RESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Begomovirus , Cromatina , Histonas , Regiões Promotoras Genéticas , Arabidopsis/virologia , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cromatina/metabolismo , Cromatina/genética , Begomovirus/genética , Begomovirus/metabolismo , Histonas/metabolismo , Histonas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Mutação , Doenças das Plantas/virologia , Doenças das Plantas/genética , Geminiviridae/genética , Geminiviridae/metabolismo , Regulação Viral da Expressão Gênica , Proteínas ViraisRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Argonautas , Resistência à Doença , Doenças das Plantas , Arabidopsis/genética , Arabidopsis/virologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Doenças das Plantas/virologia , Resistência à Doença/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Imunidade Vegetal/genética , Interferência de RNA , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
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.
Assuntos
Arabidopsis , Bacteriófagos , Doenças das Plantas , Pseudomonas syringae , Sementes , Sementes/microbiologia , Sementes/virologia , Pseudomonas syringae/virologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Doenças das Plantas/virologia , Bacteriófagos/fisiologia , Bacteriófagos/genética , Arabidopsis/virologia , Arabidopsis/microbiologia , Xanthomonas/virologia , Mucilagem Vegetal/metabolismo , Mucilagem Vegetal/química , Agentes de Controle Biológico , Ligação ViralRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição de Zíper de Leucina Básica , Doenças das Plantas , Plantas Geneticamente Modificadas , Potexvirus , Arabidopsis/virologia , Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Doenças das Plantas/virologia , Doenças das Plantas/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Potexvirus/fisiologia , Regulação da Expressão Gênica de Plantas , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Mutação/genética , Tunicamicina/farmacologia , Proteínas de Membrana , Proteínas QuinasesRESUMO
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.
Assuntos
Arabidopsis , Proteínas de Fluorescência Verde , Arabidopsis/virologia , Arabidopsis/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas de Transferência de Genes , Plasmídeos/genética , Poliaminas/química , Protoplastos/metabolismo , Nanoestruturas/química , DNA/química , DNA/administração & dosagemRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Potyvirus , Potyvirus/patogenicidade , Potyvirus/fisiologia , Arabidopsis/virologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Doenças das Plantas/virologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Replicação Viral , Nicotiana/virologia , Nicotiana/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Argonautas , Cucumovirus , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Cucumovirus/metabolismo , Cucumovirus/genética , Cucumovirus/fisiologia , Arabidopsis/metabolismo , Arabidopsis/virologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Ligação Proteica , Proteínas Virais/metabolismo , Proteínas Virais/genética , Interações Hospedeiro-Patógeno , Proteínas do Complexo da Replicase Viral/metabolismo , Proteínas do Complexo da Replicase Viral/genética , Doenças das Plantas/virologia , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/química , MetiltransferasesRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Interferência de RNA , Ribonuclease III , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Mutação , Doenças das Plantas/virologia , Doenças das Plantas/imunologia , Doenças das Plantas/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Tenuivirus , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , Tenuivirus/genéticaRESUMO
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.
Assuntos
Arabidopsis , Proteínas do Capsídeo , Cloroplastos , Mitocôndrias , Nicotiana , Proteínas de Plantas , Receptores de Superfície Celular , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas do Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , Cloroplastos/metabolismo , Cloroplastos/virologia , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Nicotiana/metabolismo , Nicotiana/virologia , Proteínas de Plantas/metabolismo , Transporte Proteico , Receptores de Superfície Celular/metabolismoRESUMO
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.
Assuntos
Gentiana , Nicotiana , Tumores de Planta , Vírus de Plantas , Fatores de Virulência , Xilema , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Gentiana/virologia , Vírus de Plantas/genética , Vírus de Plantas/patogenicidade , Nicotiana/metabolismo , Nicotiana/virologia , Xilema/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Folhas de Planta , Tumores de Planta/virologia , Transdução de Sinais , Fatores de Processamento de RNARESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fator de Iniciação 4E em Eucariotos , Doenças das Plantas , Potexvirus , Arabidopsis/metabolismo , Arabidopsis/virologia , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Doenças das Plantas/genética , Potexvirus/fisiologia , Proteínas de Arabidopsis/metabolismo , Resistência à Doença/genética , Ligação Proteica , Motivos de Aminoácidos , Deleção de Genes , Células Vegetais/virologia , Biossíntese de Proteínas/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Correpressoras , Geminiviridae , Doenças das Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Ciclopentanos/metabolismo , Geminiviridae/metabolismo , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Vírus de PlantasRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Condensados Biomoleculares , Carmovirus , Interações Hospedeiro-Patógeno , Arabidopsis/genética , Arabidopsis/virologia , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/virologia , Carmovirus/metabolismo , Núcleo Celular , Proteínas de Arabidopsis/metabolismoRESUMO
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.
Assuntos
Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Interferência de RNA/fisiologia , Infecções por Vírus de RNA/imunologia , Arabidopsis/genética , Arabidopsis/virologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Doenças das Plantas/virologia , Infecções por Vírus de RNA/virologiaRESUMO
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.
Assuntos
Begomovirus/patogenicidade , DNA Glicosilases/metabolismo , Metilação de DNA/genética , Interações Hospedeiro-Patógeno/genética , Arabidopsis/virologia , DNA Viral/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Ligação Proteica , Vírus Satélites/patogenicidade , Nicotiana/virologia , Proteínas Virais/metabolismo , VirulênciaRESUMO
Geminiviruses consist of a single-stranded DNA genome that replicates by a rolling circle (RCR) and recombination-dependent (RDR) modes of replication. The AC1 or Rep is the indispensable viral protein required for the RCR mode of replication. Since these viruses encode only a few proteins, they depend on several host factors for replication, transcription, and other physiological processes. To get insights into the repertoire of host factors influencing the replication of geminiviruses, we performed phage display experiments which led to the identification of putative mungbean yellow mosaic India virus (MYMIV) Rep interacting host proteins. These proteins might directly or indirectly participate in geminivirus biology. MCM3 was one of the Rep-interacting partners obtained in the phage display results. Using bimolecular fluorescence complementation (BiFC), the interaction of the MYMIV Rep with Arabidopsis thaliana MCM3 (AtMCM3) was confirmed. We report the involvement of AtMCM3 in the replication of MYMIV DNA through an ex vivo system. The physiological relevance of the interaction between AtMCM3 and MYMIV Rep is reflected by yeast replication assay.Communicated by Ramaswamy H. Sarma.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Geminiviridae , Componente 3 do Complexo de Manutenção de Minicromossomo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Viral/genética , DNA Viral/metabolismo , Geminiviridae/fisiologia , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Replicação Viral , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
Alighting aphids probe a new host plant by intracellular test punctures for suitability. These induce immediate calcium signals that emanate from the punctured sites and might be the first step in plant recognition of aphid feeding and the subsequent elicitation of plant defence responses. Calcium is also involved in the transmission of non-persistent plant viruses that are acquired by aphids during test punctures. Therefore, we wanted to determine whether viral infection alters calcium signalling. For this, calcium signals triggered by aphids were imaged on transgenic Arabidopsis plants expressing the cytosolic FRET-based calcium reporter YC3.6-NES and infected with the non-persistent viruses cauliflower mosaic (CaMV) and turnip mosaic (TuMV), or the persistent virus, turnip yellows (TuYV). Aphids were placed on infected leaves and calcium elevations were recorded by time-lapse fluorescence microscopy. Calcium signal velocities were significantly slower in plants infected with CaMV or TuMV and signal areas were smaller in CaMV-infected plants. Transmission tests using CaMV-infected Arabidopsis mutants impaired in pathogen perception or in the generation of calcium signals revealed no differences in transmission efficiency. A transcriptomic meta-analysis indicated significant changes in expression of receptor-like kinases in the BAK1 pathway as well as of calcium channels in CaMV- and TuMV-infected plants. Taken together, infection with CaMV and TuMV, but not with TuYV, impacts aphid-induced calcium signalling. This suggests that viruses can modify plant responses to aphids from the very first vector/host contact.
Assuntos
Afídeos/fisiologia , Cálcio/metabolismo , Folhas de Planta/parasitologia , Folhas de Planta/virologia , Vírus de Plantas/fisiologia , Animais , Arabidopsis/genética , Arabidopsis/parasitologia , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , Sinalização do Cálcio , Caulimovirus/fisiologia , Mutação/genética , Folhas de Planta/genéticaRESUMO
BACKGROUND: RNA silencing has an important role mediating sequence-specific virus resistance in plants. The complex interaction of viruses with RNA silencing involves the loading of viral small interfering RNAs (vsiRNAs) into its host ARGONAUTE (AGO) proteins. As a side effect of their antiviral activity, vsiRNAs loading into AGO proteins can also mediate the silencing of endogenous genes. Here, we analyze at the genome-wide level both aspects of the interference of cucumber mosaic virus (CMV) with the RNA silencing machinery of Arabidopsis thaliana. RESULTS: We observe CMV-derived vsiRNAs affect the levels of endogenous sRNA classes. Furthermore, we analyze the incorporation of vsiRNAs into AGO proteins with a described antiviral role and the viral suppressor of RNA silencing (VSR) 2b, by combining protein immunoprecipitation with sRNA high-throughput sequencing. Interestingly, vsiRNAs represent a substantial percentage of AGO-loaded sRNAs and displace other endogenous sRNAs. As a countermeasure, the VSR 2b loaded vsiRNAs and mRNA-derived siRNAs, which affect the expression of the genes they derive from. Additionally, we analyze how vsiRNAs incorporate into the endogenous RNA silencing pathways by exploring their target mRNAs using parallel analysis of RNA end (PARE) sequencing, which allow us to identify vsiRNA-targeted genes genome-wide. CONCLUSIONS: This work exemplifies the complex relationship of RNA viruses with the endogenous RNA silencing machinery and the multiple aspects of virus resistance and virulence that this interaction induces.
Assuntos
Arabidopsis/genética , Cucumovirus/genética , Doenças das Plantas/genética , Interferência de RNA , Arabidopsis/virologia , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Imunoprecipitação , RNA Interferente Pequeno/genética , RNA Viral/genéticaRESUMO
The P1/HC-Pro viral suppressor of potyvirus suppresses posttranscriptional gene silencing (PTGS). The fusion protein of P1/HC-Pro can be cleaved into P1 and HC-Pro through the P1 self-cleavage activity, and P1 is necessary and sufficient to enhance PTGS suppression of HC-Pro. To address the modulation of gene regulatory relationships induced by turnip mosaic virus (TuMV) P1/HC-Pro (P1/HC-ProTu), a comparative transcriptome analysis of three types of transgenic plants (P1Tu, HC-ProTu, and P1/HC-ProTu) were conducted using both high-throughput (HTP) and low-throughput (LTP) RNA-Seq strategies. The results showed that P1/HC-ProTu disturbed the endogenous abscisic acid (ABA) accumulation and genes in the signaling pathway. Additionally, the integrated responses of stress-related genes, in particular to drought stress, cold stress, senescence, and stomatal dynamics, altered the expressions by the ABA/calcium signaling. Crosstalk among the ABA, jasmonic acid, and salicylic acid pathways might simultaneously modulate the stress responses triggered by P1/HC-ProTu. Furthermore, the LTP network analysis revealed crucial genes in common with those identified by the HTP network in this study, demonstrating the effectiveness of the miniaturization of the HTP profile. Overall, our findings indicate that P1/HC-ProTu-mediated suppression in RNA silencing altered the ABA/calcium signaling and a wide range of stress responses.