Transcriptional and hormonal profiling uncovers the interactions between plant developmental stages and RNA virus infection.

Arabidopsis thaliana is more susceptible to certain viruses during its later developmental stages. The differential responses and the mechanisms behind this development-dependent susceptibility to infection are still not fully understood. Here we explored the outcome of a viral infection at different host developmental stages by studying the response of A. thaliana to infection with turnip mosaic virus at three developmental stages: juvenile vegetative, bolting, and mature flowering plants. We found that infected plants at later stages downregulate cell wall biosynthetic genes and that this downregulation may be one factor facilitating viral spread and systemic infection. We also found that, despite being more susceptible to infection, infected mature flowering plants were more fertile (i.e. produce more viable seeds) than juvenile vegetative and bolting infected plants; that is, plants infected at the reproductive stage have greater fitness than plants infected at earlier developmental stages. Moreover, treatment of mature plants with salicylic acid increased resistance to infection at the cost of significantly reducing fertility. Together, these observations support a negative trade-off between viral susceptibility and plant fertility. Our findings point towards a development-dependent tolerance to infection.

Strain-specific differences in the interactions of the cucumber mosaic virus 2b protein with the viral 1a and host Argonaute 1 proteins.

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. IMPORTANCE: 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.

Different viral effectors hijack TCP17, a key transcription factor for host Auxin synthesis, to promote viral infection.

Auxin is an important class of plant hormones that play an important role in plant growth development, biotic stress response, and viruses often suppress host plant auxin levels to promote infection. However, previous research on auxin-mediated disease resistance has focused mainly on signaling pathway, and the molecular mechanisms of how pathogenic proteins manipulate the biosynthetic pathway of auxin remain poorly understood. TCP is a class of plant-specific transcription factors, of which TCP17 is a member that binds to the promoter of YUCCAs, a key rate-limiting enzyme for auxin synthesis, and promotes the expression of YUCCAs, which is involved in auxin synthesis in plants. In this study, we reported that Tomato spotted wilt virus (TSWV) infection suppressed the expression of YUCCAs through its interaction with TCP17. Further studies revealed that the NSs protein encoded by TSWV disrupts the dimerization of TCP17, thereby inhibit its transcriptional activation ability and reducing the auxin content in plants. Consequently, this interference inhibits the auxin response signal and promotes the TSWV infection. Transgenic plants overexpressing TCP17 exhibit resistance against TSWV infection, whereas plants knocking out TCP17 were more susceptible to TSWV infection. Additionally, proteins encoded by other RNA viruses (BSMV, RSV and TBSV) can also interact with TCP17 and interfere with its dimerization. Notably, overexpression of TCP17 enhanced resistance against BSMV. This suggests that TCP17 plays a crucial role in plant defense against different types of plant viruses that use viral proteins to target this key component of auxin synthesis and promote infection.

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.

Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a ß-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.

Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.

bZIP60 and Bax inhibitor 1 contribute IRE1-dependent and independent roles to potexvirus infection.

IRE1, BI-1, and bZIP60 monitor compatible plant-potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of three IRE1 isoforms, the bZIP60U and bZIP60S, and BI-1 roles in genetic reprogramming of cells during potexvirus infection. Experiments were performed using Arabidopsis thaliana knockout lines and Plantago asiatica mosaic virus infectious clone tagged with the green fluorescent protein gene (PlAMV-GFP). There were more PlAMV-GFP infection foci in ire1a/b, ire1c, bzip60, and bi-1 knockout than wild-type (WT) plants. Cell-to-cell movement and systemic RNA levels were greater bzip60 and bi-1 than in WT plants. Overall, these data indicate an increased susceptibility to virus infection. Transgenic overexpression of AtIRE1b or StbZIP60 in ire1a/b or bzip60 mutant background reduced virus infection foci, while StbZIP60 expression influences virus movement. Transgenic overexpression of StbZIP60 also confers endoplasmic reticulum (ER) stress resistance following tunicamycin treatment. We also show bZIP60U and TGB3 interact at the ER. This is the first demonstration of a potato bZIP transcription factor complementing genetic defects in Arabidopsis. Evidence indicates that the three IRE1 isoforms regulate the initial stages of virus replication and gene expression, while bZIP60 and BI-1 contribute separately to virus cell-to-cell and systemic movement.

DNA Delivery by Virus-Like Nanocarriers in Plant Cells.

Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.

Seed coating with phages for sustainable plant biocontrol of plant pathogens and influence of the seed coat mucilage.

Pathogens resistant to classical control strategies pose a significant threat to crop yield, with seeds being a major transmission route. Bacteriophages, viruses targeting bacteria, offer an environmentally sustainable biocontrol solution. In this study, we isolated and characterized two novel phages, Athelas and Alfirin, which infect Pseudomonas syringae and Agrobacterium fabrum, respectively, and included the recently published Pfeifenkraut phage infecting Xanthomonas translucens. Using a simple immersion method, phages coated onto seeds successfully lysed bacteria post air-drying. The seed coat mucilage (SCM), a polysaccharide-polymer matrix exuded by seeds, plays a critical role in phage binding. Seeds with removed mucilage formed five to 10 times less lysis zones compared to those with mucilage. The podovirus Athelas showed the highest mucilage dependency. Phages from the Autographiviridae family also depended on mucilage for seed adhesion. Comparative analysis of Arabidopsis SCM mutants suggested the diffusible cellulose as a key component for phage binding. Long-term activity tests demonstrated high phage stability on seed surfaces and significantly increasing seedling survival rates in the presence of pathogens. Using non-virulent host strains enhanced phage presence on seeds but also has potential limitations. These findings highlight phage-based interventions as promising, sustainable strategies for combating pathogen resistance and improving crop yield.

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.

AtHVA22a, a plant-specific homologue of Reep/DP1/Yop1 family proteins is involved in turnip mosaic virus propagation.

The movement of potyviruses, the largest genus of single-stranded, positive-sense RNA viruses responsible for serious diseases in crops, is very complex. As potyviruses developed strategies to hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using split-ubiquitin membrane yeast two-hybrid assays, we screened an Arabidopsis cDNA library for interactors of TuMV6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22), which belongs to a multigenic family of transmembrane proteins, homologous to Receptor expression-enhancing protein (Reep)/Deleted in polyposis (DP1)/Yop1 family proteins in animal and yeast. HVA22/DP1/Yop1 family genes are widely distributed in eukaryotes, but the role of HVA22 proteins in plants is still not well known, although proteomics analysis of PD fractions purified from Arabidopsis suspension cells showed that AtHVA22a is highly enriched in a PD proteome. We confirmed the interaction between TuMV6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation and showed that TuMV6K2/AtHVA22a interaction occurs at the level of the viral replication compartment during TuMV infection. Finally, we showed that the propagation of TuMV is increased when AtHVA22a is overexpressed in planta but slowed down upon mutagenesis of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation and that the C-terminal tail of the protein is important in this process.

Evidence for an RNAi-independent role of Arabidopsis DICER-LIKE2 in growth inhibition and basal antiviral resistance.

Flowering plant genomes encode four or five DICER-LIKE (DCL) enzymes that produce small interfering RNAs (siRNAs) and microRNAs, which function in RNA interference (RNAi). Different RNAi pathways in plants effect transposon silencing, antiviral defense, and endogenous gene regulation. DCL2 acts genetically redundantly with DCL4 to confer basal antiviral defense. However, DCL2 may also counteract DCL4 since knockout of DCL4 causes growth defects that are suppressed by DCL2 inactivation. Current models maintain that RNAi via DCL2-dependent siRNAs is the biochemical basis of both effects. Here, we report that DCL2-mediated antiviral resistance and growth defects cannot be explained by the silencing effects of DCL2-dependent siRNAs. Both functions are defective in genetic backgrounds that maintain high levels of DCL2-dependent siRNAs, either with specific point mutations in DCL2 or with reduced DCL2 dosage because of heterozygosity for dcl2 knockout alleles. Intriguingly, all DCL2 functions require its catalytic activity, and the penetrance of DCL2-dependent growth phenotypes in dcl4 mutants correlates with DCL2 protein levels but not with levels of major DCL2-dependent siRNAs. We discuss this requirement and correlation with catalytic activity but not with resulting siRNAs, in light of other findings that reveal a DCL2 function in innate immunity activation triggered by cytoplasmic double-stranded RNA.

DICER-LIKE2 Plays a Crucial Role in Rice Stripe Virus Coat Protein-Mediated Virus Resistance in Arabidopsis.

Virus coat protein (CP)-mediated resistance is considered an effective antiviral defense strategy that has been used to develop robust resistance to viral infection. Rice stripe virus (RSV) causes significant losses in rice production in eastern Asia. We previously showed that the overexpression of RSV CP in Arabidopsis plants results in immunity to RSV infection, using the RSV-Arabidopsis pathosystem, and this CP-mediated viral resistance depends on the function of DCLs and is mostly involved in RNA silencing. However, the special role of DCLs in producing t-siRNAs in CP transgenic Arabidopsis plants is not fully understood. In this study, we show that RSV CP transgenic Arabidopsis plants with the dcl2 mutant background exhibited similar virus susceptibility to non-transgenic plants and were accompanied by the absence of transgene-derived small interfering RNAs (t-siRNAs) from the CP region. The dcl2 mutation eliminated the accumulation of CP-derived t-siRNAs, including those generated by other DCL enzymes. In contrast, we also developed RSV CP transgenic Arabidopsis plants with the dcl4 mutant background, and these CP transgenic plants showed immunity to virus infection and accumulated comparable amounts of CP-derived t-siRNAs to CP transgenic Arabidopsis plants with the wild-type background except for a significant increase in the abundance of 22 nt t-siRNA reads. Overall, our data indicate that DCL2 plays an essential, as opposed to redundant, role in CP-derived t-siRNA production and induces virus resistance in RSV CP transgenic Arabidopsis plants.

A viral protein targets mitochondria and chloroplasts by subverting general import pathways and specific receptors.

IMPORTANCE: Many plant proteins and some proteins from plant pathogens are dually targeted to chloroplasts and mitochondria, and are supposed to be transported along the general pathways for organellar protein import, but this issue has not been explored yet. Moreover, organellar translocon receptors exist as families of several members whose functional specialization in different cargos is supposed but not thoroughly studied. This article provides novel insights into such topics showing for the first time that an exogenous protein, the melon necrotic spot virus coat protein, exploits the common Toc/Tom import systems to enter both mitochondria and chloroplasts while identifying the involved specific receptors.

Identification of a novel viral factor inducing tumorous symptoms by disturbing vascular development in planta.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development. IMPORTANCE While various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

Interaction of EXA1 and eIF4E Family Members Facilitates Potexvirus Infection in Arabidopsis thaliana.

Plant viruses depend on a number of host factors for successful infection. Deficiency of critical host factors confers recessively inherited viral resistance in plants. For example, loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. However, the molecular mechanism of how EXA1 assists potexvirus infection remains largely unknown. Previous studies reported that the salicylic acid (SA) pathway is upregulated in exa1 mutants, and EXA1 modulates hypersensitive response-related cell death during EDS1-dependent effector-triggered immunity. Here, we show that exa1-mediated viral resistance is mostly independent of SA and EDS1 pathways. We demonstrate that Arabidopsis EXA1 interacts with three members of the eukaryotic translation initiation factor 4E (eIF4E) family, eIF4E1, eIFiso4E, and novel cap-binding protein (nCBP), through the eIF4E-binding motif (4EBM). Expression of EXA1 in exa1 mutants restored infection by the potexvirus Plantago asiatica mosaic virus (PlAMV), but EXA1 with mutations in 4EBM only partially restored infection. In virus inoculation experiments using Arabidopsis knockout mutants, EXA1 promoted PlAMV infection in concert with nCBP, but the functions of eIFiso4E and nCBP in promoting PlAMV infection were redundant. By contrast, the promotion of PlAMV infection by eIF4E1 was, at least partially, EXA1 independent. Taken together, our results imply that the interaction of EXA1-eIF4E family members is essential for efficient PlAMV multiplication, although specific roles of three eIF4E family members in PlAMV infection differ. IMPORTANCE The genus Potexvirus comprises a group of plant RNA viruses, including viruses that cause serious damage to agricultural crops. We previously showed that loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. EXA1 may thus play a critical role in the success of potexvirus infection; hence, elucidation of its mechanism of action is crucial for understanding the infection process of potexviruses and for effective viral control. Previous studies reported that loss of EXA1 enhances plant immune responses, but our results indicate that this is not the primary mechanism of exa1-mediated viral resistance. Here, we show that Arabidopsis EXA1 assists infection by the potexvirus Plantago asiatica mosaic virus (PlAMV) by interacting with the eukaryotic translation initiation factor 4E family. Our results imply that EXA1 contributes to PlAMV multiplication by regulating translation.

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis.

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.

CASC3 Biomolecular Condensates Restrict Turnip Crinkle Virus by Limiting Host Factor Availability.

The exon-junction complex (EJC) plays a role in post-transcriptional gene regulation and exerts antiviral activity towards several positive-strand RNA viruses. However, the spectrum of RNA viruses that are targeted by the EJC or the underlying mechanisms are not well understood. EJC components from Arabidopsis thaliana were screened for antiviral activity towards Turnip crinkle virus (TCV, Tombusviridae). Overexpression of the accessory EJC component CASC3 inhibited TCV accumulation > 10-fold in Nicotiana benthamiana while knock-down of endogenous CASC3 resulted in a > 4-fold increase in TCV accumulation. CASC3 forms cytoplasmic condensates and deletion of the conserved SELOR domain reduced condensate size 7-fold and significantly decreased antiviral activity towards TCV. Mass spectrometry of CASC3 complexes did not identify endogenous stress granule or P-body markers and CASC3 failed to co-localize with an aggresome-specific dye suggesting that CASC3 condensates are distinct from well-established membraneless compartments. Mass spectrometry and bimolecular fluorescence complementation assays revealed that CASC3 sequesters Heat shock protein 70 (Hsp70-1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), two host factors with roles in tombusvirus replication. Overexpression of Hsp70-1 or GAPDH reduced the antiviral activity of CASC3 2.1-fold and 2.8-fold, respectively, and suggests that CASC3 inhibits TCV by limiting host factor availability. Unrelated Tobacco mosaic virus (TMV) also depends on Hsp70-1 and CASC3 overexpression restricted TMV accumulation 4-fold and demonstrates that CASC3 antiviral activity is not TCV-specific. Like CASC3, Auxin response factor 19 (ARF19) forms poorly dynamic condensates but ARF19 overexpression failed to inhibit TCV accumulation and suggests that CASC3 has antiviral activities that are not ubiquitous among cytoplasmic condensates.

Geminiviruses employ host DNA glycosylases to subvert DNA methylation-mediated defense.

DNA methylation is an epigenetic mechanism that plays important roles in gene regulation and transposon silencing. Active DNA demethylation has evolved to counterbalance DNA methylation at many endogenous loci. Here, we report that active DNA demethylation also targets viral DNAs, tomato yellow leaf curl China virus (TYLCCNV) and its satellite tomato yellow leaf curl China betasatellite (TYLCCNB), to promote their virulence. We demonstrate that the ßC1 protein, encoded by TYLCCNB, interacts with a ROS1-like DNA glycosylase in Nicotiana benthamiana and with the DEMETER (DME) DNA glycosylase in Arabidopsis thaliana. The interaction between ßC1 and DME facilitates the DNA glycosylase activity to decrease viral DNA methylation and promote viral virulence. These findings reveal that active DNA demethylation can be regulated by a viral protein to subvert DNA methylation-mediated defense.

RNAi-Based Antiviral Innate Immunity in Plants.

Multiple antiviral immunities were developed to defend against viral infection in hosts. RNA interference (RNAi)-based antiviral innate immunity is evolutionarily conserved in eukaryotes and plays a vital role against all types of viruses. During the arms race between the host and virus, many viruses evolve viral suppressors of RNA silencing (VSRs) to inhibit antiviral innate immunity. Here, we reviewed the mechanism at different stages in RNAi-based antiviral innate immunity in plants and the counteractions of various VSRs, mainly upon infection of RNA viruses in model plant Arabidopsis. Some critical challenges in the field were also proposed, and we think that further elucidating conserved antiviral innate immunity may convey a broad spectrum of antiviral strategies to prevent viral diseases in the future.