The prevailing view of intracellular RNA trafficking in eukaryotic cells is that RNAs transcribed in the nucleus either stay in the nucleus or cross the nuclear envelope, entering the cytoplasm for function. However, emerging evidence illustrates that numerous functional RNAs move in the reverse direction, from the cytoplasm to the nucleus. The mechanism underlying RNA nuclear import has not been well elucidated. Viroids are single-stranded circular noncoding RNAs that infect plants. Using Nicotiana benthamiana, tomato (Solanum lycopersicum), and nuclear-replicating viroids as a model, we showed that cellular IMPORTIN ALPHA-4 (IMPa-4) is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (viroid RNA-binding protein 1 [VIRP1]) that binds both IMPa-4 and viroids. Moreover, a conserved C-loop in nuclear-replicating viroids serves as a key signal for nuclear import. Disrupting C-loop impairs VIRP1 binding, viroid nuclear accumulation, and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on VIRP1 for nuclear import. These results advance our understanding of subviral RNA infection and the regulation of RNA nuclear import.

Viroids are single‐stranded circular noncoding RNAs that mainly infect crops. Upon infection, nuclear‐replicating viroids engage host DNA‐dependent RNA polymerase II for RNA‐templated transcription, which is facilitated by a host protein TFIIIA‐7ZF. The sense‐strand and minus‐strand RNA intermediates are differentially localised to the nucleolus and nucleoplasm regions, respectively. The factors and function underlying the differential localisation of viroid RNAs have not been fully elucidated. The sense‐strand RNA intermediates are cleaved into linear monomers by a yet‐to‐be‐identified RNase III‐type enzyme and ligated to form circular RNA progeny by DNA ligase I (LIG1). The subcellular compartment for the ligation reaction has not been characterised. Here, we show that LIG1 and potato spindle tuber viroid (PSTVd) colocalise near the nucleolar region in Nicotiana benthamiana protoplasts. The colocalised region is also the highly condensed region of sense‐strand PSTVd RNA, indicating that PSTVd RNA and LIG1 form a biomolecular condensate for RNA processing. This finding expands the function of biomolecular condensates to the infection of subviral pathogens. In addition, this knowledge of viroid biogenesis will contribute to exploring thousands of viroid‐like RNAs that have been recently identified.

RNA silencing is one of the major defence activities against viral pathogens in plants. Silencing signals are initiated by Dicer‐like proteins (DCLs) to generate viral‐derived small RNAs (sRNAs). Viral sRNAs are then loaded into Argonaute proteins to form an RNA‐induced silencing complex to guide cleavage of target RNAs based on sequence homology. While the model regarding RNA silencing‐mediated defence against viral pathogens is largely established based on extensive studies using the model plant Arabidopsis thaliana , there are diverse sets of silencing components in other plants, especially in domesticated crops. Here, we tracked the expansion of solanaceous‐specific DCL2 genes during the course of evolution. We found that the DCL2a gene in tomato chromosome 6 is likely an evolutionarily new gene copy. We also found that DCL2b is more prone to be induced by viral pathogens in tomato plants, which is dependent on the combinations of cultivar and viral pathogen. Both DCL2a and DCL2b are critical to suppress the accumulation titre of a subviral agent, potato spindle tuber viroid (PSTVd). We noticed an unusually high accumulation of viral sRNAs shorter than 20 nt (16‐ to 19‐nt in length) in viroid‐infected tomato cv. Heinz 1706. Using synthetic small interfering RNAs, we demonstrated that shorter size sRNAs may also play a role in suppressing target RNAs, which can be interfered with by a viral suppressor of silencing, P19. Altogether, we provided further insights into the expansion of functional DCL2 family members in the Solanaceae family and their roles in combating viral and subviral agents.

Aphids represent a major threat to crops. Hundreds of different viruses are aphid-borne. Upon aphid attack, plants release volatile organic compounds (VOCs) as airborne alarm signals to turn on the airborne defense (AD) of neighboring plants, thereby repelling aphids as well as reducing aphid fitness and virus transmission. This phenomenon provides a critical community-wide plant protection to fend off aphids, but the underlying molecular basis remains undetermined for a long time. In a recent article, Gong et al. established the NAC2-SAMT1 module as the core component regulating the emission of methyl-salicylate (MeSA), a major component of VOCs in aphid-attacked plants. Furthermore, they showed that SABP2 protein is critical for the perception of volatile MeSA signal by converting MeSA to Salicylic Acid (SA), which is the cue to elicit AD against aphids at the community level. Moreover, they showed that multiple viruses use a conserved glycine residue in the ATP-dependent helicase domain in viral proteins to shuttle NAC2 from the nucleus to the cytoplasm for degradation, leading to the attenuation of MeSA emission and AD. These findings illuminate the functional roles of key regulators in the complex MeSA-mediated airborne defense process and a counter-defense mechanism used by viruses, which has profound significance in advancing the knowledge of plant-pathogen interactions as well as providing potential targets for gene editing-based crop breeding.

Viroids are a group of infectious noncoding RNAs that cause substantial crop diseases. Previous studies showed that both RNA silencing and plant immunity play defense roles against viroid infection. However, viroids can still achieve successful systemic infection in hosts. The underlying mechanism remains elusive. Through comparing proteomics profiles from mock and infected tomato samples, we unexpectedly observed a systematic reduction of proteins factors involved in nuclear pore complex and nuclear transport receptors. This fortuitous observation was confirmed in subsequent analyses and led us to discover the impaired NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1) nuclear import in infected samples. Transgenically increasing NPR1 expression in tomato plants or exogenously application of salicylic acid analog repressed potato spindle tuber viroid (PSTVd) infection. Altogether, our finding pinpoints the repressed expression of host nuclear transport receptors and nucleoporins as the mechanism limiting the effectiveness of plant defense against viroid, which significantly advances the understanding of plant-pathogen interactions. Significance Statement: Viroids represent a mysterious group of pathogens. Plant-viroid interactions have not been well understood. Here, we report a fortuitous observation of a wide range of protein level reductions in nucleoporins and nuclear transport receptors upon potato spindle tuber viroid (PSTVd) infection. This reduction impairs the nuclear import of a key immune factor, NPR1. Based on this finding, we increased the expression of NPR1 protein in plants or used a salicylic acid analog (acibenzolar-S-methyl/ASM) to elevate NPR1 activity, both of which limited the infection potato spindle tuber viroid. Notably, spraying ASM to prevent PSTVd infection provides the first practical measure against this internationally controlled agent.

RNA is key component of a versatile group of complexes that play diverse roles in a wide range of biological processes, including plant acclimation to ever-changing environments. From synthesis to degradation, RNA molecules interact with cognate proteins that assist in processes such as transcription, splicing, modification, trafficking, and the execution of their functions. While numerous valuable techniques exist to study RNA-protein interactions, visualizing RNAs and their associated proteins simultaneously within cells remains challenging, despite its potential to provide deeper insights into the biology of RNA-protein interactions. In this study, we adapted a modified immunofluorescence (IF) assay in combination with RNA fluorescence in situ hybridization (FISH) to successfully visualize the colocalization of endogenous as well as infectious RNAs with their cognate binding proteins in plant nucleus. This new method that combines IF and FISH will facilitate future studies on RNA and protein colocalization in various plant systems.