Intracellular bacteria in plants: Elucidation of abundant and diverse cytoplasmic bacteria in healthy plant cells using in vitro cell and callus cultures

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    Abstract

    This study was initiated to assess whether the supposedly axenic plant cell cultures harbored any cultivation-recalcitrant endophytic bacteria (CREB). Adopting live-cell imaging with bright-field, fluorescent and confocal microscopy and bacterial 16S-rRNA gene taxonomic profiling, we report the cytoplasmic association of abundant and diverse CREBs in long-term actively maintained callus and cell suspension cultures of different plant species. Preliminary bright-field live-cell imaging on grape cell cultures showed abundant intracellular motile micro-particles resembling bacteria, which proved uncultivable on enriched media. Bacterial probing employing DNA stains, transmission electron microscopy, and Eubacterial FISH indicated abundant and diverse cytoplasmic bacteria. Observations on long-term maintained/freshly established callus stocks of different plant species—grapevine, barley, tobacco, Arabidopsis, and medicinal species—indicated intracellular bacteria as a common phenomenon apparently originating from field shoot tissues.Cultivation-independent 16S rRNA gene V3/V3–V4 amplicon profiling on 40-year-old grape cell/callus tissues revealed a high bacterial diversity (>250 genera), predominantly Proteobacteria, succeeded by Firmicutes, Actinobacteria, Bacteriodetes, Planctomycetes, and 20 other phyla, including several candidate phyla. PICRUSt analysis revealed diverse functional roles for the bacterial microbiome, majorly metabolic pathways. Thus, we unearth the widespread association of cultivation-recalcitrant intracellular bacteria “Cytobacts” inhabiting healthy plant cells, sharing a dynamic mutualistic association with cell hosts.

    Original languageEnglish
    Article number269
    Number of pages27
    JournalMicroorganisms
    Volume9
    Issue number2
    DOIs
    Publication statusPublished - Feb 2021

    Keywords

    • 16S rRNA gene taxonomic profiling
    • Confocal laser scanning microscopy
    • Endophytic bacterial microbiome
    • Fluorescent in situ hybridization (FISH)
    • Metagenomics
    • Next-generation deep sequencing
    • Plant tissue culture
    • Plant-microbe interactions
    • Vitis vinifera L

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