Correlation between activity of ribonucleases and potato virus Y biosynthesis in tobacco plants M. S Ï INDELA Â R Ï OVA Â *, L. S Ï INDELA Â R Ï and L. BURKETOVA Â Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Karlovce 1, CS-160 00, Prague 6, Czech Republic (Accepted for publication September 2000 and published electronically 27 October 2000) Tobacco plants, leaf discs and protoplasts infected with two strains of the potato virus Y (PVY O and PVY N ) were studied during the period of the acute infection stage. The following characteristics of the host plants were monitored: dynamic changes in the ribonuclease (RNase) activities in relation to the virus multiplication; changes in subcellular localization of RNases, phosphomonoesterase (PME) and phosphodiesterase (PDE); and connection of RNase activities with host plants resistance. Activities of RNases in both the crude homogenates and the partially puri®ed enzyme preparations from the diseased discs were markedly increased during the experimental period when compared to the discs from healthy plants. The courses of both curves were coincident and correlated with the multiplication curve of the PVY O and PVY N strains. Changes in the content and in the subcellular localization of RNase, PME and PDE isozymes were determined in mesophyll protoplasts prepared from the healthy and PVY N infected plants of Nicotiana tabacum L. cv. Samsun. In the chloroplast and cytosol fractions of the infected protoplasts, activities of RNase as well as PME and PDE were increased. The enhanced content of the RNase isozymes was also found in the infected leaves after electrophoretic separation. A linear correlation (P 5 0 . 001) was found between the content of the potato virus Y N and the tobacco host resistance to virus multiplication. This was established on the basis of enhanced levels of RNases, using susceptible, tolerant and resistant plants. * c 2000 Academic Press Keywords: Nicotiana tabacum L. cv. Samsun; cell organelles; leaf discs; potato virus Y; protoplasts; resistance; ribonucleases; rRNA; subcellular localization; virus RNA; phosphomonoesterase; phospho- diesterase. INTRODUCTION In an infected host cell, virus RNA can be synthesized from intermediates of the reductive pentosephosphate pathway during photosynthesis or from intermediates of the oxidative pentosephosphate pathway which is active preferentially in the dark, or from intermediates released from degraded host rRNA. These three metabolic path- ways are involved in virus RNA biosynthesis, but their participation usually depends on the type of virus, host and environmental conditions [38 ]. Knowledge about the number of multiple forms, intracellular location and metabolic functions of the enzymes involved in the degradation of ribonucleic acids in plant cells is not fully clear [15 ]. To obtain insight into the regulation of cellular RNA breakdown, extensive studies were done to localize and purify the ribonucleo- lytic enzymes from the plant material [18 , 43 ]. The occurrence of an RNA-degrading enzyme activity was demonstrated in extracellular space [4 ] but up to 80 % of RNA-splitting capacity was found in intracel- lular space. Cytochemical and direct isolation studies have revealed the vacuolar location of nuclease activities [25 , 31 ]. Abel and Glund [1 ] and Boller and Kende [6 ] indicated that the major vacuolar ribonucleolytic activity was identical with that of the soluble major plant endoribonuclease. The increase in activity of ribonu- cleases (RNases) in plant tissues could be induced by wounding [3 , 12 ], by fungal infection [4 , 40 ], by chilling or osmotic stress [22 , 42 ] or by viral infections. Diener [12 ] observed stimulation of RNases in TMV- inoculated Datura stramonium and in BPMV-inoculated Phaseolus vulgaris, Wyen et al. [44 ] found increased concentration of a relatively purine speci®c endoribonu- clease in TMV-inoculated Xanthi-nc tobacco leaves, and Randles [28 ] reported that the activity of one of three host RNases rose signi®cantly at the time of rapid virus accumulation in Chinese cabbage systemically infected Physiological and Molecular Plant Pathology (2000) 57, 191±199 doi:10.1006/pmpp.2000.0296, available online at http://www.idealibrary.com on 0885-5765/00/110191+09 $35.00/00 * c 2000 Academic Press Abbreviations used in text: BPMV, bean pod mottle virus; PDE, phosphodiesterase; PME, phosphomonoesterase; PVY O , potato virus Y, ordinary strain; PVY N , potato virus Y, necrotic strain; RNase, ribonuclease; TMV, tobacco mosaic virus; TYMV, turnip yellow mosaic virus. * To whom all correspondence should be addressed. E-mail: sindelarova@ueb.cas.cz