Petr Dědič

Acid nucleases in PSTV-infected tomato (Lycopersicum esculentum L.) I. Levels of Acid Nuclease Activity in Healthy and PSTV-Infected Tomato Leaves and Callus Tissues

Summary Nuclease activities at pH 5.2 were investigated in potato spindle tuber viroid (PSTV)-infected tomato leaves and calluses. DNase activity per g fresh mass and per mg soluble protein was, approximately, two times higher in infected leaves at «early» stage of pathogenesis than in healthy controls. The activity remained enhanced also when symptoms related to PSTV infection expressed to a greater degree. The nuclease activity was higher with heat denatured ssDNA than with native dsDNA. The ratio of activities did not change significantly upon viroid infection. The activity with RNA in healthy leaves was about forty-fold higher than with dsDNA, but was enhanced only by 19% in infected leaves at the «early» stage of pathogenesis. The nuclease activity with dsDNA was several times higher in tomato calluses than in tomato leaves. Examination of the DNase activities in healthy and PSTV-infected calluses grown at different temperatures showed significant differences and their relation to PSTV pathogenesis.

Acid Nucleases in PSTV-Infected Tomato (Lycopersicum esculentum L.) II. Characterization of Sugar Non-Specific Nuclease Extracted from Healthy and PSTV-Infected Tomato Leaves

Summary The activity of acid nuclease was investigated in healthy and potato spindle tuber viroid (PSTV)-infected tomato leaves. The enzyme has some characteristics of nuclease I, exhibiting DNase, RNase and presumably 3′-nucleotidase activities. It also showed preference for heat-denatured DNA versus native DNA and was active at pH 5.2. Molecular sieving of the extract from infected tomato leaves produced one distinct nuclease peak with an apparent molecular mass of 26 kDaltons. The nuclease extracted from healthy and PSTV-infected leaves exhibited similar PAGE patterns under both native and denatured conditions. On the other hand, some quantitative and qualitative differences were observed on SDS-PAGE and from immunoelectrophoresis patterns of proteins extracted at pH 5.2 from infected and healthy leaves. Both, circular and linear forms of PSTV were found to be cleavable with the nuclease. But PSTVspecific RNA was about 900-fold more resistant substrate to cleavage with plant nucleases than highly polymerized RNA from yeast.

Characterization of Potato spindle tuber viroid (PSTVd) incidence and new variants from ornamentals

We analyzed the spreading and persistence of PSTVd variants in several ornamentals in the territory of the Czech Republic. The pool of PSTVd variants detected in Solanum jasminoides, S. muricatum, Datura sp. and Brugmansia sp. was biolistically transferred to Matricaria chamomilla, Argyranthemum frutescens and Diascia sp., species which we found as sensitive hosts for PSTVd from ornamentals. The PSTVd pool showed sequence changes and increased variation after its transfer to potato, suggesting a wide adaptation potential of PSTVd in this crop. Potato exhibited genotype-dependent leaf and spindle tuber symptoms, when inoculated with the sap from S. jasminoides infected with the predominant and sequence-stable PSTVd-S1.

Complementation analysis of triple gene block of Potato virus S (PVS) revealed its capability to support systemic infection and aphid transmissibility of recombinant Potato virus X.

Triple gene block (TGB) sequences derived from isolates of ordinary Potato virus S (PVS-O) and Chenopodium-systemic (PVS-CS) were analyzed. Although the TGB sequences did not reveal any specific difference within the 7K protein, some specific differences within the 25K and 12K ORFs were found. In order to investigate a possible functional divergence of PVS-O and PVS-CS TGB variants, these genes were propagated in chimeric Potato virus X (PVX). Both PVS TGB variants partly complemented PVX TGB in Nicotiana benthamiana. The recombinant viruses multiplied to lower titer than the wild-type PVX in N. benthamiana showed attenuated symptoms. Whereas the recombinant PVX variants were also propagated systemically in Nicotiana glutinosa, Celosia argentea, Nicotiana occidentalis and chimeric PVX bearing TGB from PVS-O in Solanum lycopersicum, neither were propagated systemically in Chenopodium quinoa nor in Nicotiana tabacum cv. Samsun nn and the PVX-resistant Solanum tuberosum cv. Szignal. The potential for recombinant viruses to be transmitted by the aphid Myzus persicae was investigated. Aphid transmission in the recombinant virus was obtained by replacing PVX TGB by TGB from the PVS-CS isolate. These results show the potential function of Carlavirus TGB in aphid transmissibility and underlines the possible biological risks from certain recombinant virus variants.

An immunochemical study of antigen expression in potato spindle tuber viroid (PSTV) - infected tomato leaves and calluses

A polyspecific antiserum against protein extracted from PSTV-infected tomato leaves was prepared and the IgGs were separated by affinity chromatography on a beaded cellulose adsorbent with an immobilized “healthy” antigen. The antibody not adsorbed entered into a preferential reaction with the antigen from PSTV-infected leaves as estimated by an enzyme-linked immunosorbent assay. The immunochemical reactions did not significantly exceed the control background, if antigens from tomato leaves infected with potato viruses X, Y and M were analyzed. By immunoblot technique we revealed, however, that several antigens not detected in healthy leaves appeared in the leaves infected either with PSTV or with viruses X and M. An accumulation of a major antigen having a molecular mass of about 70 kDa was observed in viroid-infected leaves only, suggesting the specificity for viroid infection. The antigen was found not to be an alkaline endoproteinase - the pathogenesis-related protein P-69.Some antigens with molecular masses approximately 38.0, 23.7 and 22 kDa, which occurred in PSTV-infected leaves and in healthy calluses, were not detectable in PSTV-infected calluses.No reaction exceeding the control level was observed using enzyme-linked immunosorbent assay for antigens from silver nitrate-treated tomato leaves, although such leaves showed symptoms similar to that caused by viroids.