Abstract
The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host–pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain’s virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen’s interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.
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Acknowledgments
The authors acknowledge Lone Hoj, Evan Goulden, and Rebecca Schilling for their advice and assistance. This work was partially supported by a Human Frontiers in Science Program Award (No. RGY0089RS) to RS.
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338_2015_1273_MOESM1_ESM.mp4
Video S1. Direct visualization of host–pathogen interactions showing time-lapse projections of a Pocillopora damicornis coral with inoculated, non-labeled Vibrio coralliilyticus cells. Imaging occurred under bright field GFP fluorescence. Scale bar denotes 25 μm. Link: http://youtu.be/UcVgdYZNKJM (MP4 11045 kb)
338_2015_1273_MOESM2_ESM.mp4
Video S2. Direct visualization of host/pathogen interactions showing time-lapse projections of a Pocillopora damicornis coral with inoculated, non-labeled Vibrio coralliilyticus cells. Imaging occurred under GFP fluorescence. Scale bar denotes 25 μm. Link: http://youtu.be/oprFuXg_KXk (MP4 10323 kb)
Video S3. Direct visualization of host/pathogen interactions showing time-lapse projections of a Pocillopora damicornis coral with inoculated GFP-labeled V. coralliilyticus cells. Imaging occurred under GFP fluorescence. Scale bar denotes 25 μm. Link: http://youtu.be/GhtOHIohalY (MP4 1874 kb)
Video S4. Direct visualization of host/pathogen interactions showing z-stack projections of a Pocillopora damicornis coral with inoculated, non-labeled Vibrio coralliilyticus cells. Imaging occurred under bright field fluorescence. Scale bar denotes 25 μm. Link: http://youtu.be/kVJ4_vd4B8w (MP4 84 kb)
Video S5. Direct visualization of host/pathogen interactions showing z-stack projections of a Pocillopora damicornis coral with inoculated, non-labeled V. coralliilyticus cells. Imaging occurred under GFP fluorescence. Scale bar denotes 25 μm. Link: http://youtu.be/j2VgvDN8Aq4 (MP4 144 kb)
Video S6. Direct visualization of host/pathogen interactions showing z-stack projections of a Pocillopora damicornis coral with inoculated GFP-labeled V. coralliilyticus cells. Imaging occurred under GFP fluorescence. Scale bar denotes 25 μm. Link: http://youtu.be/49Cha3rbFQY (MP4 42 kb)
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Pollock, F.J., Krediet, C.J., Garren, M. et al. Visualization of coral host–pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain. Coral Reefs 34, 655–662 (2015). https://doi.org/10.1007/s00338-015-1273-3
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DOI: https://doi.org/10.1007/s00338-015-1273-3