The Gradual Reduction of Viroid Levels in Hop Mericlones Following Heat Therapy: A Possible Role for a Nuclease Degrading dsRNA

Abstract

A high concentration of hop latent viroid (HLVd) was detected in infected mericlones of Osvald's hops grown in vitro. This concentration was about 8-fold higher than in leaves of young, field-grown plants, reaching about 30 pg/mg of fresh mass. Treatment of these in vitro-grown plants at high temperature (35 degrees C) for two weeks lead to a dramatic (about 70-90%) decrease of HLVd content. More detailed investigations performed with mericlone 6147 of Osvald 31 showed that HLVd levels decrease gradually during subsequent cycles of heat treatment. A nuclease activity capable of cleaving HLVd and fully double-stranded RNA was shown to increase significantly in hop tissues during thermotherapy cycles, or after the heat shock. The nuclease activity was found to have similar properties to those extracted earlier from tobacco anthers. This enzyme resembles a sugar-unspecific nuclease which has a maximum activity at pH 5.5. Analysis of the activity with viroid and dsRNA showed that both, endo- and exonucleolytic activities were attributable to the enzyme. A strong tissue-specific gradient of viroid (the lowest level in stem apex and the highest level in roots) was observed in young plants, showing a negative correlation with the dsRNAse activity. In senescent plants, the highest viroid concentration was observed in maturated cones and in upper stems. High nuclease activity in the upper stem tissue suggests that viroid RNA must be protected in this tissue against degradation.

Sequence Analysis of a “True” Chalcone Synthase (chs_H1) Oligofamily from hop (Humulus lupulus L.) and PAP1 Activation of chs_H1 in Heterologous Systems

“…After blotting onto Biodyne A transfer membranes (Pall, Hampshire, England), samples were hybridized to a full-length PAP1 cDNA probe labeled with [R-32 P]dCTP. Prehybridization and hybridization were carried out in a 50% formamide-based (pre)hybridization buffer (29) , which provided a concentrated and high-intensity (7000 µW cm -2 ) beam of 365 nm at a distance of 38 cm. If not otherwise stated, plant leaves were exposed to UV-A for 15 min from a distance between 35 and 40 cm.…”

Crystallization and Preliminary Diffraction Data of a Major Pollen Allergen

“…It is known that group V allergens are exported from the pollen once they are released from the inflorescence (26). Novel data presented on pollen RNases in different plant species indicate that they may function as defense proteins against viral infection since a number of pollen RNases can degrade double-stranded RNA (27). The relatively low RNase specific activity of Phl p 5b placed a question mark over biological significance, but the increase in activity caused by cleavage of the N-terminal portion suggests that the protein may play a significant role in the pollen biology.…”

Antitumor activity of apoptotic nuclease TBN1 from L. esculentum.

“…Reactions were terminated by phenolization, samples were then precipitated by ethanol precipitation, and separated by electrophoresis in a denaturating 1.5% agarose gel containing formaldehyde. After capillary blotting the samples onto Biodyne A transfer membrane (Pall, Hampshire, England), prehybridization and hybridization were carried out in 50% formamide-based (pre)hybridization buffer [30] at 32°C overnight. The washing procedure included an incubation of membranes in 2x SSC for 10 min at 0 °C followed by incubation in 2x SSC buffer containing 0.1% SDS for 30 In some experiments we used partly purified potato spindle tuber viroid (PSTVd) RNA as substrate and assayed viroid cleavage after separation of RNA in polyacrylamide denaturing gels, electroblot and hybridization as described by Matoušek et al [26].…”