The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.

Viroids are small, circular, single stranded RNA molecules that infect plants. Since they are non-coding, their structures play a critical role in their life cycles. To date, little effort has been spend on elucidating viroid structures in solution due to both the experimental difficulties and the time-consuming nature of the methodologies implicated. Recently, the technique of high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) was adapted for the probing of the members of family Avsunviroidae, all of whom replicate in the chloroplast and demonstrate ribozyme activity. In the present work, twelve viroid species belonging to four different genera of the family Pospiviroidae, whose members are characterized by the presence of a central conserved region (CCR) and who replicate in nucleus of the host, were probed. Given that the structures of five distinct viroid species from the family Pospiviroidae have been previously reported, an overview of the different structural characteristics for all genera and the beginning of a manual classification of the different viroids based on their structural features are presented here.

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen. Significance Our article presents the most comprehensive reconstruction of the evolutionary and phylogeographic history of a major plant pathogen of brassica vegetables in Eurasia. Sampling across such a large landmass poses considerable challenges, and our study attempts to describe the spatial and temporal patterns of migration for a plant pathogen on a large scale. Our phylogeographic and molecular clock analyses show that the migration pathways of turnip mosaic potyvirus retrace some of the historical trade arteries of the Silk Road. This study demonstrates how a comprehensive genetic analysis can provide a large-scale view of the epidemiology and human-mediated spread of a plant pathogen across centuries of evolutionary history.

It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (−) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3′ UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.