M SINDELAROVA¹, L BURKETOVA¹, V TABORSKY² and L SINDELAR¹

¹lnstitute of Experimental Botany, Academy of Sciences of the Czech Republic, 160 00 Praha 6, CR; ²Czech Agriculture University, 160 00 Praha 6, CR

Background and objectives
In an infected host cell, virus RNA can be synthesised from intermediates of the reductive pentose phosphate pathway during photosynthesis or from intermediates of the oxidative pentose phosphate pathway which is active preferentially in the dark, or from intermediates released from degraded host rRNA [1]. With respect to the degradation of ribonucleic acids, information about the precise number of multiple forms, intracellular location and metabolic functions of the enzymes involved is not so clear [2]. For this reason, we present a complex study of dynamic changes in ribonucleases (RNases) activities in relation to virus multiplication, changes in content and subcellular localisation of their isozymes and a connection of their activities with susceptibility, tolerance and resistance of tobacco cultivars to potato virus Y (PVY) and of sugar beet plants to beet necrotic yellow vein virus (BNYVV) biosynthesis.

Results and conclusions
Activities of RNases in both crude homogenates and partially purified enzyme preparations from diseased discs (tobacco) and leaves or roots (sugar beet) were markedly increased during the experimental period of the acute-infection stage (when compared with healthy plants), the courses of the curves were coincident and correlated with the multiplication curve of the PVY and BNYVV, respectively. It can be concluded that the increase in the activity of RNases is the consequence of enhanced de novo synthesis of the enzyme (coarse regulation) and not the result of fine regulation by low-molecular-weight effectors.

Changes in the content and in the subcellular localization of ribonuclease isozymes were determined in mesophyll protoplasts prepared from Nicotiana tabacum cv. Samsun from healthy and PVY-infected plants. Intact chloroplasts, mitochondria and soluble cytosolic proteins were obtained after protoplast disruption by means of differential centrifugation. The 1000 g pellet from healthy protoplasts contained 6.7% , the 15,000 g pellet 13.2%, and the 15,000 g supernatant 83.4% of the total activity of ribonucleases. The 1000 g pellet from infected protoplasts contained 9.9% , the 15,000 g pellet 10.1% and the 15,000 g supernatant 91.0% of the total activity of ribonucleases. In infected protoplasts, activities of these enzymes were enhanced in ruptured protoplasts preparation to 136.9%, in 1000 g pellet to 203%, in 15,000 g pellet to 104%, and in 15,000 g supernatant to 149.5% of healthy mock-inoculated protoplasts. From the results we conclude that in infected tissue of intact plants, the increased activities of these enzymes are due to the increased content of cytosolic (include vacuolar) and chloroplast isozymes.

A linear correlation was found between the content of PVY and tobacco host resistance as well as between the content of BNYVV and the resistance of sugar beet plants to virus multiplication, determined by enhanced levels of ribonucleases involved in viral RNA biosynthesis in susceptible, tolerant and resistant plants. This finding enabled us to quantitatively define the degree of resistance to virus multiplication for use in gene manipulation and breeding. We presuppose the common validity of these relations so that they could be used for the study of virus resistance of further host plants.

References
1. Sindelarova M, Sindelar L, Burketova L, 1997. Physiological and Molecular Plant Pathology 51, 99-109.
2. Green PJ 1994. Annual Review of Plant Physiology Plant Molecular Biology 45, 421-445.