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Propidium iodide enabled live imaging of Pasteuria sp.-Pratylenchus zeae infection studies under fluorescence
Francine Perrine-Walker1,2, Khoa Le3,4
1School of Life and Environmental Sciences, Life Earth and Environmental Sciences Building (F22), The University of Sydney, Sydney, NSW, 2006, Australia. marie.perrine-walker@sydney.edu.au.
Abstract:
Live imaging allows observations of cell structures and processes in real time, to monitor dynamic changes within living organisms compared to fixed organisms. Fluorescence microscopy was used to monitor the dynamic infection process of the nematode parasitic bacterium Pasteuria sp. and the sugarcane root-lesion nematode, Pratylenchus zeae. Under fluorescence microscopy, green-autofluorescent globules were observed in live control and Pasteuria sp.-infected nematodes. Only nematodes killed by Pasteuria sp. or heat treated displayed a diffuse pattern of autofluorescence. Propidium iodide (PI), used as a cell membrane integrity indicator, confirmed that the nematode's cuticle acts as an impermeable barrier. PI stained cells/DNA of heat-treated control and Pasteuria sp.-infected P. zeae. PI as a counterstain facilitated the location of Pasteuria endospores on the cuticle surface of P. zeae. No PI staining was observed in sporangia and in endospores within the nematode body. However, PI specifically stained endospores on the cuticle surface and within the cuticle carcass showing, in mature propagules, a ring-like pattern. Live imaging, combined with fluorescence microscopy and fluorescent dyes such as PI, appears useful in live studies on plant nematode interactions with nematophagous bacteria.
Insights
Live imaging and fluorescence microscopy revealed how Pasteuria sp. bacteria infect nematodes. Propidium iodide confirmed nematode cuticle impermeability and tracked bacterial endospores during infection.
Area of Science:
- Microbiology
- Nematology
- Microscopy
Background:
- Live imaging offers real-time observation of dynamic cellular processes in living organisms.
- Understanding plant-parasitic nematode interactions with beneficial bacteria is crucial for sustainable agriculture.
Purpose of the Study:
- To monitor the dynamic infection process of Pasteuria sp. in Pratylenchus zeae using live imaging.
- To assess the integrity of the nematode cuticle as a barrier during bacterial infection.
Main Methods:
- Utilized fluorescence microscopy to observe live nematodes and bacterial interactions.
- Employed propidium iodide (PI) as a cell membrane integrity indicator.
- Applied live imaging techniques to track Pasteuria endospores on and within the nematode.
Main Results:
- Green-autofluorescent globules were observed in live nematodes.
- Nematode cuticle integrity was confirmed; PI did not penetrate intact cuticles.
- PI staining revealed Pasteuria endospores on the cuticle surface and within the nematode carcass, with a ring-like pattern in mature propagules.
Conclusions:
- Live imaging combined with fluorescence microscopy and PI is effective for studying plant nematode-nematophagous bacteria interactions.
- The nematode cuticle serves as an impermeable barrier, with PI staining indicating compromised integrity or bacterial presence.
- This approach provides insights into the dynamics of bacterial infection in nematodes.
