Protein arginine methyltransferase 6 mediates antiviral immunity in plants.

Viral suppressor RNA silencing (VSR) is essential for successful infection. Nucleotide-binding and leucine-rich repeat (NLR)-based and autophagy-mediated immune responses have been reported to target VSR as counter-defense strategies. Here, we report a protein arginine methyltransferase 6 (PRMT6)-mediated defense mechanism targeting VSR. The knockout and overexpression of PRMT6 in tomato plants lead to enhanced and reduced disease symptoms, respectively, during tomato bush stunt virus (TBSV) infection. PRMT6 interacts with and inhibits the VSR function of TBSV P19 by methylating its key arginine residues R43 and R115, thereby reducing its dimerization and small RNA-binding activities. Analysis of the natural tomato population reveals that two major alleles associated with high and low levels of PRMT6 expression are significantly associated with high and low levels of viral resistance, respectively. Our study establishes PRMT6-mediated arginine methylation of VSR as a mechanism of plant immunity against viruses.

Transposon and Transgene Tribulations in Mosquitoes: A Perspective of piRNA Proportions.

Mosquitoes, like Drosophila, are dipterans, the order of "true flies" characterized by a single set of two wings. Drosophila are prime model organisms for biomedical research, while mosquito researchers struggle to establish robust molecular biology in these that are arguably the most dangerous vectors of human pathogens. Both insects utilize the RNA interference (RNAi) pathway to generate small RNAs to silence transposons and viruses, yet details are emerging that several RNAi features are unique to each insect family, such as how culicine mosquitoes have evolved extreme genomic feature differences connected to their unique RNAi features. A major technical difference in the molecular genetic studies of these insects is that generating stable transgenic animals are routine in Drosophila but still variable in stability in mosquitoes, despite genomic DNA-editing advances. By comparing and contrasting the differences in the RNAi pathways of Drosophila and mosquitoes, in this review we propose a hypothesis that transgene DNAs are possibly more intensely targeted by mosquito RNAi pathways and chromatin regulatory pathways than in Drosophila. We review the latest findings on mosquito RNAi pathways, which are still much less well understood than in Drosophila, and we speculate that deeper study into how mosquitoes modulate transposons and viruses with Piwi-interacting RNAs (piRNAs) will yield clues to improving transgene DNA expression stability in transgenic mosquitoes.

Viral Recognition and Evasion in Plants.

Viruses, causal agents of devastating diseases in plants, are obligate intracellular pathogens composed of a nucleic acid genome and a limited number of viral proteins. The diversity of plant viruses, their diminutive molecular nature, and their symplastic localization pose challenges to understanding the interplay between these pathogens and their hosts in the currently accepted framework of plant innate immunity. It is clear, nevertheless, that plants can recognize the presence of a virus and activate antiviral immune responses, although our knowledge of the breadth of invasion signals and the underpinning sensing events is far from complete. Below, I discuss some of the demonstrated or hypothesized mechanisms enabling viral recognition in plants, the step preceding the onset of antiviral immunity, as well as the strategies viruses have evolved to evade or suppress their detection.

RH20, a phase-separated RNA helicase protein, facilitates plant resistance to viruses.

RNA silencing, a conserved gene regulatory mechanism, is critical for host resistance to viruses. Liquid-liquid phase separation (LLPS) is an important mechanism in regulating various biological processes. Emerging studies suggest RNA helicases play important roles in microRNA (miRNA) production through LLPS. In this study, we investigated the functional role of RNA helicase 20 (RH20), a DDX5 homolog in Arabidopsis thaliana, in RNA silencing and plant resistance to viruses. Our findings reveal that RH20 localizes in both the cytoplasm and nucleus, with puncta formation in the cytoplasm exhibiting liquid-liquid phase separation behavior. We demonstrate that RH20 plays positive roles in plant immunity against viruses. Further study showed that RH20 interacts with Argonaute 2 (AGO2), a key component of the RNA silencing pathway. Moreover, RH20 promotes the accumulation of both endogenous and exogenous small RNAs (sRNAs). Overall, our study identifies RH20 as a novel phase separation protein that interacting with AGO2, influencing sRNAs accumulation, and enhancing plant resistance to viruses.

Deficiency of multiple RNA silencing-associated genes may contribute to the increased susceptibility of Nicotiana benthamiana to viruses.

KEY MESSAGE: Recently published high-quality reference genome assemblies indicate that, in addition to RDR1-deficiency, the loss of several key RNA silencing-associated genes may contribute to the hypersusceptibility of Nicotiana benthamiana to viruses.

Analysis of the TID-I and TID-L Splice Variants' Expression Profile under In Vitro Differentiation of Human Mesenchymal Bone Marrow Cells into Osteoblasts.

Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the TID splice variants (TID-L, TID-I) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the TID gene on the expression of ALPL and SPP1 was also investigated. The TID proteins and the expression of TID splice variants were detected. After differentiation, the expression of TID-L and TID-I increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of SPP1. Three days after transfection, the expression of SPP1 increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of TID-L and TID-I changes under differentiation of B-MSCs into osteoblasts and may influence the expression of SPP1. However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.

microRNA-guided immunity against respiratory virus infection in human and mouse lung cells.

Viral infectivity depends on multiple factors. Recent studies showed that the interaction between viral RNAs and endogenous microRNAs (miRNAs) regulates viral infectivity; viral RNAs function as a sponge of endogenous miRNAs and result in upregulation of its original target genes, while endogenous miRNAs target viral RNAs directly and result in repression of viral gene expression. In this study, we analyzed the possible interaction between parainfluenza virus RNA and endogenous miRNAs in human and mouse lungs. We showed that the parainfluenza virus can form base pairs with human miRNAs abundantly than mouse miRNAs. Furthermore, we analyzed that the sponge effect of endogenous miRNAs on viral RNAs may induce the upregulation of transcription regulatory factors. Then, we performed RNA-sequence analysis and observed the upregulation of transcription regulatory factors in the early stages of parainfluenza virus infection. Our studies showed how the differential expression of endogenous miRNAs in lungs could contribute to respiratory virus infection and species- or tissue-specific mechanisms and common mechanisms could be conserved in humans and mice and regulated by miRNAs during viral infection.

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Role of the prefrontal cortical protease TACE/ADAM17 in neurobehavioral responses to chronic stress during adolescence.

INTRODUCTION: Chronic adolescent stress profoundly affects prefrontal cortical networks regulating top-down behavior control. However, the neurobiological pathways contributing to stress-induced alterations in the brain and behavior remain largely unknown. Chronic stress influences brain growth factors and immune responses, which may, in turn, disrupt the maturation and function of prefrontal cortical networks. The tumor necrosis factor alpha-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17) is a sheddase with essential functions in brain maturation, behavior, and inflammatory responses. This study aimed to determine the impact of stress on the prefrontal cortex and whether TACE/ADAM17 plays a role in these responses. METHODS: We used a Lewis rat model that incorporates critical elements of chronic psychosocial stress, such as uncontrollability, unpredictability, lack of social support, and re-experiencing of trauma. RESULTS: Chronic stress during adolescence reduced the acoustic startle reflex and social interactions while increasing extracellular free water content and TACE/ADAM17 mRNA levels in the medial prefrontal cortex. Chronic stress altered various ethological behavioral domains in the observation home cages (decreased ingestive behaviors and increased walking, grooming, and rearing behaviors). A group of rats was injected intracerebrally either with a novel Accell™ SMARTpool TACE/ADAM17 siRNA or a corresponding siRNA vehicle (control). The RNAscope Multiplex Fluorescent v2 Assay was used to visualize mRNA expression. Automated puncta quantification and analyses demonstrated that TACE/ADAM17 siRNA administration reduced TACE/ADAM17 mRNA levels in the medial prefrontal cortex (59% reduction relative to control). We found that the rats that received prefrontal cortical TACE/ADAM17 siRNA administration exhibited altered eating patterns (e.g., increased food intake and time in the feeding zone during the light cycle). CONCLUSION: This study supports that the prefrontal cortex is sensitive to adolescent chronic stress and suggests that TACE/ADAM17 may be involved in the brain responses to stress.

Heritable epigenetic changes are constrained by the dynamics of regulatory architectures.

Interacting molecules create regulatory architectures that can persist despite turnover of molecules. Although epigenetic changes occur within the context of such architectures, there is limited understanding of how they can influence the heritability of changes. Here, I develop criteria for the heritability of regulatory architectures and use quantitative simulations of interacting regulators parsed as entities, their sensors, and the sensed properties to analyze how architectures influence heritable epigenetic changes. Information contained in regulatory architectures grows rapidly with the number of interacting molecules and its transmission requires positive feedback loops. While these architectures can recover after many epigenetic perturbations, some resulting changes can become permanently heritable. Architectures that are otherwise unstable can become heritable through periodic interactions with external regulators, which suggests that mortal somatic lineages with cells that reproducibly interact with the immortal germ lineage could make a wider variety of architectures heritable. Differential inhibition of the positive feedback loops that transmit regulatory architectures across generations can explain the gene-specific differences in heritable RNA silencing observed in the nematode Caenorhabditis elegans. More broadly, these results provide a foundation for analyzing the inheritance of epigenetic changes within the context of the regulatory architectures implemented using diverse molecules in different living systems.

Selecting genes for analysis using historically contingent progress: from RNA changes to protein-protein interactions.

Progress in biology has generated numerous lists of genes that share some property. But, advancing from these lists of genes to understanding their roles is slow and unsystematic. Here we use RNA silencing in C. elegans to illustrate an approach for prioritizing genes for detailed study given limited resources. The partially subjective relationships between genes forged by both deduced functional relatedness and biased progress in the field was captured as mutual information and used to cluster genes that were frequently identified yet remain understudied. Studied genes in these clusters suggest regulatory links connecting RNA silencing with other processes like the cell cycle. Many proteins encoded by the understudied genes are predicted to physically interact with known regulators of RNA silencing. These predicted influencers of RNA-regulated expression could be used for feedback regulation, which is essential for the homeostasis observed in all living systems. Thus, among the gene products altered when a process is perturbed are regulators of that process, providing a way to use RNA sequencing to identify candidate protein-protein interactions. Together, the analysis of perturbed transcripts and potential interactions of the proteins they encode could help prioritize candidate regulators of any process.

Straightforward and affordable agroinfiltration with RUBY accelerates RNA silencing research.

Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.

Advances in the mechanisms and applications of RNA silencing in crop protection.

RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes, which plays vital roles in plant development and response to biotic and abiotic stresses. The discovery of trans-kingdom RNAi and interspecies RNAi provides a theoretical basis for exploiting RNAi-based crop protection strategies. Here, we summarize the canonical RNAi mechanisms in plants and review representative studies associated with plant-pathogen interactions. Meanwhile, we also elaborate upon the principles of host-induced gene silencing, spray-induced gene silencing and microbe-induced gene silencing, and discuss their applications in crop protection, thereby providing help to establish novel RNAi-based crop protection strategies.

Understanding Citrus Viroid Interactions: Experience and Prospects.

Citrus is the natural host of at least eight viroid species, providing a natural platform for studying interactions among viroids. The latter manifests as antagonistic or synergistic phenomena. The antagonistic effect among citrus viroids intuitively leads to reduced symptoms caused by citrus viroids, while the synergistic effect leads to an increase in symptom severity. The interaction phenomenon is complex and interesting, and a deep understanding of the underlying mechanisms induced during this viroid interaction is of great significance for the prevention and control of viroid diseases. This paper summarizes the research progress of citrus viroids in recent years, focusing on the interaction phenomenon and analyzing their interaction mechanisms. It points out the core role of the host RNA silencing mechanism and viroid-derived siRNA (vd-siRNA), and provides suggestions for future research directions.

Plant Immunity against Tobamoviruses.

Tobamoviruses are a group of plant viruses that pose a significant threat to agricultural crops worldwide. In this review, we focus on plant immunity against tobamoviruses, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI), the RNA-targeting pathway, phytohormones, reactive oxygen species (ROS), and autophagy. Further, we highlight the genetic resources for resistance against tobamoviruses in plant breeding and discuss future directions on plant protection against tobamoviruses.

RNAi-mediated silencing of the neverland gene inhibits molting in the migratory locust, Locusta migratoria.

7-dehydrocholesterol (7-DHC) is a key intermediate product used for biosynthesis of molting hormone. This is achieved through a series of hydroxylation reactions catalyzed by the Halloween family of cytochrome P450s. Neverland is an enzyme catalyzes the first reaction of the ecdysteroidogenic pathway, which converts dietary cholesterol into 7-DHC. However, research on the physiological function of neverland in orthopteran insects is lacking. In this study, neverland from Locusta migratoria (LmNvd) was cloned and analyzed. LmNvd was mainly expressed in the prothoracic gland and highly expressed on days 6 and 7 of fifth instar nymphs. RNAi-mediated silencing of LmNvd resulted in serious molting delays and abnormal phenotypes, which could be rescued by 7-DHC and 20-hydroxyecdysone supplementation. Hematoxylin and eosin staining results showed that RNAi-mediated silencing of LmNvd disturbed the molting process by both promoting the synthesis of new cuticle and suppressing the degradation of the old cuticle. Quantitative real-time PCR results suggested that the mRNA expression of E75 early gene and chitinase 5 gene decreased and that of chitin synthase 1 gene was markedly upregulated after knockdown of LmNvd. Our results suggest that LmNvd participates in the biosynthesis process of molting hormone, which is involved in regulating chitin synthesis and degradation in molting cycles.

Manifestation of agronomically valuable traits in the progeny of a sorghum mutant carrying the genetic construct for RNA silencing of the γ-kafirin gene.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T2-T4 generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T2, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T3 and T4 plants, the genetic construct was present in 2-4 copies. In T3, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5-7 %) was selected.

Knockout of SlDCL2b attenuates the resistance of tomato to potato spindle tuber viroid infection.

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.