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We developed a new method for simultaneously visualizing RNAs and proteins in plant cells. This method works well for endogenous as well as infectious RNAs and their cognate binding proteins. 

ABSTRACT 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 inNicotiana benthamianaprotoplasts. 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. 

Abstract RNA comprises a versatile group of biomolecules that play diverse roles in a wide range of biological processes. From synthesis to degradation, RNAs 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, observing RNAs and their associated proteins simultaneously within cells remains a challenge, despite its potential to provide deeper insights into RNA-protein interactions. In this study, we adapted a modified immunofluorescence (IF) assay combined with RNA fluorescence in situ hybridization (FISH) to successfully visualize the colocalization of potato spindle tuber viroid with RNA polymerase II in the nucleus. This new method that combines IF and FISH will facilitate future studies on RNA and protein colocalization in various plant systems. 

Abstract 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 theNAC2-SAMT1module 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, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription. 

Abstract 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.