TRNA-derived RNA fragments (tRFs) are 18–26 nucleotide small RNAs that are not random degradation products, but are rather specifically cleaved from mature tRNA transcripts. Abundant in stressed or viral-infected cells, the function and potential targets of tRFs are not known. We identified that in the unstressed wild-type male gamete containing pollen of flowering plants, and analogous reproductive structure in non-flowering plant species, tRFs accumulate to high levels. In the reference plant Arabidopsis thaliana, tRFs are processed by Dicer-like 1 and incorporated into Argonaute1 (AGO1), akin to a microRNA. We utilized the fact that many plant small RNAs direct cleavage of their target transcripts to demonstrate that the tRF–AGO1 complex acts to specifically target and cleave endogenous transposable element (TE) mRNAs produced from transcriptionally active TEs. The data presented here demonstrate that tRFs are bona-fide regulatory microRNA-like small RNAs involved in the regulation of genome stability through the targeting of TE transcripts.

Summary: Chloride (Cl⁻) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation–Cl⁻ cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub‐family of animal K⁺:Cl⁻ cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co‐ordinated symport of K⁺, Na⁺ and Cl⁻, and this transport activity was inhibited by the ‘loop’ diuretic bumetanide, a specific inhibitor of vertebrate Na⁺:K⁺:Cl⁻ cotransporters, indicating that At CCC encodes for a bona fide Na⁺:K⁺:Cl⁻ cotransporter. Analysis of At CCC promoter‐β‐glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T‐DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl⁻ homeostasis under high salinity, as they accumulated higher and lower Cl⁻ amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long‐distance Cl⁻ transport. Compelling evidence is provided on the occurrence of cation–chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl⁻ homeostasis.

Viroids are small self-replicating RNAs that infect plants. How these noncoding pathogenic RNAs interact with hosts to induce disease symptoms is a long-standing unanswered question. Recent experimental data have led to the suggestive proposal of a pathogenic model based on the RNA silencing mechanism. However, evidence of a direct relation between key components of the RNA silencing pathway and symptom expression in infected plants remains elusive. To address this issue, we used a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes dimeric forms of Hop stunt viroid (HSVd). These plants were analyzed under different growing temperature conditions and were used as stocks in grafting assays with the rdr6i-Nb line, in which the RNA-dependent RNA polymerase 6 (RDR6) is constitutively silenced. Here, we show that the symptom expression in N. benthamiana plants is independent of HSVd accumulation levels but dependent on an active state of the viroid-specific RNA silencing pathway. The scion of rdr6i-Nb plants remained asymptomatic when grafted onto symptomatic plants, despite an accumulation of a high level of mature forms of HSVd, indicating the requirement of RDR6 for viroid-induced symptom production. In addition, the RDR6 requirement for symptom expression was also observed in wild-type N. benthamiana plants mechanically infected with HSVd. These results provide biological evidence of the involvement of the viroid-specific RNA silencing pathway in the symptom expression associated with viroid pathogenesis.