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Published on: June 19, 2013
Purification of fungal haustoria from infected plant tissue by flow cytometry
Diana P Garnica1, John P Rathjen
1The Australian National University, Research School of Biology, Linnaeus Building #134, Linnaeus Way, Acton, ACT, 0200, Australia.
Abstract:
A hallmark of biotrophy in many fungal plant pathogens is the formation of a specialized pathogenic structure called the haustorium from infectious hyphae. This is the major parasitic structure, where nutrients are taken up from the host and pathogenicity factors are exported to the host tissue. Obligate biotrophic fungi can typically be cultured in vivo only to a limited extent and do not produce haustoria under these conditions. This has hampered the application of classic molecular biology techniques to haustoria-forming pathogens. The lectin Concanavalin A (Con A), which binds specifically to sugars present on the exterior of rust haustoria, was first used in a column-based affinity purification procedure in 1992 (Hahn and Mendgen, Protoplasma 170:95-103, 1992). Here we describe a new technique where we combine initial gradient purification of haustoria with flow-sorting based on labeling of haustoria with fluorescent Con A. Our method allows haustorial isolation with purity above 98 % and yields ten times more isolated haustoria in a single experiment than the previous procedure.
Insights
Researchers developed a new method to isolate fungal haustoria, crucial for plant pathogen research. This technique significantly improves purity and yield, enabling better study of these vital structures.
Area of Science:
- Plant Pathology
- Mycology
- Molecular Biology
Background:
- Haustoria are specialized structures formed by biotrophic fungal plant pathogens for nutrient uptake and pathogenicity factor export.
- Obligate biotrophic fungi are difficult to culture and lack haustoria in vitro, hindering molecular studies.
- Previous methods for haustoria isolation, like lectin affinity purification, have limitations in efficiency and yield.
Purpose of the Study:
- To develop an improved technique for isolating fungal haustoria from host plants.
- To enhance the purity and yield of isolated haustoria for molecular analysis.
- To overcome limitations of previous methods in studying haustoria-forming pathogens.
Main Methods:
- Combined gradient purification with fluorescence-activated flow sorting (FACS).
- Utilized fluorescently labeled Concanavalin A (Con A) to specifically bind and label haustoria.
- Optimized the procedure for high-purity isolation of haustoria.
Main Results:
- Achieved haustorial isolation purity exceeding 98%.
- Obtained a tenfold increase in isolated haustoria yield per experiment compared to previous methods.
- Demonstrated the efficacy of the new combined purification and flow-sorting technique.
Conclusions:
- The novel technique provides a highly efficient and pure method for isolating fungal haustoria.
- This advancement facilitates the application of molecular biology techniques to obligate biotrophic pathogens.
- Improved haustoria isolation will advance research into fungal pathogenicity and host-pathogen interactions.
