Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis

Abstract

Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

Antitumor activity of apoptotic nuclease TBN1 from L. esculentum.

“…The plants were grown under natural light with supplementary illumination [90 µmol m -2 s -1 PAR] to keep a 16 h-day period. Nuclease TBN1 cDNA (GenBank accession number AM238701) [25] was used for construction of the plant expression vector. This cDNA was re-amplified using primers Xho5'TBN1 (5'gacCTCGAGGGCCCATGTTGAGGTTAAC TTTATTAAGC 3') and Xba3'TBN1 (5'aatTCTAGATTAAG TTGCAACAACTGAATCTTC3') (restriction ends designed for construction of a plant vector suitable for the expression of the recombinant TBN1 nuclease are underlined and added nucleotides are designated by small letters).…”

“…Sequences of nuclease I class designated as bifunctional nucleases [20] were reported, for instance, for barley [21,22], Arabidopsis [20], zinnia [20,23] daylily [24], tomato [25] and hop [26]. Bifunctional plant nucleases are closely related, forming a cluster of amino acid sequences with high identity [25].…”

“…Bifunctional plant nucleases are closely related, forming a cluster of amino acid sequences with high identity [25]. Some of bifunctional plant nucleases are activated under stress conditions and play an important role in the cell apoptosis [22,23].…”

“…In our previous work, we identified and cloned cDNA of bifunctial nuclease TBN1 from tomato sufferred from viroid disease [25]. It was proposed that disease-induced upregulation of this nuclease leads to apoptosis and collapse of the Plants as producers of new engineered pharmaceuticals are becoming a very attractive tool in attempts to improve human health [1,2].…”

“…In this respect, it is of interest to analyze nucleolytic enzymes of plant origin for their potential antitumorigenic effects and biological activities. plant vascular system [25]. In the present work we analyzed degradative potential of TBN1 nuclease-dsRNAse and its targeting as a cytostatic agent against human malignant tumors grown on nude mice.…”