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Intracellular Bacteria in Plants: Elucidation of Abundant and Diverse Cytoplasmic Bacteria in Healthy Plant Cells Using In Vitro Cell and Callus Cultures

1
Thomas Biotech & Cytobacts Centre for Biosciences, Amruthahalli, Bengaluru 560092, India
2
Department of Medical Biotechnology, College of Medicine & Public Health, Flinders University, Bedford Park, Adelaide, SA 5042, Australia
*
Authors to whom correspondence should be addressed.
Formerly Address: Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Bengaluru 560089, India.
Academic Editor: Damien F. Meyer
Microorganisms 2021, 9(2), 269; https://doi.org/10.3390/microorganisms9020269
Received: 31 December 2020 / Revised: 21 January 2021 / Accepted: 23 January 2021 / Published: 28 January 2021
(This article belongs to the Special Issue Host-Intracellular Bacteria Interactions)
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. View Full-Text
Keywords: 16S rRNA gene taxonomic profiling; confocal laser scanning microscopy; endophytic bacterial microbiome; fluorescent in situ hybridization (FISH); metagenomics; next-generation deep sequencing; plant-microbe interactions; plant tissue culture; Vitis vinifera L. 16S rRNA gene taxonomic profiling; confocal laser scanning microscopy; endophytic bacterial microbiome; fluorescent in situ hybridization (FISH); metagenomics; next-generation deep sequencing; plant-microbe interactions; plant tissue culture; Vitis vinifera L.
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MDPI and ACS Style

Thomas, P.; Franco, C.M.M. Intracellular Bacteria in Plants: Elucidation of Abundant and Diverse Cytoplasmic Bacteria in Healthy Plant Cells Using In Vitro Cell and Callus Cultures. Microorganisms 2021, 9, 269. https://doi.org/10.3390/microorganisms9020269

AMA Style

Thomas P, Franco CMM. Intracellular Bacteria in Plants: Elucidation of Abundant and Diverse Cytoplasmic Bacteria in Healthy Plant Cells Using In Vitro Cell and Callus Cultures. Microorganisms. 2021; 9(2):269. https://doi.org/10.3390/microorganisms9020269

Chicago/Turabian Style

Thomas, Pious, and Christopher M.M. Franco 2021. "Intracellular Bacteria in Plants: Elucidation of Abundant and Diverse Cytoplasmic Bacteria in Healthy Plant Cells Using In Vitro Cell and Callus Cultures" Microorganisms 9, no. 2: 269. https://doi.org/10.3390/microorganisms9020269

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