Additional file 1 of Arbuscular mycorrhizal fungi and Streptomyces: brothers in arms to shape the structure and function of the hyphosphere microbiome in the early stage of interaction
Creators: name: Jin, Zexing
name: Jiang, Feiyan
name: Wang, Letian
name: Declerck, Stéphane
name: Feng, Gu
name: Zhang, Lin
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: Additional file 1. Methods: Biological material - Extraction of bacterial suspensions from soil. Isolation and identification of bacteria from the extraradical hyphae network of R. irregularis MUCL 43194. Whole genome sequencing of Streptomyces sp. D1 and Pseudomonas sp. H2. Competition of Streptomyces sp. D1 and Pseudomonas sp. H2 in the hyphosphere. Carbon assimilation profiles of bacterial isolates. Pot system set up to study the impact of Streptomyces sp. D1 on organic P utilization by R. irregularis MUCL 43194 and stimulation of gene expression in plants. Liquid M-MSR medium in the hyphal compartment of the in vitro culture system. Quantification of alkaline phosphatase activity of the bacterial isolates. Competition of Streptomyces sp. and Pseudomonas sp. on hyphae. Identification of antibacterial activity of Streptomyces sp. D1. RNA extraction for RT-PCR and RT-PCR protocol. RNA extraction for transcriptome and transcriptome protocol. DNA extraction. 16S rRNA gene sequence analysis. Figure S1. Growth curves of 20 bacterial isolates grown in M9 minimal salts medium with 6 carbon sources (i.e., fructose, glucose, inositol, citric acid, trehalose, or succinic acid) as sole carbohydrate source. Error bars represent the standard deviation of four independent replicates. Figure S2. Reconstruction of (A) the Streptomyces sp. D1 and (B) Pseudomonas sp. H2 major carbohydrate metabolic pathway and phosphate metabolic pathway mapped with transcriptomic data. The reconstructed metabolic pathways of the two bacteria were based on KEGG genes annotations and their relative differential expression profiles of triplicates (each replicate was a mix of 5 culture plates). The genes with a significant (P &lt; 0.05) differential expression of &gt;1 |log2FC| were indicated with an arrow (pathway) and a star (heatmap) in green (up-regulated in absence of the extraradical hyphae), red (up-regulated in the presence of the extraradical hyphae). The number in the pathways correspond to the number in the heatmaps. Gray arrow represents the absence of genes identified in the pathway. Figure S3. Reconstruction of the Streptomyces sp. D1 albaflavenone synthesis pathway mapped with transcriptomic data. The reconstructed metabolic pathway was based on KEGG genes annotations and their relative differential expression profiles of triplicates (each replicate was a mix of 5 culture plates, 15 plates for every treatment). The pathways with genes significant (P &lt; 0.05) differential expression of |log2FC| &gt; 1 are indicated with pathway in green (down-regulated in the presence of the extraradical hyphae), red (up-regulated in absence of the extraradical hyphae). Figure S4. Bi-compartmented in vitro cultivation system. (A) Ri T-DNA transformed root of carrot developing single in the root compartment (RC) and bacterial community plated in the hyphal compartment (HC), representing the control treatment (RC/HC–AMF). (B) Ri T-DNA transformed root of carrot associated with an AM fungus in the RC, with extraradical hyphae (ERH) and spores (in blue) extending in the HC in contact with the bacterial community, representing the ERH treatment (RC/HC+AMF). (C) Detailed picture of the bi-compartmented in vitro cultivation system with a carrot root and AM fungus in the RC and ERH crossing the plastic barrier separating the RC from the HC and developing profusely in the HC in contact with a suspension of bacteria. Figure S5. Schematic representation of the hyphal compartment (HC). The HC was cuboid (6.8 cm in length, 6.0 cm in width, 5.8 cm in height) and made of PVC plates. In the plate on the top of the HC, a hole (6 mm in diameter) was made and a plastic tube (5 mm in diameter, 50 cm in length) was inserted and fixed with PVC glue. One centimeter below the top plate, another PVC plate with holes (1 mm in diameter) was fixed and covered with a 30 μm nylon mesh. On one side of the HC, a PVC plate with holes (4 mm in diameter) and covered with 30 μm or 0.45 μm nylon mesh was fixed to allow or not the AM fungal hyphae to grow into the HC. Two HCs were buried in the soil at both sides of the pots containing three maize plants associated to an AM fungus. The soil composition in the pot and HCs is detailed (see Methods in Additional file 1). The bacterial suspension was then injected with a syringe through the plastic tube, allowing the bacteria to diffuse homogeneously in the soil of the HC in contact or not with the ERH. Figure S6. (A) Schematic representation of the three-compartmented pot experimental system. The inner compartment contains the roots and fungal hyphae (RC), the middle compartment is a buffer compartment (BC) and the outer compartment is the hyphal compartment (HC) allowing only the extraradical hyphae (ERH) to extend. The BC, with a width of 1 cm, was placed between the two compartments to prevent the movement of soluble organic P from HC to RC. A nylon mesh of 30 μm separating the RC from the BC and the BC from the HC was used to avoid roots to extend from RC to HC, while allowing the hyphae to extend from RC to HC. The bacterium was inoculated in the HC in contact with the ERH of Rhizophagus irregularis MUCL 43194. Each treatment consisted of four replicates with three Medicago truncatula plants per RC. Red lines, extraradical hyphae of R. irregularis MUCL 43194; red dots, spores of R. irregularis MUCL 43194. (B) Detailed picture of the three-compartmented pot system. Table S1. Mean relative abundance of 20 bacterial isolates of all hyphal samples. Table S2. Trehalose metabolism and transporter genes in the genome of Streptomyces sp. D1 identified with KEGG. Table S3. Phosphate metabolism genes in the genome of Streptomyces sp. D1 based on KEGG gene annotations. Table S4. Phosphate metabolism genes in the genome of Pseudomonas sp. H2 based on KEGG gene annotations. Table S5. Number of genes significantly differentially expressed for each KEGG metabolic pathways in Pseudomonas sp. H2. Table S6. Secondary metabolites gene clusters in the genome of Streptomyces sp. D1 detected by antiSMASH6 pipeline. Table S7. Physico-chemical properties of the soils used to extract bacterial suspensions. Table S8. Summary of primers used in the study. Table S9. Phosphate, polyphosphate and associated genes expression significantly increased in Rhizophagus irregularis MUCL 43194 inoculated with Streptomyces sp. D1 as compared to the fungus grown in absence of the bacteria.