Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of viroid “thermomutants” to tomato and Brassica species

Abstract

Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the "pathogenicity" domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid "thermomutants" progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa, where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1, sde2, and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis, a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid "thermomutants"; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in "hairpin II" domain (C-->A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50-80 days pi) when infection reached 70-90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.

Screening of Chrysanthemum Cultivars with Resistance to Chrysanthemum Stunt Viroid

“…It was also suggested that there are at least two types of resistance among chrysanthemum cultivars; however, the possibility cannot be ruled out that these resistant types may be CSVd sequence specific. Accumulating evidence suggests that viroid systemic infections in particular hosts are strongly affected by the viroid sequence and that even a single point mutation can change their compatibilities (Matoušek et al, 2004;Zhong et al, 2007). The data presented in this study confirmed that the resistance to these four sequence types of CSVd infecting P4 plants maintained for more than seven years; however, the resistance of 'Sei no Issei' and 'Mari Kazaguruma' should be evaluated more carefully with respect to the various CSVd sequence types.…”

Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thaliana

“…In the antisense strand of our sequences, we detected no hammerhead or hairpin ribozyme motif, nor was the antisense RNA of the consensus sequence catalytically active (data not shown). Furthermore, Arabidopsis is naturally not infected by viroid species carrying HHRzs, and recent data using this plant as an artificial host indicate that a viroid of the family Avsunviroidae is poorly processed in planta (Daros and Flores, 2004;Matousek et al, 2004). This argues against an evolutionary origin of the HHRzs in Arabidopsis by incorporation of the cDNA of a hammerhead-like viroid RNA.…”

Experimental Transmission ofPospiviroidPopulations to Weed Species Characteristic of Potato and Hop Fields

“…Compared to the standard experimental inoculation method for viroids with Carborundum as an abrasive agent, biolistic inoculation using a gene gun is highly advantageous (25,26). For example, individual sequences in a viroid population that can be coprecipitated to microprojectiles and delivered at once as a mixture to individual cells have comparative starting conditions for propagation.…”