH pathway has a supportive role for the net formaldehyde oxidation in addition to the H4MPT pathway that has primary importance. Studies on methylotrophy in Methylobacterium species have a long history, and this study provides further insights into genetic and physiological diversity and the differences in methylotrophy within the plant-colonizing methylotrophs.

High-Quality Draft Genome Sequence of Fusarium oxysporum f. sp. cubense Strain 160527, a Causal Agent of Panama Disease.

Fusarium oxysporum f. sp. cubense is the causal agent of banana Fusarium wilt, also known as Panama disease. Here, we present a high-quality genome sequence of F. oxysporum f. sp. cubense strain 160527. The genome assembly is composed of 12 contigs with a total assembly length of 51,139,495 bp (N 50 contig length, 4,884,632 bp).

Mapping of quantitative trait loci related to primary rice root growth as a response to inoculation with Azospirillum sp. strain B510.

Azospirillum sp. strain B510 has been known as the plant growth-promoting endophyte; however, the growth-promotion effect is dependent on the plant genotype. Here, we aimed to identify quantitative trait loci (QTL) related to primary root length in rice at the seedling stage as a response to inoculation with B510. The primary root length of "Nipponbare" was significantly reduced by inoculation with B510, whereas that of "Kasalath" was not affected. Thus, we examined 98 backcrossed inbred lines and four chromosome segment substitution lines (CSSL) derived from a cross between Nipponbare and Kasalath. The primary root length was measured as a response to inoculation with B510, and the relative root length (RRL) was calculated based on the response to non-inoculation. Three QTL alleles, qRLI-6 and qRLC-6 on Chromosome (Chr.) 6 and qRRL-7 on Chr. 7 derived from Kasalath increased primary root length with inoculation (RLI), without inoculation, (RLC) and RRL and explained 20.2%, 21.3%, and 11.9% of the phenotypic variation, respectively. CSSL33, in which substitution occurred in the vicinity region of qRRL-7, showed a completely different response to inoculation with B510 compared with Nipponbare. Therefore, we suggest that qRRL-7 might strongly control root growth in response to inoculation with Azospirillum sp. strain B510.

Lanthanide-Dependent Regulation of Methylotrophy in Methylobacteriumaquaticum Strain 22A.

Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca2+-dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF. This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La3+. We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La3+; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides. IMPORTANCE Lanthanides have been considered unimportant for biological processes. In methylotrophic bacteria, however, a methanol dehydrogenase (MDH) encoded by xoxF was recently found to be lanthanide dependent, while the classic-type mxaFI is calcium dependent. XoxF-type MDHs are more widespread in diverse bacterial genera, suggesting their importance for methylotrophy. Methylobacterium species, representative methylotrophic and predominating alphaproteobacteria in the phyllosphere, contain both types and regulate their expression depending on the availability of lanthanides. RNA-seq analysis showed that the regulation takes place not only for MDH genes but also the subsequent formaldehyde oxidation pathways and respiratory chain, which might be due to the direct oxidation of methanol to formate by XoxF. In addition, a considerable number of genes of unknown function, including AT-rich genes, were found to be upregulated in the presence of lanthanides. This study provides first insights into the specific reaction of methylotrophic bacteria to the presence of lanthanides, emphasizing the biological relevance of this trace metal.