# Genome Sequence of the Streptomyces Strain AgN23 Revealed Expansion and Acquisition of Gene Repertoires Potentially Involved in Biocontrol Activity and Rhizosphere Colonization

Damien D. Gayrard, Clément Nicolle, Marine Veyssière, Kévin Adam, Yves Martinez, Céline Vandecasteele, Marie Vidal, Bernard Dumas, Thomas Rey

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Damien D. Gayrard, Clément Nicolle, Marine Veyssière, Kévin Adam, Yves Martinez, et al.. Genome Sequence of the Streptomyces Strain AgN23 Revealed Expansion and Acquisition of Gene Repertoires Potentially Involved in Biocontrol Activity and Rhizosphere Colonization. PhytoFrontiers, 2023, 10.1094/PHYTOFR-11-22-0131-R. hal-04244412

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Research

# Genome Sequence of the *Streptomyces* Strain AgN23 Revealed Expansion and Acquisition of Gene Repertoires Potentially Involved in Biocontrol Activity and Rhizosphere Colonization

Damien Gayrard<fnref n="1" />,<fnref n="2" /> | Clément Nicolle<fnref n="1" /> | Marine Veyssière<fnref n="1" /> | Kévin Adam<fnref n="1" /> | Yves Martinez<fnref n="3" /> |
Céline Vandecasteele<fnref n="4" /> [D] | Marie Vidal<fnref n="4" /> | Bernard Dumas<fnref n="1" />,⁺ | Thomas Rey<fnref n="1" />,<fnref n="2" />,⁺[D] |

Accepted for publication 22 January 2023.

**Author contributions:** D.G. performed the research and wrote the manuscript. M. Veyssière, C.N., K.A., Y.M., C.V., and M. Vidal performed the research. B.D. and T.R. designed the research and wrote the manuscript.

D. Gayrard and C. Nicolle contributed equally.

## Funding

This work was funded by the Fond Unique Interministériels (NEOPROTEC project), the Fonds Européen de Développement Économique et Régional (FEDER), the Agence Nationale de la Recherche (LabCom BioPlantProtec ANR-14-LAB7-0001 and STREPTOCONTROL ANR-17-CE20-0030), and the Région Occitanie (projet GRAINE-BioPlantProducts). The Laboratoire de Recherche en Sciences Végétales (LRSV) belongs to the TULIP Laboratoire d'Excellence (ANR-10-LABX-41) and benefits from the "Ecole Universitaire de Recherche (EUR)" TULIP-GS (ANR-18-EURE-0019). Work performed in the GeT core facility, Toulouse, France (https://get.genotoul.fr) was supported by the France Génomique National infrastructure, funded as part of the "Investissement d'Avenir" program managed by the Agence Nationale de la Recherche (contract ANR-10-INBS-09) and by the GET-PACBIO program (FEDER Programme opérationnel FEDER-FSE MIDI-PYRENEES ET GARONNE 2014-2020). D. Gayrard was funded by Agence Nationale de la Recherche Technique, with the Convention Industrielle de Formation par la Recherche and Association Nationale de la Recherche et de la Technologie (grant number 2016/1297). C. Nicolle was funded by the Ministère de l'Enseignement Supérieur et de la Recherche (PhD grant).

**e-Xtra:** Supplementary material is available online.

The following information may be seen as competing interests. B.D. is one of the inventors of the patent WO2015044585A1 relating to the use of AgN23 in agriculture. T.R. and D.G. are full-time researchers at the AgChem company De Sangosse (Pont-Du-Casse, France), which registers and markets crop-protection products and owns the patent WO2015044585A1. M. Veyssière, M. Vidal, C.N., K.A., Y.M., and C.V. do not declare any competing interests.

## Abstract

Streptomycetes are gram-positive actinobacteria largely represented in the plant root microbiota. The genetic determinants involved in the adaption of *Strepto-myces* in the rhizosphere environment are mostly unknown but can rely on the ability to release phytohormones, degrade plant cell-wall polysaccharides, and produce specialized metabolites notably involved in microbial competition. Here, we sequenced the genome of the rhizospheric and plant defense-stimulating strain *Streptomyces* sp. AgN23. We found that it belongs to the soil- and plant root-dwelling *S. violaceusniger* clade. The genome annotation of AgN23 revealed the genetic potential of the bacterium to degrade the plant cell wall with a large repertoire of carbohydrate degrading enzymes, to synthesize auxin, a major regulator of plant development, and to produce antimicrobials (rustmicin, mediomycin, niphimycin, nigericin) and plant bioactive compounds (nigericin, echosides, elaiophylin) through a set of biosynthetic gene clusters. We also found that these genomic features are well conserved among members of the *S. violaceusniger* clade. In addition, AgN23 displays original events of biosynthetic gene cluster acquisitions and losses, which may account for its interaction with plants. Taken together, our work supports the hypothesis that evolution of a large set of conserved hydrolytic enzymes directed against plant polymers and specialized metabolite repertoires can mediate the adaptation of *S. violaceusniger* strains to the rhizospheric ecological niches.

**Keywords:** biosynthetic gene clusters, carbohydrate-active enzymes, long-read sequencing, phylogenomic, specialized metabolites, *Streptomyces*, transcriptome

Streptomycetes are aerobic and gram-positive actinobacteria forming branched vegetative mycelia before developing aerial hyphae-bearing spores (Bush et al. 2015). These bacteria have received considerable attention from a biotechnological point of view, notably regarding their enzymatic repertoire (Book et al. 2016) and in the drug discovery

<footnote n="1">¹ Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, Toulouse INP, Auzeville-Tolosane, France</footnote>

<footnote n="2">² De Sangosse, Bonnel, Pont-Du-Casse, France</footnote>

<footnote n="3">³ CNRS, Plateforme Imagerie-Microscopie, Fédération de Recherche FR3450, Castanet-Tolosan, France</footnote>

<footnote n="4">⁴ INRAE, US 1426, GeT-PlaGe, Genotoul, Castanet-Tolosan, France</footnote>

<footnote>† Corresponding authors: T. Rey, reyt@desangosse.com, and B. Dumas; bernard.dumas@univ-tlse3.fr</footnote>
field, leading to the structure elucidation of more than 6,000 spe-
cialized metabolites (Barka et al. 2016; Moumbock et al. 2021).
Their tremendous ability to produce antimicrobial compounds
relies on the wealth and diversity of biosynthetic gene clusters
(BGCs) found in their genomes (Lee et al. 2020). The recent
soaring of microbial metabarcoding approaches have highlighted
*Streptomyces* species' prominent abundance in plant root mi-
crobiota (Fitzpatrick et al. 2018; Lundberg et al. 2012). Plants
release 11 to 40% of total fixed carbon (C) through photosynthe-
sis into the surrounding soil root environment. Thus, the rhizo-
sphere is notably enriched in complex carbohydrate compounds,
meaning that access to this resource depends on the ability to
degrade them. Other factors that can favor the adaptation of mi-
crobial species to this environment rely on the competition with
other microbial strains. Because several of the metabolite strepto-
mycetes produced display strong antimicrobial activities, *Strep-
tomyces* recruitment in the rhizosphere and inside root tissues
presumably help them to fight against competitors and protect the
plant from pathogens. Consequently, *Streptomyces*-based prod-
ucts have been developed for agriculture, but these strains only
cover a subset of plant-colonizing *Streptomyces* species (Hamedi
and Mohammadipanah 2015; Vurukonda et al. 2018). Notwith-
standing, little is known regarding the biological function of
these bacteria in the plant environment and the gene families
involved in their adaptation to this ecological niche (Rey and
Dumas 2017; Viaene et al. 2016). More than 100 fully assembled
genome sequences of *Streptomyces* strains are currently avail-
able, paving the way for genome-based phylogeny and compar-
isons of BGC content across *Streptomyces* clades (Kautsar et al.
2021a; Medema 2021; Tracanna et al. 2017). This knowledge
opens inroads to rationalize and investigate the potential use of
*Streptomyces* strains in agriculture.

Here, we report a gapless assembly of *Streptomyces* sp. AgN23 (AgN23), previously isolated from grapevine rhizosphere and identified as a strong inducer of plant defenses (Vergnes et al. 2019). We used this high-quality assembly to position AgN23 in the *S. violaceusniger* genospecies. We then dissected the original features in the CAZyme and BGC content of AgN23 and other *S. violaceusniger* strains and shed light on specifici- ties in the specialized metabolism and carbohydrate degradation capabilities of this lineage. This comparative genomic approach also led us to identify original features in the genome of AgN23, such as putative BGC losses and acquisition by horizontal gene transfer, which could account for the bacteria adaptation in the rhizospheric niche.

MATERIALS AND METHODS

AgN23 cultivation and high molecular weight DNA extraction

The AgN23 strain was cultivated as described previously
(Errakhi et al. 2007; Vergnes et al. 2019). In brief, the strain was
grown on solid modified Bennet medium (D-glucose 10 g/liter;
soybean peptones 2.5 g/liter; yeast extract 1.5 g/liter; agar
16 g/liter) or International Streptomyces Project media ISP2,
ISP3, ISP4, and ISP5 (Shirling and Gottlieb 1966; Kieser et al.
2000). To produce the spore inoculum, we incubated Bennet
plates for 2 weeks at 22°C in the darkness before filling them
with 10 ml of sterile water. The mycelium was scraped with a
spreader, and the resulting solution was filtered in a 50-ml sy-
ringe filled with nonabsorbent cotton wool. For DNA extraction,
the AgN23 mycelium was grown at 28°C and under 250 rpm
in 250-ml Erlenmeyer flasks containing 50 ml of liquid Ben-
net. Approximately 100 mg of AgN23 pellets was collected by
centrifugation at 11,000 × g and flash frozen in liquid nitrogen.

Genomic DNA was isolated using the Nucleobond RNA/DNA kit (Macherey-Nagel) according to the manufacturer's instructions.

Library preparation for genome sequencing

Library preparation and sequencing were performed at the GeT-PlaGe core facility (Castanet-Tolosan), according to the PacBio instructions, with a 15-kb size cutoff (“20 kb Template Preparation Using BluePippin Size Selection system”). At each step, DNA was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies). DNA purity was tested using the Nano-Drop (Thermo Fisher), and size distribution and degradation were assessed using the High-Sensitivity Large Fragment 50-kb Analysis Kit with a fragment analyzer (AATI). Purification steps were performed using 0.45 × AMPure PB beads (PacBio). A total of 10 µg of DNA was purified then sheared at 40 kb using the Megaruptor system (Diagenode). Single-molecule real-time (SMRT) sequencing was performed using SMRTBell Template Prep Kit 1.0 (PacBio), and a DNA and END damage repair step was performed on 5 µg of sample. Then, blunt hairpin adapters were ligated to the library. The library was treated with an exonuclease cocktail to digest unligated DNA fragments. A size selection step using a 10-kb cutoff was performed on the BluePippin Size Selection system (Sage Science) with 0.75% agarose cassettes, Marker S1 high Pass 15-20 kb. Conditioned Sequencing Primer V2 was annealed to the size-selected SMRTbell. The annealed library was then bound to the P6-C4 polymerase using a 10:1 ratio of polymerase to SMRTbell. Then, after a magnetic bead-loading step (OCPW), SMRTcell libraries were sequenced on two SMRT cells on an RSII instrument at 0.18 to 0.23 nM with a 360-min movie. The initially generated raw sequencing reads were evaluated in terms of the average quality score at each position, GC content distribution, quality distribution, base composition, and other metrics. Sequencing reads with low quality were also filtered out before the genome assembly and annotation of gene structure. Finally, microbial DNA potential contamination was excluded after comparison by BLAST of the draft assembly of the first SMRT cell against a 16S ribosomal RNA sequences data bank (Bacteria and Archaea).

Genome assembly, annotation, and comparative genomics

The subreads were assembled with PacBio's SMRT analysis
software version 2.3.0 using default settings with a minimum
subread length of 3 kb to exclude smaller sequenced reads and
a read score of better than 0.8 to enrich in reads with a low
error rate. The single unitig obtained by long-read sequencing
was corrected with Mi-Seq (Illumina) data using Pilon (version
1.21), resulting in 165 substitutions and two deletions of 44 and
5 bases. This final genome assembly was retained for subsequent
analysis. The gene annotation was performed on the MicroScope
Microbial Genome Annotation & Analysis Platform (Vallenet
et al. 2020). Genome completion was checked with CheckM and
BUSCO (Parks et al. 2015; Simão et al. 2015). CAZy genes an-
notation was obtained by running the HMMER tool (e-value <
1e-15, coverage > 0.35) on the dbCAN2 meta server (Yin et al.
2012). For the annotation of auxin biosynthesis genes, we re-
trieved the protein sequences from UniProt corresponding to the
KEGG ontologies associated with the different biosynthetic steps
described in Zhang et al. (2019). These sequences were systemat-
ically used as queries for BLASTx analysis against AgN23 chro-
mosome sequences. The position on the AgN23 chromosome
of any hits showing more than 70% identity and 40% coverage
with one query was used to determine the corresponding AgN23
gene model (Zhang et al. 2019). antiSMASH 6.0 was used to
detect BGC-containing regions in the AgN23 chromosome and annotate detected sequences based on the MIBiG 2.0 repository (Blin et al. 2019; Epstein et al. 2018). The comparative genomics were performed with BiG-SCAPE using default parameters with the antiSMASH 6.0-predicted region-containing BGCs as input data to produce similarity networks (Navarro-Muñoz et al. 2020). Complementarily, the BiG-SLiCE software (1.0.0) was used to identify BGCs showing similarity to AgN23 regions annotated by antiSMASH among the 1,225,071 BGCs stored in the BiG-FAM database (Kautsar et al. 2021b). BiG-SLICE was used with standard parameters, including the arbitrary clustering threshold (T = 900.0).

## Phylogenetic and phylogenomic analysis

The phylogenetic analysis of AgN23 was performed using genome sequences previously assigned to the *S. violaceusniger* clade (*Streptomyces* sp. M56, *Streptomyces* sp. 11-1-2, *S. rapamycinicus* NRRL 5491, *S. malaysiensis* DSM4137, *S. antimycoticus* NBRC 100767, *S. sabulosicollis* PRKS01-29, *S. albi-flaviniger* DSM 41598, *S. rhizosphaericus* NRRL B-24304, *S. javensis* DSM 41764, and *S. milbemycinicus* NRRL (Díaz-Cruz et al. 2022; Goodfellow et al. 2007; Klassen et al. 2019; Kumar et al. 2007; Kusuma et al. 2021). In addition, we introduced as input the genomes of the model strain *S. coelicolor* A(3)2 and two biocontrol strains, *S. lydicus* WYEC108 and *S. griseoveridis* K61, as well as *Frankia alni* ACN14a as an outgroup (Supplementary Information S1). The *Streptomyces* phylogeny was built using a multi-locus sequence typing (MLST) strategy with the de novo workflow of autoMLST (Alanjary et al. 2019). The concatenated alignment of 85 single-copy conserved genes was built using the Fast alignment mode (MAFFT FFT-NS-2), and the IQ-TREE Ultrafast Bootstrap analysis was performed with 1,000 replicates (Supplementary Information S2) (Alanjary et al. 2019).

Finally, a whole-genome-based phylogenetic tree based on fully assembled chromosomes of isolates from the *S. violaceusniger* clade was inferred with FastME 2.1.6.1 using the distance calculated by genome blast distance phylogeny (GBDP) available on the TYGS platform (Lefort et al. 2015; Meier-Kolthoff and Göker 2019). The branch lengths were scaled in terms of GBDP distance formula d5. The tree had an average branch support of 96.9% after 100 bootstrap replicates and was rooted at midpoint.

## Library preparation for transcriptome sequencing and expression analysis

AgN23 was cultivated in liquid Bennett medium at 28°C for 48 h to reach the exponential phase of the growth. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. Three replicates were prepared for the construction of the libraries. rRNA depletion was performed using the Ribozero rRNA Removal Kit (Illumina). RNA sequencing library preparation was completing using the NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer's recommendations (NEB). RNAs were fragmented to generate double-stranded cDNA and subsequently ligated to a universal adapter, followed by index addition and library enrichment with limited-cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies) and quantified by the Qubit 2.0 Fluorometer (Invitrogen), as well as quantitative PCR (Applied Biosystems). RNA-Seq experiments were performed on an Illumina HiSeq4000 using a paired-end read length of 2 × 150 bp with the Illumina HiSeq4000 sequencing kits. The mapping and statistical analysis were performed using the bioinformatics pipeline implemented in the MicroScope Platform (Vallenet et al. 2020) (Supplementary Information S3). Raw reads of each sample (R1 fastq files from the paired-end run) were mapped onto the AgN23 reference genome with BWA-MEM (v.0.7.4) (Li 2013). An alignment score equal to at least half of the read was required for a hit to be retained. SAMtools (v.0.1.8) was then used to extract reliable alignments with a mapping quality (MAPQ) ≥ 1 from SAM formatted files (Li et al. 2009). The number of reads matching each genomic object of the reference sequence was subsequently counted with the toolset BEDTools (v.2.10.1) (Quinlan and Hall 2010). The mean transcripts per million (TPM) for each gene was then calculated from the three independent samples (Supplementary Information S3). The BGC expression levels were determined by calculating the mean expression of biosynthetic genes for each BGC as defined by antiSMASH and listed in Supplementary Information S4. A mean expression for each BGC across the three biological repetitions was then determined.

## Scanning electron microscopy

The observation of AgN23 mycelium and spore development by scanning electron microscopy was performed on a Quanta 250 FEG (FEI). Agar plugs of 2-week-old AgN23 cultures were placed on micrometric platen, frozen in liquid nitrogen, and finally metallized with platinum. The samples were observed micromanipulatively at an accelerating voltage of 5.00 kV.

## RESULTS AND DISCUSSION

### Chromosome-scale assembly of AgN23 and genome-based taxonomic assignation to the *S. violaceusniger* clade

We performed PacBio RSII long-read sequencing and obtained a linear chromosome of 10.86 Mb for AgN23. This genome sequence was polished using Illumina MiSeq sequences to produce the final assembly. The final assembly was uploaded to the MicroScope Platform (Vallenet et al. 2020). The genome annotation retrieved 10,514 protein coding sequences, 10,458 of them being supported by RNA-seq with expression levels comprising 0.09 to 20,661 TPM and mean and median values of 94 and 21 TPM, respectively (Supplementary Information S3) (Table 1). A checkM analysis was performed using 455 genomes and 315 lineage-specific markers and validated the completeness of the assembly and the annotation (Table 1) (Parks et al. 2015). Complementarily, the completeness was also supported by a BUSCO analysis that detected 99.7% of the genes that are expected in the genome of an actinobacterium (Table 1) (Simão et al. 2015).

<table><caption>TABLE 1</caption><caption>Summary of the assembly and the annotation of <i>Streptomyces</i> sp. AgN23 complete chromosome obtained by PacBio and Illumina sequencing</caption><thead><tr><th>Assembly statistics</th><th><i>Streptomyces</i> sp. AgN23</th></tr></thead><tbody><tr><td>Genome size (bp)</td><td>10,858,739</td></tr><tr><td>Coverage (PacBio)</td><td>77x</td></tr><tr><td>G + C content (%)</td><td>70.9</td></tr><tr><td>Number of protein-coding genes</td><td>10,514</td></tr><tr><td>CheckM completeness</td><td>100%</td></tr><tr><td>Contamination</td><td>1.84%</td></tr><tr><td>BUSCO</td><td>451/452</td></tr><tr><td>CAZymes</td><td>278</td></tr><tr><td>Number of biosynthetic gene clusters (antiSMASH 6.0)</td><td>47</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>FIGURE 1</figcaption></figure>

Multilocus sequence typing assigned AgN23 to the *Streptomyces violaceusniger* clade. Phylogenetic tree based on the multiple alignment of 85 single-copy homologous genes selected from genomic sequences with AutoMLST. The green node highlights the isolates considered to be from the same species (average nucleotide identity [ANI] > 95%). The black node highlights the clade formed by isolates with ANI > 90% as compared with AgN23. The black squares highlight AgN23 and the 18 strains that were used for the biosynthetic gene cluster (BGC) conservation study. The green logo indicates plant-isolated strains. *Frankia alni* ACN14a was used as the outgroup, bootstrap = 100.
More than 500 *Streptomyces* species have been described based on their 16S rRNA sequences. Previous sequencing of AgN23 16S rRNA showed the strongest conservation with representatives of the *S. violaceusniger* clade, most notably *S. castarensis* (Vergnes et al. 2019). However, lack of variation in 16S rRNA may confound strains belonging to different species (Labeda et al. 2012). Recent development of long-read technologies and massive sequencing of *Streptomyces* have leveraged genome-based phylogenies (Lee et al. 2020). Thus, we decided to consolidate the taxonomic affiliation of AgN23 with the gapless assembly of the AgN23 chromosome. We performed an autoMLST approach based on publicly available *Streptomyces* sequences. In brief, 85 conserved housekeeping genes showing neutral dN/dS were concatenated and aligned as a basis for tree building (Fig. 1, Supplementary Information S2) (Alanjary et al. 2019).

As a result, using AgN23 as the query, we affiliated the strain to a clade containing six other strains showing an average nucleotide identity (ANI) higher than 95% with AgN23, which is conventionally considered a threshold for species delimitation (Jain et al. 2018). It contains the closely related *S. melanosporofaciens* DSM 40318, *S. antimycoticus* NBRC 100767, *S. violaceusniger* NRRL F-8817, *S. violaceusniger* Tu 4113, *S. hygroscopicus* XM201, and *Streptomyces* sp. 11-1-2 (Caicedo-Montoya et al. 2021; Komaki and Tamura 2020). In total, 28 isolates harbored an ANI > 85% with *Streptomyces* sp. AgN23. Notably, this group contains representative species of the *S. violaceusniger* clade, such as *S. hygroscopicus*, *S. sparsogenes*, *S. malaysiensis* (Goodfellow et al. 2007), *S. himastatinicus*, *S. rapamycinicus*, and other close species *S. autolyticus* (Yin et al. 2017), *S. antioxidans* (Ser et al. 2016), *S. iranensis* (Hamedi et al. 2010), *S. albiflaviniger*, *S. rhizosphaericus*, *S. sabulosicollis*, *S. javensis*, and *S. milbemycinicus* (Kusuma et al. 2021) (Supplementary Information S5). Interestingly, *Streptomyces* strain RT-d22 (Chagas et al. 2016), *Streptomyces* sp. strain PRh5 (Yang et al. 2014), *S. hygroscopicus* Osish-2 (Cao et al. 2021; Gao et al. 2021; Xu et al. 2017, 2019; Zeng et al. 2018), *Streptomyces* sp. NBRC 109436 (Komaki et al. 2016), and *S. rhizosphaericus* NRRL B-24304 (Goodfellow et al. 2007) were isolated from the rhizosphere of diverse plants across the world. Similarly, *Streptomyces* sp. 11-1-2 was isolated from the scab of a potato tuber (Bown and Bignell 2017; Díaz-Cruz et al. 2022). This suggests frequent interactions with plants of the strains from the *S. violaceusniger* clade. Finally, morphological characterization of AgN23 on a range of ISP media confirmed that AgN23 displays typical phenotypes of the *S. violaceusniger* clade regardless of the ISP media tested, with a whitish colony that turns gray during the sporulation process, resulting in the formation of spiraled chains of rugose-ornamented spores (Fig. 2) (Goodfellow et al. 2007; Kumar and Goodfellow 2008; Kumar et al. 2007).

## AgN23 exhibits a wide repertoire of CAZymes related to plant cell wall degradation

Streptomycetes display extensive abilities to degrade polysaccharides, notably plant polysaccharides derived from the cell wall, such as cellulose, hemicellulose, and pectins based on their rich repertoires of carbohydrate-active enzymes (CAZymes) (Book et al. 2016; Yin et al. 2012). We annotated the AgN23 CAZymes with dbCAN2 and found 278 CAZymes representing 2.6% of AgN23 coding sequences (Supplementary Information S6). In *Streptomyces*, the amount of CAZymes typically ranges from 1.5 to 3% of the proteome (Book et al. 2016). For example, CAZymes represents 2.9% of coding sequences of the cellulolytic *Streptomyces* sp. SirexAA-E; thus, the AgN23 genome harbors a rather large repertoire of CAZymes among the *Streptomyces* gen-

era (Book et al. 2016). To investigate whether AgN23 displays an expanded repertoire in particular CAZymes families, we compared it with the repertoire of CAZymes from the highly cellulolytic *Streptomyces* sp. SirexAA-E and the 50 genomes used in the phylogenomic analysis, which comprised *S. violaceusniger* representatives, as well as outgroup strains (Fig. 3).

We found that AgN23 and other members of the *S. violaceusniger* clade possess two PL9 polysaccharide lyases involved in pectin degradation that are absent from 20 of the 22 *Streptomyces* outgroup genomes. Similarly, the AgN23 genome harbors seven GH6 (glycoside hydrolases) that are endoglucanases related to cellulose degradation. Such a large repertoire of GH6 is frequent in the *S. violaceusniger* clade (ANI > 85%), whereas zero to four genes were found in the outgroup strains tested. A similar observation could be drawn for GH43 and GH95 involved in hemicellulose catabolism. Strikingly, the AgN23 genome encodes nine GH43, whereas the other *S. violaceusniger* spp. possess up to 15 of these genes, and eight strains among the outgroup (ANI < 85%) had none.

Finally, we analyzed the expression pattern of AgN23 CAZymes, consistently with the whole-genome expression analysis, and only three CAZymes showed no expression in the Bennett liquid culture dataset (Supplementary Information S6). Strikingly, two genes of the PL9 family (AgN23_3063 and AgN23_1031), which is largely restricted to the *S. violaceusniger* group, showed a strong expression at 526 and 279 TPM, respectively. These values rank the two genes among the 10% most expressed CAZymes of AgN23. By contrast, the PL1 representatives of AgN23_3657 and AgN23_1017 have 9 and 8 TPM, respectively. Regarding the GH6 and GH43 families, we no-

<figure><img src="image_4.png" /><figcaption>FIGURE 2</figcaption></figure>

AgN23 harbors typical phenotypic features of the *Streptomyces violaceusniger* clade. A, AgN23 forms a white mycelium that turns gray at the onset of sporulation as observed on a range of ISP media; the Petri plates are 9 cm in diameter, and the scale bar is 1 mm. B, Scanning electron microscopy observation of spiraled chains of spores (white arrow) formed by AgN23 (scale bar = 6 µm). C, Scanning electron microscopy observation of AgN23 spores showing a rugose-ornamented surface (scale bar = 6 µm).
ticed that they are dominated by the expression of one single gene, respectively AgN23_4221 (TPM = 199) and AgN23_1548 (TPM = 537), which also belongs to the top 10% in terms of CAZyme expression in AgN23. Taken together, these genomic and transcriptomic data support the hypothesis that AgN23 could degrade plant cell wall cellulose, hemicelluloses, and pectins to use them as carbon sources, a feature which could represent a major advantage in the rhizosphere niche. This hypothesis should be strengthened by investigating CAZyme gene expression patterns in the frame of plant co-cultivation with AgN23.

## The AgN23 genome encodes the biosynthesis of antifungal and plant bioactive metabolites

Specialized metabolites are presumed to play key roles in the adaption of streptomyces to their environment. We used antiSMASH 6.0 to detect and annotate AgN23 BGCs according to their similarity to reference clusters deposited in the MIBiG database (Blin et al. 2019; Epstein et al. 2018). Forty-seven BGCs

were detected in the AgN23 genome, all of them being expressed during AgN23 cultivation in the Bennett liquid medium except for region 47 (Table 2, Supplementary Information S4). This result is somehow surprising because it has been shown in many *Streptomyces* strains that the expression of BGCs is tightly regulated, and only a few of them are expressed in standard cultivation media. This suggests particular regulatory mechanisms of the expression of AGN23 BGCs. Only region 20, putatively involved in pigment biosynthesis, stood out in terms of expression with 3,330 TPM, all the others ranging from a few to 200 TPM. Twenty BGCs of AgN23 showed at least 50% similar gene content with MIBiG reference BGCs (Table 2). These candidate BGCs are notably involved in the biosynthesis of volatile terpenes (regions 3 and 10), including geosmin (region 23), indoles (region 21), ribosomally synthesized and post-translationally modified peptide-like (RiPP-like, regions 5 and 28), ochronotic pigment (region 20), and a bicyclomycin-like antibacterial (region 45). Other BGCs are potentially involved in the production of siderophores, including coelichelin and desferrioxamin (regions

<figure><img src="image_5.png" /><figcaption>FIGURE 3</figcaption></figure>

Plant cell wall-associated carbohydrate-active enzymes (CAZymes) repertoire is expanded in *Streptomyces violaceusniger* sp. as compared with *S. coelicolor* and *Streptomyces* sp. SirexAA-E. CAZymes families with at least two genes in AgN23 are ordered according to their target polymers and the type of enzymatic activity: glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and auxiliary activities (AAs). The transcriptions of AgN23 CAZymes are displayed as the mean transcript per million (TPM) reads for each family (n = 3).
24, 27, and 44), or osmotic and cold-stress protectants (ectoin, region 15). Additional BGCs are likely involved in the regulation of the bacterium life cycle, such as spore pigment (region 31), hopene (region 32), and butyrolactone (region 37). BGCs encoded in regions 2, 17, 41, and 42 are similar to the ones belonging to the biosynthesis pathways of the antifungal compounds rustmicin, also known as galbonolide A, mediomycin A, nigericin, and niphimycins C-E, respectively (Cai et al. 2007; Harvey et al. 2007; Hu et al. 2018; Kim et al. 2014). In addition, echoside (region 26), elaiophylin (region 40), and nigericin (region 41) are structural analogues of terfestatin, pteridic acid, and monensin, respectively, three compounds affecting plant immunity and development (Hayashi et al. 2008; Igarashi et al. 2002; Maintz et al. 2018; Wang et al. 2015; Yamazoe et al. 2005).

In addition to the BGC-mediated production of specialized metabolites, some *Streptomyces* spp. produce auxin, a phytohormone with a strong impact on root growth (Rashad et al. 2015). We undertook the annotation of auxin biosynthesis genes of AgN23 by retrieving protein sequences associated with KEGG ontologies, leading to the biosynthesis of indole-3 acetic acid (IAA), the most common plant auxin, from its precursor tryptophane through the indole-3-acetamide (IAM) pathway, the indole-3-acetaldoxime/indole-3-acetonitrile (IAN/IAOx) pathway, and the tryptamine (TAM) or indole-3-pyruvate (IPyA) pathway, both leading to indole-3-acetaldehyde (IAAld) before its conversion to IAA (Zhang et al. 2019). As a result, we identified a complete biosynthetic route for the IAM pathway (AgN23_8393 and AgN23_8392), the TAM-IAAld pathway (AgN23_3181, AgN23_0775, AgN23_0524, and AgN23_0525), and the IPyA

<table><caption>TABLE 2
antiSMASH annotation of AgN23 chromosomal regions coding for biosynthetic gene clusters (BGCs)<sup>a</sup></caption><thead><tr><th>Region</th><th>BGC type</th><th>Putative compound (MIBiG similarity score)</th><th>MIBiG accession</th><th>Species</th><th>Transcripts per million</th></tr></thead><tbody><tr><td>1</td><td>NRPS</td><td>Atratumycin (7%)</td><td>BGC0001975</td><td>S. atratus</td><td>13</td></tr><tr><td>2</td><td>PKS-like, terpene</td><td>Rustmicin (20%)</td><td>BGC0000065</td><td>S. galbus KCCM 41354</td><td>15</td></tr><tr><td>3</td><td>Terpene</td><td>Tiancilactone (17%)</td><td>BGC0002019</td><td>Streptomyces sp. CB03234</td><td>7</td></tr><tr><td>4</td><td>NRPS</td><td>Dechlorocuracomycin (8%)</td><td>BGC0001569</td><td>S. noursei ATCC 11455</td><td>74</td></tr><tr><td>5</td><td>RiPP-like</td><td></td><td></td><td></td><td>13</td></tr><tr><td>6</td><td>T1PKS, hglE-KS</td><td>Leinamycin (2%)</td><td>BGC0001101</td><td>S. atroolivaceus</td><td>8</td></tr><tr><td>7</td><td>NRPS-like</td><td></td><td></td><td></td><td>6</td></tr><tr><td>8</td><td>T1PKS, NRPS</td><td>Meridamycin (52%)</td><td>BGC0001011</td><td>Streptomyces sp. NRRL 30748</td><td>14</td></tr><tr><td>9</td><td>Terpene</td><td>2-methylisoborneol (100%)</td><td>BGC0000658</td><td>S. griseus NBRC 13350</td><td>31</td></tr><tr><td>10</td><td>Terpene</td><td>Pristinol (100%)</td><td>BGC0001746</td><td>S. pristinaespiralis ATCC 25486</td><td>10</td></tr><tr><td>11</td><td>NRPS-like, T1PKS</td><td>Amipurimycin (90%)</td><td>BGC0001957</td><td>S. novoguineensis</td><td>26</td></tr><tr><td>12</td><td>T1PKS, T3PKS, NRPS-like, NRPS, betalactone</td><td>Totopotensamide A / totopotensamide B (43%)</td><td>BGC0001807</td><td>S. pactum SCSI0 02999</td><td>22</td></tr><tr><td>13</td><td>Lanthipeptide-class-iii</td><td>Lipopolysaccharide (43%)</td><td>BGC0000774</td><td>Xanthomonas campestris</td><td>18</td></tr><tr><td>14</td><td>T1PKS, siderophore</td><td>Apoptolidin (23%)</td><td>BGC0000021</td><td>Nocardiopsis sp. FU 40</td><td>24</td></tr><tr><td>15</td><td>Ectoine</td><td>Ectoine (100%)</td><td>BGC0000853</td><td>S. anulatus</td><td>43</td></tr><tr><td>16</td><td>Terpene</td><td></td><td></td><td></td><td>260</td></tr><tr><td>17</td><td>T1PKS</td><td>Mediomycin A (68%)</td><td>BGC0001932</td><td>Kitasatospora mediocidica</td><td>10</td></tr><tr><td>18</td><td>RRE-containing</td><td>Granaticin (10%)</td><td>BGC0000227</td><td>S. violaceoruber</td><td>25</td></tr><tr><td>19</td><td>Ladderane</td><td>Atratumycin (31%)</td><td>BGC0001975</td><td>Streptomyces atratus</td><td>14</td></tr><tr><td>20</td><td>NRPS</td><td>Ochronotic pigment (75%)</td><td>BGC0000918</td><td>S. avermitilis</td><td>3,330</td></tr><tr><td>21</td><td>Indole</td><td></td><td>BGC0001483</td><td>Streptomyces sp. RM-5-8</td><td>4</td></tr><tr><td>22</td><td>NRPS</td><td>RP-1776 (46%)</td><td>BGC0000429</td><td>Streptomyces sp. Acta 2897</td><td>87</td></tr><tr><td>23</td><td>Terpene</td><td>Geosmin (100%)</td><td>BGC0001181</td><td>S. coelicolor A3(2)</td><td>182</td></tr><tr><td>24</td><td>Siderophore</td><td>Desferrioxamin B (100%)</td><td>BGC0000941</td><td>S. griseus NBRC 13350</td><td>8</td></tr><tr><td>25</td><td>Terpene</td><td>Carotenoid (63%)</td><td>BGC0000633</td><td>S. avermitilis</td><td>155</td></tr><tr><td>26</td><td>NRPS-like</td><td>Echosides (100%)</td><td>BGC0000340</td><td>Streptomyces sp. LZ35</td><td>33</td></tr><tr><td>27</td><td>Siderophore</td><td></td><td></td><td></td><td>17</td></tr><tr><td>28</td><td>RiPP-like</td><td></td><td></td><td></td><td>13</td></tr><tr><td>29</td><td>Redox-cofactor</td><td></td><td></td><td></td><td>37</td></tr><tr><td>30</td><td>NRPS</td><td>Formicamycins A-M (18%)</td><td>BGC0001590</td><td>Streptomyces sp. KY5</td><td>32</td></tr><tr><td>31</td><td>T2PKS</td><td>Spore pigment (83%)</td><td>BGC0000271</td><td>S. avermitilis</td><td>141</td></tr><tr><td>32</td><td>Terpene</td><td>Hopene (76%)</td><td>BGC0000663</td><td>S. coelicolor A3(2)</td><td>47</td></tr><tr><td>33</td><td>Lanthipeptide-class-i</td><td>Steffimycin D (16%)</td><td>BGC0000273</td><td>S. steffisburgensis</td><td>29</td></tr><tr><td>34</td><td>Other</td><td>Mitomycin (16%)</td><td>BGC0000915</td><td>S. lavendulae</td><td>71</td></tr><tr><td>35</td><td>NRPS</td><td>Cadaside A / cadaside B (14%)</td><td>BGC0001968</td><td>Uncultured bacterium</td><td>67</td></tr><tr><td>36</td><td>T1PKS</td><td></td><td></td><td></td><td>80</td></tr><tr><td>37</td><td>Butyrolactone</td><td></td><td></td><td></td><td>6</td></tr><tr><td>38</td><td>Hserlactone</td><td>Daptomycin (3%)</td><td>BGC0000336</td><td>S. filamentosus NRRL 11379</td><td>76</td></tr><tr><td>39</td><td>Redox-cofactor</td><td></td><td></td><td></td><td>42</td></tr><tr><td>40</td><td>T1PKS</td><td>Elaiophylin (87%)</td><td>BGC0000053</td><td>Unknown</td><td>49</td></tr><tr><td>41</td><td>T1PKS</td><td>Nigericin (100%)</td><td>BGC0000114</td><td>S. violaceusniger</td><td>55</td></tr><tr><td>42</td><td>T1PKS</td><td>Niphimycins C-E (87%)</td><td>BGC00001700</td><td>Streptomyces sp. IMB7-145</td><td>13</td></tr><tr><td>43</td><td>NRPS-like</td><td>BD-12 (67%)</td><td>BGC0001379</td><td>S. luteicolor</td><td>5</td></tr><tr><td>44</td><td>NRPS</td><td>Coelichelin (90%)</td><td>BGC0000325</td><td>S. coelicolor A3(2)</td><td>12</td></tr><tr><td>45</td><td>CDPS</td><td>Bicyclomycin (100%)</td><td>BGC0001468</td><td>S. cinnamoneus</td><td>5</td></tr><tr><td>46</td><td>Betalactone</td><td></td><td></td><td></td><td>13</td></tr><tr><td>47</td><td>NRPS-like</td><td>Naphthyridinomycin (7%)</td><td>BGC0000394</td><td>Streptomyces lusitanus</td><td>0</td></tr></tbody></table>

<footnote><sup>a</sup> The functional category of each BGC was determined by antiSMASH. The BGC type and best hit in the MIBiG database as its percentage of similarity to the query are indicated, along with the bacterial strain from which the cluster was described. The expression level of each BGC was determined by determining the mean of the expression level in transcripts per million (TPM) of the core biosynthetic genes of each BGC from the RNA-seq data.</footnote>
to IAAld pathway (AgN23_1600), as well as enzymes converting IAN to IAM (AgN23_1182 and AgN23_1183) (Fig. 4). All those enzymes were found to be expressed in our transcriptome analysis of the bacteria pure culture (Supplementary Information S7). Taken together, these data reveal that AgN23 likely produces specialized metabolites and auxins with a potential to regulate host plant biology along with its root microbiota.

## A core set of BGCs conserved across S. violaceusniger species

The *S. violaceusniger* clade hosts several strains possessing BGCs involved in the synthesis of antifungal polyene macrolides such as nigericin, elaiophylin, and geldanamycin (Chen et al. 2013; Goodfellow et al. 2007; Hayakawa et al. 2004; Hayashi et al. 1992; Kang et al. 2010; Kumar and Goodfellow 2008; Nagpure et al. 2014; Riclea et al. 2014). However, strains sharing phylogenetic vicinity may differ in their specialized metabolism due to variations in their BGCs (Choudoir et al. 2018; Martinet et al. 2020). To assess the diversity of BGCs within the *S. violaceusniger* clade, we set up a comparative genomic approach based on the chromosome-scale assemblies used for our phylogenetic analysis (Supplementary Information S1). antiSMASH retrieved 873 BGC-containing regions in the genomes of AgN23 and the 18 other selected genomes. These sequences were introduced into BiG-SCAPE, which grouped them into 415 gene cluster families (GCFs) and built a sequence similarity network.

To support our BiG-SCAPE analysis, the sequences of the 47 AgN23 BGCs were then compared with the 1,225,071 BGCs of the BiG-FAM database using the BiG-SLICE algorithm (Kautsar et al. 2021a, b).

We first observed that BiG-SCAPE distributed the AgN23 BGCs among four categories comprising *S. violaceusniger*-specific BGCs (ANI > 85%) (Fig. 5A), BGCs found in the out-group (ANI < 85%) (Fig. 5B), and AgN23 BGCs not involved in any GCFs (Fig. 5C). In addition, BiG-SCAPE identified GCFs involving BGCs of other *S. violaceusniger* strains but not from AgN23 (Fig. 5D). To back up the identification of BGCs specific to *S. violaceusniger* (Fig. 5A) or unique to AgN23 (Fig. 5C), we inspected any potential hits of the corresponding AgN23 BGCs in the BiG-FAM database (Supplementary Information S8).

As a result, six BGCs found to be *S. violaceusniger* specific in BiG-SCAPE (Fig. 5A) actually had outgroup hits in BiG-FAM. Additionally, one BGC with no hits in BiG-SCAPE had a similar BGC in Big-FAM. The results of BiG-SCAPE and Big-FAM analyses are consolidated in Figure 6.

These analyses revealed that BGCs involved in the biosynthesis of geldanamycin, a largely conserved phytotoxic, antifungal, and antibacterial compound of *S. violaceusniger* strains, were detected in six strains but neither in AgN23 nor its closest neighbor *S. antimycoticus* (Fig. 5D) (Heisey and Putnam 1986). Additionally, BiG-SCAPE analysis clustered regions containing rustmicin-like BGCs from four *S. violaceusniger* strains, but nei-

<figure><img src="image_6.png" /><figcaption>FIGURE 4</figcaption></figure>

Overview of tryptophan-dependent indole-3 acetic acid (IAA) biosynthesis pathways in bacteria. Putative AgN23 genes encoding for enzymes of the KEGG pathways are indicated. Compound abbreviations: tryptophan (Trp), indole-3-acetaldoxime (IAOx), indole-3-acetonitrile (IAN), indole-3-acetamide (IAM), indole-3-pyruvate (IPyA), indole-3-acetaldehyde (IAAld), and tryptamine (TAM).
ther connected region 2 of AgN23 to it although that region con-
tains a BGC annotated as rustmicin by antiSMASH (Table 2).
Because the organization of the five BGCs is very similar to the
subcluster GalA-E of *S. galbus* responsible for the biosynthesis
of the macrolactone ring of the compound, we propose that the
biosynthesis of a rustmicin-like compound is commonly found
within the *S. violaceusniger* clade (Kim et al. 2014) (Fig. 7).

Importantly, both BiG-SCAPE and BiG-FAM confirmed the conservation of BGCs involved in antifungal activity (regions 2, 17, 41, and 42) and plant bioactive metabolites (regions 26, 40, and 41) that we identified in AgN23 and the *S. violaceusniger* clade. This suggests that this specialization could have arisen during the *Streptomyces* genus radiation and may result, or have participated, in the *S. violaceusniger* clade adaptation to its differ-

ent ecological niche (Chater 2016; Doroghazi and Buckley 2010, 2014).

BGCs synteny among *S. violaceusniger* spp. and putative BGC horizontal transfers

*Streptomyces* chromosome organization consists of a central conserved genome, whereas terminal sequences contain more variable gene content described as an accessory genome (Kim et al. 2015; Lioy et al. 2021; Szafran et al. 2021). This accessory genome undergoes frequent rearrangement, amplification, and deletion events, as well as interspecies homologous recombination (Andam et al. 2016; Cheng 2016; Choudoir and Buckley 2018; Doroghazi and Buckley 2010; McDonald and Currie 2017;

A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

C

D

<figure><img src="image_9.png" /><figcaption>FIGURE 5</figcaption></figure>

Twenty-eight biosynthetic gene cluster (BGC)-containing regions in the *Streptomyces* sp. AgN23 genome are specifically found within *S. violaceusniger* strains. **A**, The BiG-SCAPE similarity network analysis of the genomes of AgN23 and 18 other isolates corrected by BiGFAM reveals that 28 of AgN23 BGC-containing regions are similar to regions from the genomes of at least one other member of the *S. violaceusniger* clade. **B**, Twelve are broadly spread and **C**, seven do not match with any other tested sequence. **D**, AgN23 does not show similarity with any of the 14 BGC-containing regions that are found in at least three other members of the *S. violaceusniger* clade. Among them, the largely conserved geldanamycin, herbimycin, and hygrocin biosynthesis pathways are absent. Each node represents a BGC-containing region from one of the compared genomes, and they are clustered within regions displaying similar organization. They are colored as follows: BGC-containing regions from the genome of the same species as AgN23 are in green (average nucleotide identity [ANI] > 95%), other members of the *S. violaceusniger* clade are in purple (95%>ANI > 85%), and phylogenetically distant isolates are in gray. Green triangles represent AgN23 BGC sequences, and blue squares represent curated sequences from the MiBIG database. Dot frames highlight the results that differ from the BiG-SCAPE output based on manual annotation or BiG-FAM analysis.
Tidjani et al. 2019; Zhang et al. 2022). We decided to investigate commonalities and differences in the BGC organization across the *S. violaceusniger* clade and to pinpoint original features of our newly sequenced AgN23 strain.

We found that the core set of 26 BGCs found in the *S. violaceusniger* clade is syntenically positioned in the “left” and “right” arms of the chromosome or in the central region in AgN23, *Streptomyces* sp. 11-1-2, *S. antimycoticus* NBRC10767, *S. autolyticus* CGMCC0516, *S. malaysiensis* DSM4137, and *Strep-

tomyces* sp. M56 (Fig. 6). By contrast, discrepancies in BGC positions exist between this latter group of genomes and *S. violaceusniger* Tü 4113, as well as *S. hygroscopicus* XM201, suggesting that these strains underwent drastic chromosome rearrangements but still retained BGCs connected to the synthesis of plant and fungal bioactive metabolites.

The regions 1, 46, and 47, which harbor original BGCs according to antiSMASH (<10% similarity with MIBiG) and the BiG-SCAPE analysis, are located on the extremities (left and

<figure><img src="image_10.png" /><figcaption>**FIGURE 6**</figcaption></figure>

Synteny analysis of the biosynthetic gene cluster (BGC)-containing regions in genomes of the *Streptomyces violaceusniger* clade. The squares each represent BGC-containing regions that are linked together between different genomes by lines. They are colored in green if they have been shown to be specific to the *S. violaceusniger* clade, gray if they are widespread among *Streptomyces* spp., and orange if no similar regions have been detected in other genomes. The tree is inferred from GBDP distances calculated from fully assembled chromosomes of isolates from the *S. violaceusniger* clade. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications.

<figure><img src="image_11.png" /><figcaption>**FIGURE 7**</figcaption></figure>

The rustmicin ABCDE biosynthetic gene cluster is conserved across the *Streptomyces violaceusniger* clade. Comparison of the rustmicin BGC sequence from MIBiG (BGC0000065) with genomic regions that are similar in AgN23 and four other *S. violaceusniger* genomes. Colored genes are putative homologs according to antiSMASH, with ClusterBLAST e-value < 1E-05, identity > 30%, and query coverage > 25%.
right arms) of the AgN23 chromosome and syntenic strains. Region 45 contains a BGC with 100% similarity to the biosynthetic pathway of the antimicrobial bicyclomycin, which is absent from other *S. violaceusniger* strains but is found outside of the clade (Table 2, Supplementary Information S8). Thus, region 45 might have been acquired by AgN23 through horizontal gene transfer. In conclusion, our synteny analysis is consistent with a preferential accumulation of unique or horizontally transferred BGCs on the chromosome extremities of AgN23. Conversely, the biosynthesis of specialized metabolites such as niphimycin or galbonolide was reported outside the *S. violaceusniger* clade, suggesting that some of these specific BGCs might have been horizontally transferred outside of the clade (Hu et al. 2018; Kim et al. 2014). Taken together, these data support the view that AgN23 accessory BGCs are concentrated at chromosome extremities.

## CONCLUSIONS

Plant roots recruit abundant and diverse consortia of microorganisms while exploring the soil. The relevance of this root microbiota in plant nutrition and resistance to stresses is being unveiled through metabarcoding studies of plant microbiota. The *Streptomyces* genus constitutes one of the most prominent bacterial genera colonizing plant roots (Lundberg et al. 2012). Streptomyces are filamentous gram-positive bacteria universally found around and within host plant tissues. These actinobacteria have been extensively investigated for their tremendous ability to produce diverse specialized metabolites (e.g., antibiotics). By contrast, their impact on host plant physiology is widely neglected. Additionally, few studies have addressed the species diversity of plant-associated *Streptomyces* and the genomic features enabling their colonization of the plant root environment. Therefore, genome sequencing of root-associated *Streptomyces* is an important step toward the description of their molecular interaction with the host plant. Here, we produced a complete chromosome sequence of *Streptomyces* sp. AgN23, a strain isolated from grapevine rhizosphere and shown to elicit plant defense responses (Vergnes et al. 2019). This reference sequence enabled us to perform a multilocus sequence typing approach and to position AgN23 in the *S. violaceusniger* clade from which several representatives have been isolated worldwide from the rhizosphere of unrelated plants. Comparative genomic studies suggest that *S. violaceusniger* spp. produce a large repertoire of CAZymes with expansion of plant cell wall-degrading enzyme families and a conserved specialized metabolism acting on plants and microorganisms. These genomic features may underlie adaptation to the rhizospheric niche of some of the *S. violaceusniger* spp., such as AgN23. In addition, this phylogenetic lineage is considered to have a high potential in terms of specialized metabolism as its members have a large genome between 10.7 and 12.7 Mb and possess from 45 to 55 BGCs (Chung et al. 2021). The wealth of genomic data available in this clade allowed us to unveil common trends in the BGCs specifically found in *S. violaceusniger* isolates. The ability of these strains to produce antifungal compounds (nigericin, niphimycin, mediomycin, and rustmicin), as well as nigericin, which has phytotoxic or plant defense-elicitor activity, suggests that these metabolites play an important role in protection from pathogens. In addition, echosides and elaiophylin, from which structural analogues have been shown to interfere with auxin-like responses, could interfere with plant development (Igarashi et al. 2002; Wang et al. 2015). Thorough comparisons of the BGC content and chromosome organization of AgN23 with other *S. violaceusniger* spp. also enabled us to pinpoint BGC gains (i.e., bicyclomycin, an antibacterial product) and losses (i.e., geldanamycin, a phytotoxin), that could account

for specific activities of the strain in the rhizosphere microbiota and colonization of plant tissues, respectively.

## Availability of data and materials

The PacBio raw read sequences of the AgN23 genome are available at NCBI on the Sequence Read Archive “SRA” repository, SRR13990229. The MiSeq raw read sequences of the AgN23 genome are available at NCBI on the Sequence Read Archive “SRA” repository, SRR14028548. The genome assembly of AgN23 is available on the NCBI “Nucleotide” repository, NZ_CP007153.1. The RNA-seq raw reads of AgN23 are available at NCBI on the “BioProject” repository, PRJNA745930.

## ACKNOWLEDGMENTS

We thank Olivier Bouchez for helpful discussions on the next-generation sequencing strategy throughout the project. We are grateful to Sylvie Lautru and Jean-Luc Pernodet (I2BC, Paris) for helpful discussions. We thank Thierry Grollier and Valérie Arnal for helpful comments on earlier versions of the manuscript.

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