1.3.38
IMMUNOCYTOCHEMICAL LOCALIZATION OF CYSTEINE PROTEINASE INHIBITORS IN VIRUS-FREE AND PVYNTN-INFECTED POTATO

M RAVNIKAR1, M POLJSAK-PRIJATELJ2, J BRZIN3 and B STRUKELJ3

1National Institute of Biology, Vedna pot 111, 1000 Ljubljana, Slovenia; 2lnstitute of Microbiology and Immunology, Medical Faculty, University of Ljubijana, Zaloska 4, 1000 Ljubljana, Slovenia; 3Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia

Background and objectives
Many proteinase inhibitors are synthesized in response to wounding, severe mechanical damage, infection with pathogenic fungi or insect attack. It could be concluded that proteinase inhibitors are involved in the defence mechanism of plants. Recently it was shown that a drastic increase of aspartic protein inhibitors (API) [1] and cysteine proteinase inhibitors [2] could be induced by jasmonic acid - a messenger molecule in the signal transduction pathway of plant stress response. In the present study we tried to establish a possible involvement of cysteine proteinase inhibitors in virus infection. Immunocytochemical localization studies were performed to observe the sorting and accumulation of cysteine proteinase inhibitors (PCPIs) in cells of virus-free and PVYNTN-infected potato plants.

Materials and methods
Virus-tested plants of potato (Solanum tuberosum L. cv. Igor) were propagated in vitro and transferred to the soil for 4 weeks of growth. After that, they were sap-inoculated by PVYNTN and after 4 days leaves were dissected and fixed in 0.1% glutaraldehyde and 2% paraformaldehyde. They were then dehydrated in a series of ethanol solutions, and embedded in acrylic hydrophilic resin HM 20, and polymerized under UV light. Tissue sections were incubated with antibody solution (rabbit polyclonal antibodies against the homogeneous potato inhibitor with pl 6.6 and 9.4), diluted 1:30 and then incubated with protein-A-gold complex, diluted 1:50. Sections were stained with 1% Reynold's lead citrate and 1% uranyl acetate, and inspected under a Jeol 1200 EX 11 transmission electron microscope. Controls with non-immune rabbit antiserum were used. Gold particles were not found on these sections.

Results and conclusions
Comparing cells of virus-free and virus-infected plants, we observed differential expression of PCPIs. Regarding a response to viral infection, the expression of PCPI 6.6 seems to be unspecific. It was possible to observe moderate gold labelling in cytoplasma and intense labelling of protein bodies in vacuoles in cells of virus-free (unstressed) plants, while PCPI 9.4 accumulated only in cells of virus-infected plants. These first observations showed that virus infection could somehow induce the expression of some PCPIs. To confirm the involvement of PCPIs in plant response to virus attack, additional experiments which include molecular techniques are in progress.

References
1. Kreft S, Ravnikar M, Mesko P et al., 1997. Phytochemistry 44(6), 1001-1006.
2. Gruden K, Strukeij B, Ravnikar M et al., 1997. Plant Molecular Biology 34, 317-323.