Grapevine leafroll-associated virus 3 (GLRaV-3) is a formidable threat to the stability of the global grape and wine industries. It is the primary etiological agent of grapevine leafroll disease (GLD) and significantly impairs vine health, fruit quality, and yield. GLRaV-3 is a member of the genus Ampelovirus, Closteroviridae family. Viral genes within the 3\' proximal unique gene blocks (UGB) remain highly variable and poorly understood. The UGBs of Closteroviridae viruses include diverse open reading frames (ORFs) that have been shown to contribute to viral functions such as the suppression of the host RNA silencing defense response and systemic viral spread. This study investigates the role of GLRaV-3 ORF8, ORF9, and ORF10, which encode the proteins p21, p20A, and p20B, respectively. These genes represent largely unexplored facets of the GLRaV-3 genome. Here, we visualize the subcellular localization of wildtype and mutagenized GLRaV-3 ORFs 8, 9, and 10, transiently expressed in Nicotiana benthamiana. Our results indicate that p21 localizes to the cytosol, p20A associates with microtubules, and p20B is trafficked into the nucleus to carry out the suppression of host RNA silencing. The findings presented herein provide a foundation for future research aimed at the characterization of the functions of these ORFs. In the long run, it would also facilitate the development of innovative strategies to understand GLRaV-3, mitigate its spread, and impacts on grapevines and the global wine industry.

Mycoviruses are viruses that infect fungi and are widespread across all major fungal taxa, exhibiting great biological diversity. Since their discovery in the 1960s, researchers have observed a myriad of fungal phenotypes altered due to mycoviral infection. In this review, we examine the nuanced world of mycoviruses in the context of the medically and agriculturally important fungal genus, Aspergillus. The advent of RNA sequencing has revealed a previous underestimate of viral prevalence in fungi, in particular linear single-stranded RNA viruses, and here we outline the diverse viral families known to date that contain mycoviruses infecting Aspergillus. Furthermore, we describe these novel mycoviruses, highlighting those with peculiar genome structures, such as a split RNA dependent RNA polymerase gene. Next, we delineate notable mycovirus-mediated phenotypes in Aspergillus, in particular reporting on observations of mycoviruses that affect their fungal host\'s virulence and explore how this may relate to virus-mediated decreased stress tolerance. Furthermore, mycovirus effects on microbial competition and antifungal resistance are discussed. The factors that influence the manifestation of these phenotypes, such as temperature, fungal life stage, and infection with multiple viruses, among others, are also evaluated. In addition, we attempt to elucidate the molecular mechanisms that underpin these phenotypes, examining how mycoviruses can be targets, triggers, and even suppressors of RNA silencing and how this can affect fungal gene expression and phenotypes. Finally, we highlight the potential therapeutic applications of mycoviruses and how, in an approach analogous to bacteriophage therapy, their ability to produce hypovirulence in Aspergillus might be used to attenuate invasive aspergillosis infections in humans.

Viral pathogens not only threaten the health and life of humans and animals but also cause enormous crop yield losses and contribute to global food insecurity. To defend against viral pathogens, plants have evolved an intricate immune system to perceive and cope with such attacks. Although most of the fundamental studies were carried out in model plants, more recent research in crops has provided new insights into the antiviral strategies employed by crop plants. We summarize recent advances in understanding the biological roles of cellular receptors, RNA silencing, RNA decay, hormone signaling, autophagy, and ubiquitination in manipulating crop host-mediated antiviral responses. The potential functions of circular RNAs, the rhizosphere microbiome, and the foliar microbiome of crops in plant-virus interactions will be fascinating research directions in the future. These findings will be beneficial for the development of modern crop improvement strategies.

Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology.

The cucumber mosaic virus (CMV) 2b protein is a potent counter-defense factor and symptom determinant that inhibits antiviral silencing by titrating short double-stranded RNAs. Expression of the CMV subgroup IA strain Fny-CMV 2b protein in transgenic Arabidopsis thaliana plants disrupts microRNA-mediated cleavage of host mRNAs by binding Argonaute 1 (AGO1), leading to symptom-like phenotypes. This also triggers AGO2-mediated antiviral resistance and resistance to CMV\'s aphid vectors. However, in authentic viral infections, the Fny-CMV 1a protein modulates 2b-AGO1 interactions, inhibiting induction of AGO2-mediated virus resistance and aphid resistance. Contrastingly, 2b proteins encoded by the subgroup II strain LS-CMV and the recently discovered subgroup IA strain Ho-CMV induce no symptoms. Confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation showed that Fny-CMV and Ho-CMV 2b proteins interact with Fny-CMV and LS-CMV 1a proteins, while the CMV-LS 2b protein cannot. However, Fny-CMV, Ho-CMV, and LS-CMV 2b proteins, all interacted with AGO1, but while AGO1-Fny2b complexes occurred in the nucleus and cytoplasm, corresponding AGO1-2b complexes for LS-CMV and Ho-CMV accumulated almost exclusively in nuclei. AGO2 transcript accumulation was used to assess the inhibition of AGO1-mediated mRNA degradation. Fny-CMV 2b induced a fivefold increase in AGO2 accumulation, but LS-CMV and Ho-CMV 2b proteins induced only twofold increases. Thus, these 2b proteins bind AGO1 but are less effective at inhibiting AGO1 activity. We conclude that the intracellular localization of 2b-AGO1 complexes influences the degree to which a 2b protein inhibits microRNA-mediated host mRNA degradation and that cytoplasmic AGO1 has the strongest influence on miRNA-mediated cellular mRNA turnover.
OBJECTIVE: The cucumber mosaic virus (CMV) 2b protein was among the first discovered viral suppressors of RNA silencing. It has additional pro-viral functions through effects on plant defensive signaling pathways mediated by salicylic acid and jasmonic acid, the abscisic acid pathway and virus-induced drought resistance, and on host plant interactions with insect vectors. Many of these effects occur due to interaction with the important host RNA silencing component Argonaute 1 (AGO1). It was thought that only 2b proteins of \"severe\" CMV strains interacted with AGO1 and inhibited its microRNA-mediated \"slicing\" of cellular mRNAs and that the lack of interaction with AGO1 explained the moderate symptoms typically seen in plants infected with mild CMV strains. Our work overthrows this paradigm by showing that mild strain CMV 2b proteins can interact with AGO1, but their in vivo localization prevents them from interacting with AGO1 molecules present in the infected cell cytoplasm.

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.

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.

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.

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.

CONCLUSIONS: 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.