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The Use of High Pressure Freezing and Freeze Substitution to Study Host–Pathogen Interactions in Fungal Diseases of Plants

Published online by Cambridge University Press:  21 November 2003

C.W. Mims ,
Gail J. Celio  and
Elizabeth A. Richardson
C.W. Mims
Affiliation:
Department of Plant Pathology, University of Georgia, Athens, GA 30602, USA
Gail J. Celio
Affiliation:
Department of Plant Pathology, University of Georgia, Athens, GA 30602, USA
Elizabeth A. Richardson
Affiliation:
Department of Plant Biology, University of Georgia, Athens, GA 30602, USA
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Abstract

This article reports on the use of high pressure freezing followed by freeze substitution (HPF/FS) to study ultrastructural details of host–pathogen interactions in fungal diseases of plants. The specific host–pathogen systems discussed here include a powdery mildew infection of poinsettia and rust infections of daylily and Indian strawberry. The three pathogens considered here all attack the leaves of their hosts and produce specialized hyphal branches known as haustoria that invade individual host cells without killing them. We found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host–pathogen systems. Preservation of fungal and host cell membranes was particularly good and greatly facilitated the detailed study of host–pathogen interfaces. In some instances, HPF/FS provided information that was not available in samples prepared for study using conventional chemical fixation. On the other hand, we did encounter various problems associated with the use of HPF/FS. Examples included freeze damage of samples, inconsistency of fixation in different samples, separation of plant cell cytoplasm from cell walls, breakage of cell walls and membranes, and splitting of thin sections. However, we believe that the outstanding preservation of ultrastructural details afforded by HPF/FS significantly outweighs these problems and we highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.

Keywords

transmission electron microscopycryofixationfungal haustoria
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 9 , Issue 6 , December 2003 , pp. 522 - 531
Copyright
© 2003 Microscopy Society of America

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References

REFERENCES

Aist, J.A. & Bushnell, W.R. (1991). Invasion of plants by powdery mildew fungi, and cellular mechanisms of resistance. In The Fungal Spore and Disease Initiation in Plants and Animals, Cole, G.T. & Hoch, H.C. (Eds.), pp. 321–345. New York: Plenum.
Bourett, T.M., Czymmek, K.J., & Howard, R.J. (1999). Ultrastructure of chloroplast protuberances in rice leaves preserved by high pressure freezing. Planta 208, 472–479.Google Scholar
Bracker, C.E. (1968). Ultrastructure and cytochemical characterization of the haustorial apparatus of Erysiphe graminis and its relation to the epidermal cell of barley. Phytopathology 58, 12–30.Google Scholar
Celio, G.J., Richardson, E.A., & Mims, C.W. (2000). Ultrastructure of the infection of poinsettia by Oidium sp. using high pressure freezing and freeze substitution. Microsc Microanal Proc. 6, 684–685.Google Scholar
Coffey, M.D., Palevitz, B.A., & Allen, P.J. (1972). The fine structure of two rust fungi, Puccinia helianthi and Melampsora lini. Can J Bot 50, 231–240.Google Scholar
Dahl, R. & Staehelin, L.A. (1989). High-pressure freezing for the preservation of biological structure: Theory and practice. J Electr Micro Tech 13, 165–174.Google Scholar
Dahmen, H. & Hobot, J.A. (1986). Ultrastructural analysis of Erysiphe graminis haustoria and subcuticular stroma of Venturia inaequalis using cryosubstitution. Protoplasma 131, 92–102.Google Scholar
Ding, B., Turgeon, R., & Parthasarthy, M.W. (1992). Effect of high pressure freezing on plant microfilaments bundles. J Microsco 165, 367–376.Google Scholar
Gil, F. & Gay, J.L. (1977). Ultrastructure and physiological properties of the host interfacial components of haustoria of Erysiphe pisi in vivo and in vitro. Physiol Plant Pathol 10, 1–12.Google Scholar
Gilkey, J.C. & Staehelin, L.A. (1986). Advances in ultrarapid freezing for the preservation of cell ultrastructure. J Electr Microsc Tech 3, 177–210.Google Scholar
Harder, D.E. & Chong, J. (1984). Structure and physiology of haustoria. In The Cereal Rusts, Bushnell, W.R. & Roelfs, A.P. (Eds.), Vol. 1, pp. 431–476. New York: Academic Press.
Harder, D.E. & Chong, J. (1991). Rust haustoria. In Electron Microscopy of Plant Pathogens, Mendgen, K. & Lesemann, D.E. (Eds.), pp. 236–250. Berlin: Springer-Verlag.
Hippie, S. (1985). Ultrastructure of haustoria of Erysiphe graminis f.sp. hordei preserved by freeze substitution. Protoplasma 129, 52–61.Google Scholar
Honegger, R. (1985). Scanning electron microscopy of the fungus-plant cell interface: A simple preparative technique. Trans Br Mycol Soc 84, 530–533.Google Scholar
Hyde, G.J., Lancelle, S., Hepler, P.K., & Hardham, A.R. (1991). Sporangial structure is disrupted after high pressure freezing in Phytophthora. Protoplasma 165, 201–208.Google Scholar
Kiss, J.Z., Giddings, T.H., Jr., Staehelin, L.A., & Sack, F.D. (1990). Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted roots of Nicotiana and Arabidopsis. Protoplasma 157, 64–74.Google Scholar
Knauf, G.M., Welter, K., Müller, M., & Mendgen, K. (1989). The haustorial host–parasite interface in rust infected bean leaves after high pressure freezing. Physio Mol Plant Path 34, 519–530.Google Scholar
Mackie, A.J., Roberts, A.M., Callow, J.A., & Green, J.R. (1991). Molecular differentiation in pea powdery-mildew haustoria. Planta 183, 399–408.Google Scholar
Mackie, A.J., Roberts, A.M., Green, J.R., & Callow, J.A. (1993). Glycoproteins recognized by monoclonal antibodies UB7, UB8 and UB10 are expressed early in the development of pea powdery mildew haustoria. Physiol Mol Plant Pathol 43, 135–146.Google Scholar
Manners, J.M. & Gay, J.L. (1983). The host–parasite interface and nutrient transfer in biotrophic parasitism. In Biochemical Plant Pathology, Callow, J.A. (Ed.), pp. 163–195. Chichester: John Wiley and Sons.
Mendgen, K., Welter, K., Scheffold, F., & Knauf-Beiter, G. (1991). High pressure freezing of rust infected leaves. In Electron Microscopy of Plant Pathogens, Mendgen, K. & Leseman, D.E. (Eds.), pp. 31–42. Berlin: Springer-Verlag.
Mims, C.W. (1991). Using electron microscopy to study plant pathogenic fungi. Mycologia 83, 1–19.Google Scholar
Mims, C.W. & Richardson, E.A. (2003). Ultrastructure of the zoosporangia of Albugo ipomoeae-panduratae as revealed by conventional chemical fixation and high pressure freezing followed by freeze substitution. Mycologia 95, 1–10.Google Scholar
Mims, C.W., Rodriguez-Lothar, C., & Richardson, E.A. (2001). Ultrastructure of the host–pathogen interaction in leaves of Duchesnea indica infected by the rust fungus Frommeëlla mexicana var. indicae as revealed by high pressure freezing. Can J Bot 79, 49–57.Google Scholar
Mims, C.W., Rodriguez-Lother, C., & Richardson, E.A. (2002). Ultrastructure of the host–pathogen interface in daylily leaves infected by the rust fungus Puccinia hemerocallidis. Protoplasma 219, 221–226.Google Scholar
Moor, H. (1987). Theory and practice of high pressure freezing. In Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. & Ziehold, K. (Eds.), pp. 175–191. New York: Springer-Verlag.
Richardson, E.A. & Mims, C.W. (2001). A simple technique for the removal of plant cell protoplasm to facilitate scanning electron microscopy of fungal haustoria and plant cell wall features. Micros Today 01-3, 14–15.Google Scholar
Roberts, A.M., Mackie, A.J., Hathaway, V., Callow, J.A., & Green, J.R. (1993). Molecular differentiation in the extrahaustorial membrane of pea powdery mildew haustoria at early and late stages of development. Physiol Mol Plant Pathol 43, 147–160.Google Scholar
Rowley, C.R. & Moran, D.T. (1975). A simple technique for mounting wrinkle-free sections on formvar-coated slot grids. Ultramicrotomy 1, 151–155.Google Scholar
Spencer-Phillips, P.T.N. & Gay, J.L. (1980). Electron microscope autoradiography of 14C photosynthate distribution at the haustorium–host interface in powdery mildew of Pisum sativum. Protoplasma 103, 131–154.Google Scholar
Stüder, D., Hennecke, H., & Müller, M. (1992). High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure. Planta 188, 155–163.Google Scholar
Taylor, J. & Mims, C.W. (1991). Fungal development and host cell responses to the rust fungus Puccinia substriata var. indica in seedlings and mature leaves of susceptible and resistant pearl millet. Can J Bot 69, 1207–1219.Google Scholar
Thijssen, M.H., Mittempergher, F., Van Aelst, A.C., & Van Went, J.L. (1997). Improved preservation of Petunia and Brassica ovules and embryos by high pressure freezing and freeze substitution. Protoplasma 197, 199–209.Google Scholar
Welter, K., Müller, M., & Mendgen, K. (1988). The hyphae of Uromyces appendiculatus within leaf tissue after high pressure freezing and freeze substitution. Protoplasma 147, 91–99.Google Scholar