Some viroids-single-stranded, non-coding, circular RNA parasites of plants-are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (-) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming "comets" on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.

• Keywords
• AFCVd and CBCVd propagation and eradication, Nicotiana tabacum, TUDOR S-nuclease, male gametophyte, recombinant AGO, small RNA, strand-specific viroid RT-qPCR, viroid degradation, viroid replication,
• MeSH
• Phenotype MeSH
• Host-Pathogen Interactions MeSH
• Nucleic Acid Conformation MeSH
• Plant Diseases virology   MeSH
• Pollen virology   MeSH
• Virus Replication MeSH
• RNA, Viral MeSH
• Nicotiana virology   MeSH
• Viroids * MeSH
• Viral Load MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Viral MeSH

Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250-400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than [Formula: see text], quite comparable to the value of [Formula: see text] reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.

• Keywords
• Error rate, Pospiviroid, error threshold, quasispecies, sequence network mapping, sequencing error, viroid-specific small RNA,
• MeSH
• Genome, Viral * MeSH
• Mutation MeSH
• Reassortant Viruses genetics   MeSH
• Virus Replication MeSH
• RNA, Viral genetics   MeSH
• Viroids genetics   MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH