Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria.

Pious Thomas,Aparna Chandra Sekhar

doi:10.1093/aobpla/plu002

Abstract

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50-100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed 'Brownian motion'-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as 'Cytobacts' and 'Peribacts', respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.

Highlights

The term endophyte denotes microorganisms including bacteria and fungi that colonize plants internally without any apparent adverse effects on the host (Hallmann2001; Bacon et al 2002; Reinhold-Hurek and Hurek 2011)
A comparison with the pure cultures of endophytic bacteria previously isolated from banana indicated similarity in size, shape and motility to these intracellular particles [see Supporting Information—Movie 02]
The motile units in fresh tissues sections varied in their abundance from cell to cell or part of the tissue segment, ranging from high numbers towards the base of the leaf sheath to low abundance in the upper region

Summary

Introduction

The term endophyte denotes microorganisms including bacteria and fungi that colonize plants internally without any apparent adverse effects on the host (Hallmann2001; Bacon et al 2002; Reinhold-Hurek and Hurek 2011). The term endophyte denotes microorganisms including bacteria and fungi that colonize plants internally without any apparent adverse effects on the host Endophytic bacteria are known to be associated with diverse plant species and organs Thomas and Sekhar—Intracellular-colonizing endophytic bacteria in banana et al 2007; Sun et al 2008). As per the earlier understanding, endophytes were considered to colonize the host predominantly in roots (Hallmann 2001; Bacon et al 2002; Bulgarelli et al 2012), and be present in low abundance compared with the general rhizoplane colonizers (Hallmann 2001; Compant et al 2010). Studies adopting microscopic examination of tissue homogenate, on the other hand, have indicated that the organisms are present in substantial numbers; their general non-cultivability being the prime reason for them escaping the attention of biologists (Thomas and Soly 2009; Thomas 2011; Thomas and Reddy 2013)

Objectives

Methods

Results

Discussion

Conclusion