Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT 2601, Australia

Background and objectives
Melampsora lini is a biotrophic fungal pathogen of flax (Linum usitatissimum), which must establish a stable relationship with living plant cells in order to grow and reproduce. Like other rust fungi, after invading the plant leaf M. lini penetrates the cell wall of host mesophyll cells and forms specialized feeding structures known as haustoria. The fungus does not breach the host cell plasma membrane (the extrahaustorial membrane), and the haustoria remain surrounded by the plant plasma membrane during the biotrophic phase. The haustorium-plant interface becomes a major site of signal exchange between host and pathogen. The plant cell can detect the presence of the invading fungus on its cell surface and can initiate a defence response that may render the plant resistant to the pathogen. The fungus is also able to induce changes in the structural organization and metabolism within infected cells of susceptible plants such that the changes benefit the invading pathogen. Because of the importance of the haustorium-plant interface in host-pathogen interactions in biotrophic fungi, we have studied haustorium development in compatible interactions and have sought evidence of haustorial specializations that might play a role in haustorium function or in communication between plant and pathogen, including possible mechanisms to avoid detection by the host plant. We have used an immunocytochemical approach and have obtained evidence for specialization of the haustorial cell wall.

Materials and methods
Haustorial complexes were isolated from infected flax leaves following the method of Hahn and Mendgen [1], and monoclonal antibodies were generated using a co-immunization regime [2]. The immunogen consisted of a preparation enriched in haustorial complexes but also containing other fungal and plant cells and cell debris, mixed with polyclonal serum raised against a haustorium-depleted sample. Antibody production by hybridoma cell lines was screened by immunofluorescence microscopy using 10 Ám sections of infected flax leaves embedded in butyl-methyl methacrylate [3]. Ultrastructural localizations of antigens were obtained by immunogold labelling.

Results and conclusions
Many of the antibodies produced reacted with the walls of all M. lini cells. One of these, ML4, was cloned and characterized. Three hybridoma cell lines, ML1, ML2 and ML3, produced antibodies that reacted only with the walls of M. lini haustoria. These antibodies did not react with the walls of other fungal infection structures or with plant cell walls. The epitopes labelled by the four antibodies were carbohydrate in nature. In addition, three commercially available monoclonal antibodies directed towards different epitopes on the calcium-binding protein, calmodulin, were found to bind to haustorial cell walls but not to the walls of other fungal or plant cells. The antigens labelled by these seven antibodies are present from the beginning of haustorium development through to haustorium maturity. They were present in the walls of virulent and avirulent races of M. lini, but not in the walls of infection structures of maize or wheat rust. A diverse range of solubilization regimes failed to release the antigens from the cell walls, indicating that they are an integral part of the fungal cell walls. These results thus provide evidence for the specialization of the haustorial wall of the flax rust fungus. Future studies will be aimed at identifying the chemical nature of the wall component detected by the monoclonal antibodies.

1. Hahn M, Mendgen K, 1992. Protoplasma 170, 95-103.
2. Pain NA, O'Connell RJ, Mendgen K, Green JR, 1994. New Phytologist 127, 233-242.
3. Gubler F, 1989. Cell Biology International Reports 13, 137-145.