Inter- and intracellular colonization of Arabidopsis roots by endophytic actinobacteria and the impact of plant hormones on their antimicrobial activity

Abstract

Many actinobacteria live in close association with eukaryotes such as fungi, insects, animals and plants. Plant-associated actinobacteria display (endo)symbiotic, saprophytic or pathogenic life styles, and can make up a substantial part of the endophytic community. Here, we characterised endophytic actinobacteria isolated from root tissue of Arabidopsis thaliana (Arabidopsis) plants grown in soil from a natural ecosystem. Many of these actinobacteria belong to the family of Streptomycetaceae with Streptomyces olivochromogenes and Streptomyces clavifer as well represented species. When seeds of Arabidopsis were inoculated with spores of Streptomyces strain coa1, which shows high similarity to S. olivochromogenes, roots were colonised intercellularly and, unexpectedly, also intracellularly. Subsequent exposure of endophytic isolates to plant hormones typically found in root and shoot tissues of Arabidopsis led to altered antibiotic production against Escherichia coli and Bacillus subtilis. Taken together, our work reveals remarkable colonization patterns of endophytic streptomycetes with specific traits that may allow a competitive advantage inside root tissue.

Keywords: Cryptic antibiotics; Electron microscopy; Plant hormone; Plant–microbe interactions; Streptomyces.

Fig. 1

Relative abundance of the actinobacteria in the endophytic compartment of A. thaliana Msl. a Amplicon sequencing data show that actinobacteria represent 22% of the total endophytic compartment (EC), while in soil and rhizosphere (RS) communities and those in association with a toothpick (TP) inserted in the soil, they take up roughly 2% of the total bacterial community. b The enrichment of actinobacteria is predominately driven by Streptomycetaceae OTU 48 and 137. OTU 48 and 137 make up 10% of the total EC and almost 50% of all the actinobacteria present in the EC. These OTUs are not as much enriched in the samples derived from non-endophytic origins

Fig. 2

Characterization of Streptomyces endophytes and their taxonomic distribution. a Several isolates show highest similarity with Streptomyces clavifer or Streptomyces olivochromogenes on the basis of 16S rDNA analysis. The majority of the isolates showed a wide variation in closest species assigned, indicating a diverse endophytic compartment. ‘Unclassified’ means that these actinobacteria could not be classified at the species level based on the 16S rDNA sequence. b Scanning electron micrograph of Streptomyces sp. MOS18, which produces spiny spores. Scale bar 3 μM. c Scanning electron micrograph of Streptomyces sp. MOS38 showing poor sporulation. Scale bar 5 μM. Strains were grown on SFM media for 6 days

Fig. 3

Streptomyces endophytes display a wide range of morphologies. Strains shown above are grown on SFM agar plates for 6 days. S. coelicolor M145 and S. griseus DSM40236 are shown as reference strains. a S. coelicolor M145, b S. griseus DSM40236 and the endophytic streptomycetes MOS18 (c), MOS38 (d), MOS14 (e), MOS32 (f), MOS25 (g), MOS31 (h) and MOS35 (i). Scale bar 2 mm

Fig. 4

Scanning electron micrograph of Streptomyces sp. MOS31. Images of mycelial sheets as is seen in Fig. 3H. a Thick layers of vegetative mycelium made up of hyphae and extracellular matrix turn away from the inner part of the colony. Scale bar 100 μM. b Hyphae extending from the mycelial sheets, thereby growing away from the vegetative mycelium. Scale bar 30 μM. Streptomyces sp. MOS31 was grown on SFM agar plates for 6 days

Fig. 5

Colonization of Arabidopsis by Streptomyces endophyte coa1. a Confocal micrograph of a colonised lateral root. The sample is stained with propidium iodide, resulting in red fluorescence of both bacterial and plant cells. Coa1 attaches to the root as a dense pellet. Scale bar 50 μM. b Confocal micrograph of the border of the leaf. Single hyphae are growing over the leaf surface (arrowheads). Scale bar 15 μM

Fig. 6

Sections of Arabidopsis roots invaded by Streptomyces coa1. a Toluidine stained section of an Arabidopsis root invaded by Streptomyces coa1. Coa1 enters the root via the epidermis (EP) cells and colonises the root in between the cortex cells (CO) and EP cells. Scale bar 10 μM. Boxed part of the image is shown as magnification on the right. b, c Transmission electron micrographs of Arabidopsis roots invaded by cao1. Coa1 colonises the root intracellularly. The bacterium can be found in between the CO- and EP cells, and in between the endodermis (ED) and CO cells (image B). In addition, in EP cells intracellular growth of Streptomyces coa1 was observed (image C). Scale bar 2 μM