Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

HYL1's multiverse: A journey through miRNA biogenesis and beyond canonical and non-canonical functions of HYL1.

A delicate balance in gene expression, a process highly controlled by post-transcriptional gene silencing mediated by miRNAs, is vital during plant growth and responses to stress. Within the miRNA biogenesis pathway, HYL1 is one of the most important proteins, initially recognized for its role as a cofactor of DCL1. Yet, HYL1's functions extend beyond miRNA processing, encompassing transcriptional regulation and protein translation between other recently discovered functions. This review comprehensively examines our current knowledge of HYL1 functions in plants, looking at its structure, the complex biochemistry behind it, and its involvement in a variety of cellular processes. We also explored the most compelling open questions regarding HYL1 biology and the further perspectives in its study. Unraveling HYL1 functional details could better understand how plants grow, face environmental stresses, and how the miRNA pathway adapts its outcome to the plant growing conditions.

The HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15-HISTONE DEACETYLASE9 complex associates with HYPONASTIC LEAVES 1 to modulate microRNA expression in response to abscisic acid signaling.

The regulation of microRNA (miRNA) biogenesis is crucial for maintaining plant homeostasis under biotic and abiotic stress. The crosstalk between the RNA polymerase II (Pol-II) complex and the miRNA processing machinery has emerged as a central hub modulating transcription and cotranscriptional processing of primary miRNA transcripts (pri-miRNAs). However, it remains unclear how miRNA-specific transcriptional regulators recognize MIRNA loci. Here, we show that the Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15)-HISTONE DEACETYLASE9 (HDA9) complex is a conditional suppressor of miRNA biogenesis, particularly in response to abscisic acid (ABA). When treated with ABA, hos15/hda9 mutants show enhanced transcription of pri-miRNAs that is accompanied by increased processing, leading to overaccumulation of a set of mature miRNAs. Moreover, upon recognition of the nascent pri-miRNAs, the ABA-induced recruitment of the HOS15-HDA9 complex to MIRNA loci is guided by HYPONASTIC LEAVES 1 (HYL1). The HYL1-dependent recruitment of the HOS15-HDA9 complex to MIRNA loci suppresses expression of MIRNAs and processing of pri-miRNA. Most importantly, our findings indicate that nascent pri-miRNAs serve as scaffolds for recruiting transcriptional regulators, specifically to MIRNA loci. This indicates that RNA molecules can act as regulators of their own expression by causing a negative feedback loop that turns off their transcription, providing a self-buffering system.

Polymorphic inverted repeats near coding genes impact chromatin topology and phenotypic traits in Arabidopsis thaliana.

Transposons are mobile elements that are commonly silenced to protect eukaryotic genome integrity. In plants, transposable element (TE)-derived inverted repeats (IRs) are commonly found near genes, where they affect host gene expression. However, the molecular mechanisms of such regulation are unclear in most cases. Expression of these IRs is associated with production of 24-nt small RNAs, methylation of the IRs, and drastic changes in local 3D chromatin organization. Notably, many of these IRs differ between Arabidopsis thaliana accessions, causing variation in short-range chromatin interactions and gene expression. CRISPR-Cas9-mediated disruption of two IRs leads to a switch in genome topology and gene expression with phenotypic consequences. Our data show that insertion of an IR near a gene provides an anchor point for chromatin interactions that profoundly impact the activity of neighboring loci. This turns IRs into powerful evolutionary agents that can contribute to rapid adaptation.

Cotranscriptional RNA processing and modification in plants.

The activities of RNA polymerases shape the epigenetic landscape of genomes with profound consequences for genome integrity and gene expression. A fundamental event during the regulation of eukaryotic gene expression is the coordination between transcription and RNA processing. Most primary RNAs mature through various RNA processing and modification events to become fully functional. While pioneering results positioned RNA maturation steps after transcription ends, the coupling between the maturation of diverse RNA species and their transcription is becoming increasingly evident in plants. In this review, we discuss recent advances in our understanding of the crosstalk between RNA Polymerase II, IV, and V transcription and nascent RNA processing of both coding and noncoding RNAs.

Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

The eINTACT system dissects bacterial exploitation of plant osmosignalling to enhance virulence.

Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts.

Keeping up with the miRNAs: current paradigms of the biogenesis pathway.

For many years we have studied the processes involved in producing miRNAs in plants and the numerous differences from their metazoan counterpart. A well-defined catalytic process, mostly carried out by the RNase III enzyme DICER-LIKE1 (DCL1), it was identified early after the discovery of RNAi and was followed by the isolation of a plethora of miRNA biogenesis cofactors. The production of miRNAs, which later are loaded in ARGONAUTE (AGO) proteins to perform their RNA silencing functions both within the cell and non-cell autonomously, appears to be a highly regulated and dynamic process. Many regulatory events during miRNA biogenesis require the action of specific proteins. However, in recent years, many post-transcriptional modifications, structural features, and coupling with other cellular processing emerged as critical elements controlling the production of miRNA and, thus, a plant's physiology. This review discusses new evidence that has changed the way we understand how miRNAs are produced in plants. We also provide an updated view of the miRNA biogenesis pathways, focusing on the gaps in our knowledge and the most compelling questions that remain open.