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

Thomas, Pious; Franco, Christopher M. M.

doi:10.3390/microorganisms9020269

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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

Pious Thomas

^1,*,†

and

Christopher M. M. Franco

^2,*

Thomas Biotech & Cytobacts Centre for Biosciences, Amruthahalli, Bengaluru 560092, India

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)

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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. 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.

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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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Author Biographies

Pious Thomas

Dr. Pious Thomas is the CEO & Director of Thomas Biotech & Cytobacts Centre for Biosciences, an R&D Company, established in Bangalore, India in 2019 with a prime focus on Agri-Biotech. Prior to that, he held a research position at ICAR—Indian Institute of Horticultural Research—Bengaluru for 28 years as Scientist/Senior Scientist/Principal Scientist. Dr. Thomas did his masters in Horticulture at Kerala Agricultural University, Thrissur, his Ph.D at the Indian Agricultural Research Institute, New Delhi, and his Post-Doctorate at the University of Sheffield, UK. His broad research interests included Horticulture and Plant Biology/Biotechnology. Specific research areas include plant tissue culture, micropropagation, gene expression, molecular breeding and microbiology. Endophytic Microbiology is the thrust research area, leading to the discovery of Cytobacts, which is considered a ubiquitous intracellular bacteria in vascular plants. He is a fellow of National Academy of Agricultural Sciences, New Delhi, and a recipient of several national awards.

Christopher M. M. Franco

Dr Christopher Franco is a Professor and International Dean at the Faculty of Medicine at Flinders University, Adelaide. With the Masters in Applied Microbiology from Edinburgh and a Ph.D from Glasgow, he has been associated with the Flinders University from 1993. Prof. Franco’s research career combines both industrial and academic spheres. For 10 years he led a successful drug discovery and development group in an international biopharmaceutical company and holds patents for 11 compound families. He is Australia's leading actinobacteriologist and continues to discover novel actinobacteria and new bioactive from algae, plant cell culture, mammalian cells and marine organisms. He has extensive international experience, working in the UK, Germany, India, Indonesia, Singapore and China. As International Dean, Faculty of Medicine, Nursing & Health Sciences and the School of Medicine, he has been managing the faculty's international collaborations.

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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

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