Novel cultivated endophytic Verrucomicrobia reveal deep-rooting traits of bacteria to associate with plants

Despite the relevance of complex root microbial communities for plant health, growth and productivity, the molecular basis of these plant-microbe interactions is not well understood. Verrucomicrobia are cosmopolitans in the rhizosphere, nevertheless their adaptations and functions are enigmatic since the proportion of cultured members is low. Here we report four cultivated Verrucomicrobia isolated from rice, putatively representing four novel species, and a novel subdivision. The aerobic strains were isolated from roots or rhizomes of Oryza sativa and O. longistaminata. Two of them are the first cultivated endophytes of Verrucomicrobia, as validated by confocal laser scanning microscopy inside rice roots after re-infection under sterile conditions. This extended known verrucomicrobial niche spaces. Two strains were promoting root growth of rice. Discovery of root compartment-specific Verrucomicrobia permitted an across-phylum comparison of the genomic conformance to life in soil, rhizoplane or inside roots. Genome-wide protein domain comparison with niche-specific reference bacteria from distant phyla revealed signature protein domains which differentiated lifestyles in these microhabitats. Our study enabled us to shed light into the dark microbial matter of root Verrucomicrobia, to define genetic drivers for niche adaptation of bacteria to plant roots, and provides cultured strains for revealing causal relationships in plant-microbe interactions by reductionist approaches.


Supplementary Note 1 -Placement of strain LW23 in novel subdivision 8
The following data suggest that strain LW23 belongs to a novel subdivision 8: belonging to subdivision 6 neighbouring to strain LW23, were with a total length of 2.3 and 2.4 Mb at the lower range of this phylum, suggesting limited potential for metabolic and life style diversity. Also the G+C contents of both genomes were low (40.9 % and 45.5 %) 2,3 compared to strain LW23. Although Verrucomicrobium sp. strain GAS474 has a higher G+C content of 65.8% than methylotrophs and no methane monooxygenase gene, the genome size is also small (3,7 Mb) 4 . (iii) Physiological characteristics of the methanotrophic strains with low pH (1-3.5) and high temperature optima 1 did not correlate with growth characteristics of strain LW23 (Supplementary Table S2). (iv) Metagenomes of verrucomicrobia identified in a brackish microbiome (Verrucomicrobia BACL9 MAG) were also not related to LW23, because they clustered as a sister lineage of subdivision 6 5 or were proposed as a novel subdivision according to concatenated household protein sequences 6 .
They cluster with environmental sequences according to 16S rRNA gene phylogeny as second sister lineage of methylotrophs (Fig. 1A). (v) To reveal phylogenetic relationships with an independent whole-genome-based method, CVTree 7 was applied that uses peptide patterns, with K-values of 5-6 being most suitable for prokaryotes 7 . Verrucomicrobial subdivisions were well-separated from each other (Fig. 1B), and members of both lineages of methylotrophs were placed in the same subdivision 6. In contrast, isolate LW23 was only distantly related to them but clustered with Verrucomicrobiaceae bacterium GAS474 placed with methylotrophs in 16S rRNA analysis. This isolate from forest soil had been proposed as a novel branch 4 , but shared the small genome size with methylotrophs despite a relatively high G+C content (Supplementary Table S1). (vi) Genomes of both isolates did not harbour typical genes of methanotrophs encoding AmoA. Therefore, we propose description of a novel subdivision 8 that includes a large number of environmental bacteria, strain LW23 and probably strain GAS474.
The following data suggest that Astrumicrobium roseum strain LW23 belongs to a novel subdivision 8: (i) Methanotrophic strains (Methylacidimicrobium tartaophylax, M. cyclophantes and M. fagopyrum) from volcanic soil 1 in subdivision 6 were only distantly related according to phylogenetic reconstruction of almost complete 16S rDNA sequences (85-86 % sequence identity, supported by high bootstrap values) (Fig. 1A). (ii) Genome sizes and G+C contents in subdivision 6 strains (Methylacidiphilum infernorum V4 and M. fumariolicum strain SolV) were much lower (Supplementary Table 1). (iii) Physiological characteristics of the methanotrophic strains with low pH (1-3.5) and high temperature optima 1 did not correlate with growth characteristics of strain LW23 (Supplementary Table   2). (iv) Metagenomes of verrucomicrobia identified in a brackish microbiome (Verrucomicrobia BACL9 MAG) clustered as a sister lineage of subdivision 6 5 or were proposed as a novel subdivision according to concatenated household protein sequences 6 .
They cluster with environmental sequences according to 16S rRNA gene phylogeny as second sister lineage of methylotrophs (Fig. 1A). (v) To reveal phylogenetic relationships with an independent whole-genome-based method, CVTree 7 was applied that uses peptide patterns, with K-values of 5-6 being most suitable for prokaryotes 7 . Verrucomicrobial subdivisions were well-separated from each other ( Figure 1B), and members of both lineages of methylotrophs were placed in the same subdivision 6. In contrast, isolate Astrumicrobium roseum LW23 was only distantly related to them but clustered with Verrucomicrobiaceae bacterium GAS474 placed with methylotrophs in 16S rRNA analysis.
This isolate from forest soil had been proposed as a novel branch 4 , but shared the small genome size with methylotrophs despite a relatively high G+C content (Supplementary Tables S1, S3). (vi) Genomes of both isolates did not harbour typical genes of methanotrophs encoding AmoA. Therefore, we propose description of a novel subdivision 8 that includes a large number of environmental bacteria, strain LW23 and probably strain GAS474.

Supplementary Note 2 -Some physiological and plant-microbeinteraction related traits of verrucomicrobial isolates Nitrogen cycling
Microbial nitrogen cycling is instrumental for fertile soils and plant growth, and the new Verrucomicrobia may participate in several steps. The capability for nitrogen fixation was detected in only few cultivated members of the Verrucomicrobia, although their nitrogenase (nifH) genes are common in many habitats [8][9][10] . Only strains EW11 and LR76 were found to be equipped with the nifHDK cluster essential for nitrogen fixation, but did not possess the alternative anfHDK cluster. Most or all strains harboured genes for nitrate/nitrite transport and assimilation, and parts of denitrification pathway (Supplementary Table S7). Strains LW23 and ER46 possessed valine and leucine arylamidase (Supplementary Table S8), probably involved in the release of amino acids from soil organic matter.

Protein secretion systems
Bacterial protein secretion systems are involved in transport of enzymes into the environment or for the interaction with other prokaryotic or eukaryotic cells. Like in many other sequenced Verrucomicrobia, evidence for components of the Twin-arginine translocation (Tat) and Type II secretion pathway, the (Sec-SRP pathway) was found in all four genomes (Supplementary Table S7). In addition, two strains LW23 and EW11 harboured genes for the Type III secretion system, unusual for most other sequenced Verrucomicrobia (Supplementary Table S7). As it commonly secretes effector proteins into host cells, it is likely linked to the plant-associated lifestyle. a After assembly, the draft genomes were annotated by RAST 1-3 , and results were compared with data derived by the NCBI prokaryotic genome annotation pipeline (PGAP) 4 . Metabolic pathways were revised by the KEGG Automatic Annotation Server (KAAS (Moriya et al., 2007)) and metabolic pathway mapper. It was found that some annotations were unique to the RAST assignments and not identified by the PGAP. Generally, small differences in annotation results obtained from different pipelines are common 5 .    . Also the production of organic acids, e.g. citric acid can be efficient in solubilizing mineral phosphates. All strains possessed genes encoding a citrate synthase (see also Supplementary  Table S7). Genes encoding phosphate transporters (pstABC) were present in all draft genomes. The plate assay evaluating the capability to solubilize phosphate 2 , only showed a positive result for strain EW11 and a weak result for strain LR76. However, also the other strains might be able to solubilize phosphate in their natural environment since phosphate solubilisation is highly complex and dependent on different factors like the nutritional and growth condition of the culture 3 . Auxin production: Among the plant-growth promoting traits, synthesis of plant hormones such as indole-3-acetic acid (IAA) as a predominant auxin can lead e.g. to increased root development. Several bacterial pathways for IAA biosynthesis are described so far. One important precursor for the synthesis is L-tryptophan, which is present in root exudates 4,5 . A colorimetric assay based on the reaction of iron(III)-chloride and perchloric acid in presence of IAA formed from the added precursor L-tryptophan 6 , showed different results for the tested isolates. Weak or strong reactions were detected for strain LR76 or strains EW11 and ER46, respectively. However, genes for the classical tryptophan-dependent IAA synthesis (ipdC/ppdC coding for phenylpyruvate decarboxylase and 3-pyruvate decarboxylase) were not found in either of the draft genomes. Iron uptake: An universal chrome azurol S plate assay 7 , which indicates the production of siderophores by a colour change, showed a positive or a weak reaction for strains EW11 and LW23, respectively. Genomic analyses indicated the presence of genes for TonB-dependent receptor proteins for all strains, see also Supplementary Table S7. The specific gene for a TonBdependent siderophore receptor (fiu) was only found in strains LW23 and EW11