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1.
Nucleic Acids Res ; 49(15): 8900-8922, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34370034

RESUMO

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/virologia , Proteínas de Arabidopsis/fisiologia , Proteínas do Capsídeo/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Núcleo Celular/metabolismo , Doenças das Plantas/virologia , RNA Viral/química , DNA Polimerase Dirigida por RNA/metabolismo
2.
Nucleic Acids Res ; 49(12): 6908-6924, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34133725

RESUMO

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2ß and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2ß, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Iniciação Traducional da Cadeia Peptídica , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Caulimovirus , Fator de Iniciação 2B em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Fosforilação , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo
3.
Commun Biol ; 4(1): 511, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931721

RESUMO

Cell-to-cell movement is an important step for initiation and spreading of virus infection in plants. This process occurs through the intercellular connections, termed plasmodesmata (PD), and is usually mediated by one or more virus-encoded movement proteins (MP) which interact with multiple cellular factors, among them protein kinases that usually have negative effects on MP function and virus movement. In this study, we report physical and functional interaction between MP of Tobacco mosaic virus (TMV), the paradigm of PD-moving proteins, and a receptor-like kinase BAM1 from Arabidopsis and its homolog from Nicotiana benthamiana. The interacting proteins accumulated in the PD regions, colocalizing with a PD marker. Reversed genetics experiments, using BAM1 gain-of-function and loss-of-function plants, indicated that BAM1 is required for efficient spread and accumulation the virus during initial stages of infection of both plant species by TMV. Furthermore, BAM1 was also required for the efficient cell-to-cell movement of TMV MP, suggesting that BAM1 interacts with TMV MP to support early movement of the virus. Interestingly, this role of BAM1 in viral movement did not require its protein kinase activity. Thus, we propose that association of BAM1 with TMV MP at PD facilitates the MP transport through PD, which, in turn, enhances the spread of the viral infection.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vírus do Mosaico do Tabaco/fisiologia , Tabaco/virologia , Proteínas Virais/metabolismo , Replicação Viral , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Tabaco/metabolismo , Proteínas Virais/genética
4.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799566

RESUMO

Potato virus X (PVX) belongs to genus Potexvirus. This study characterizes the cellular transcriptome responses to PVX infection in Russet potato at 2 and 3 days post infection (dpi). Among the 1242 differentially expressed genes (DEGs), 268 genes were upregulated, and 37 genes were downregulated at 2 dpi while 677 genes were upregulated, and 265 genes were downregulated at 3 dpi. DEGs related to signal transduction, stress response, and redox processes. Key stress related transcription factors were identified. Twenty-five pathogen resistance gene analogs linked to effector triggered immunity or pathogen-associated molecular pattern (PAMP)-triggered immunity were identified. Comparative analysis with Arabidopsis unfolded protein response (UPR) induced DEGs revealed genes associated with UPR and plasmodesmata transport that are likely needed to establish infection. In conclusion, this study provides an insight on major transcriptional regulatory networked involved in early response to PVX infection and establishment.


Assuntos
Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Imunidade Vegetal/genética , Potexvirus/genética , Solanum tuberosum/genética , Fatores de Transcrição/genética , Transcriptoma , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/virologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Padrões Moleculares Associados a Patógenos/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Potexvirus/crescimento & desenvolvimento , Potexvirus/patogenicidade , Transdução de Sinais , Solanum tuberosum/imunologia , Solanum tuberosum/virologia , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo , Transcrição Genética , Resposta a Proteínas não Dobradas
5.
Plant J ; 107(1): 256-267, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33899980

RESUMO

Mutations in the eukaryotic translation initiation factors eIF4E and eIF(iso)4E confer potyvirus resistance in a range of plant hosts. This supports the notion that, in addition to their role in translation of cellular mRNAs, eIF4E isoforms are also essential for the potyvirus cycle. CERES is a plant eIF4E- and eIF(iso)4E-binding protein that, through its binding to the eIF4Es, modulates translation initiation; however, its possible role in potyvirus resistance is unknown. In this article, we analyse if the ectopic expression of AtCERES is able to interfere with turnip mosaic virus replication in plants. Our results demonstrate that, during infection, the ectopic expression of CERES in Nicotiana benthamiana promotes the development of a mosaic phenotype when it is accumulated to moderate levels, but induces veinal necrosis when it is accumulated to higher levels. This necrotic process resembles a hypersensitive response (HR)-like response that occurs with different HR hallmarks. Remarkably, Arabidopsis plants inoculated with a virus clone that promotes high expression of CERES do not show signs of infection. These final phenotypical outcomes are independent of the capacity of CERES to bind to eIF4E. All these data suggest that CERES, most likely due to its leucine-rich repeat nature, could act as a resistance protein, able to promote a range of different defence responses when it is highly overexpressed from viral constructs.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/virologia , Fatores de Iniciação em Eucariotos/genética , Doenças das Plantas/virologia , Tabaco/genética , Tabaco/virologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Necrose , Fenótipo , Folhas de Planta/virologia , Plantas Geneticamente Modificadas , Potyvirus/patogenicidade , Potyvirus/fisiologia , Isoformas de Proteínas/metabolismo , Replicação Viral
6.
Plant Cell Environ ; 44(5): 1399-1416, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33554358

RESUMO

Compatible plant viral infections are a common cause of agricultural losses worldwide. Characterization of the physiological responses controlling plant water management under combined stresses is of great interest in the current climate change scenario. We studied the outcome of TuMV infection on stomatal closure and water balance, hormonal balance and drought tolerance in Arabidopsis. TuMV infection reduced stomatal aperture concomitantly with diminished gas exchange rate, daily water consumption and rosette initial dehydration rate. Infected plants overaccumulated salicylic acid and abscisic acid and showed altered expression levels of key ABA homeostasis genes including biosynthesis and catabolism. Also the expression of ABA signalling gene ABI2 was induced and ABCG40 (which imports ABA into guard cells) was highly induced upon infection. Hypermorfic abi2-1 mutant plants, but no other ABA or SA biosynthetic, signalling or degradation mutants tested abolished both stomatal closure and low stomatal conductance phenotypes caused by TuMV. Notwithstanding lower relative water loss during infection, plants simultaneously subjected to drought and viral stresses showed higher mortality rates than mock-inoculated drought stressed controls, alongside downregulation of drought-responsive gene RD29A. Our findings indicate that despite stomatal closure triggered by TuMV, additional phenomena diminish drought tolerance upon infection.


Assuntos
Arabidopsis/fisiologia , Secas , Estômatos de Plantas/fisiologia , Estômatos de Plantas/virologia , Potyvirus/fisiologia , Estresse Fisiológico , Ácido Abscísico/metabolismo , Arabidopsis/virologia , Mutação/genética , Doenças das Plantas/virologia , Ácido Salicílico/metabolismo , Transdução de Sinais , Água/metabolismo
7.
Virus Genes ; 57(2): 233-237, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33599903

RESUMO

Turnip mosaic virus (TuMV), belonging to the genus Potyvirus (family Potyviridae), has a large host range and consists of a single-stranded positive sense RNA genome encoding 12 proteins, including the P1 protease. This protein which is separated from the polyprotein by cis cleavage at its respective C-terminus, has been attributed with different functions during potyviral infection of plants. P1 of Turnip mosaic virus (P1-TuMV) harbors an FGSF-motif and FGSL-motif at its N-terminus. This motif is predicted to be a binding site for the host Ras GTPase-activating protein-binding protein (G3BP), which is a key factor for stress granule (SG) formation in mammalian systems and often targeted by viruses to inhibit SG formation. We therefore hypothesized that P1-TuMV might interact with G3BP to control and regulate plant SGs to optimize cellular conditions for the production of viral proteins. Here, we analyzed the co-localization of the Arabidopsis thaliana G3BP-2 with the P1 of two TuMV isolates, namely UK 1 and DEU 2. Surprisingly, P1-TuMV-DEU 2 co-localized with AtG3BP-2 under abiotic stress conditions, whereas P1-TuMV-UK 1 did not. AtG3BP-2::RFP showed strong SGs formation after stress, while P1-UK 1::eGFP maintained a chloroplastic signal under stress conditions, the signal of P1-DEU 2::eGFP co-localized with that of AtG3BP-2::RFP. This indicates a specific interaction between P1-DEU 2 and the AtG3BP family which is not solely based on the canonical interaction motifs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/virologia , Potyvirus/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Arabidopsis/metabolismo , Brassica napus/virologia , Raphanus/virologia
8.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526695

RESUMO

Environmental conditions are an important factor driving pathogens' evolution. Here, we explore the effects of drought stress in plant virus evolution. We evolved turnip mosaic potyvirus in well-watered and drought conditions in Arabidopsis thaliana accessions that differ in their response to virus infection. Virus adaptation occurred in all accessions independently of watering status. Drought-evolved viruses conferred a significantly higher drought tolerance to infected plants. By contrast, nonsignificant increases in tolerance were observed in plants infected with viruses evolved under standard watering. The magnitude of this effect was dependent on the plant accessions. Differences in tolerance were correlated to alterations in the expression of host genes, some involved in regulation of the circadian clock, as well as in deep changes in the balance of phytohormones regulating defense and growth signaling pathways. Our results show that viruses can promote host survival in situations of abiotic stress, with the magnitude of such benefit being a selectable trait.


Assuntos
Arabidopsis/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Vírus de Plantas/genética , Simbiose/genética , Adaptação Fisiológica , Arabidopsis/virologia , Brassica napus/genética , Brassica napus/virologia , Secas , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Doenças das Plantas/virologia , Reguladores de Crescimento de Plantas/genética , Vírus de Plantas/patogenicidade , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/virologia , Potyvirus/genética , Potyvirus/patogenicidade , Estresse Fisiológico/genética
9.
Sci Rep ; 11(1): 2187, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500425

RESUMO

The Arabidopsis thaliana genome encodes several genes that are known or predicted to participate in the formation of stress granules (SG). One family of genes encodes for Ras GTPase-activating protein-binding protein (G3BP)-like proteins. Seven genes were identified, of which one of the members was already shown to interact with plant virus proteins in a previous study. A phylogenetic and tissue-specific expression analysis, including laser-dissected phloem, by qRT-PCRs was performed and the sub-cellular localization of individual AtG3BP::EYFP fluorescent fusion proteins expressed in Nicotiana benthamiana epidermal cells was observed. Individual AtG3BP-protein interactions in planta were studied using the bimolecular fluorescence complementation approach in combination with confocal imaging in living cells. In addition, the early and late induction of G3BP-like expression upon Turnip mosaic virus infection was investigated by RNAseq and qRT-PCR. The results showed a high divergence of transcription frequency in the different plant tissues, promiscuous protein-protein interaction within the G3BP-like gene family, and a general induction by a viral infection with TuMV in A. thaliana. The information gained from these studies leads to a better understanding of stress granules, in particular their molecular mode of action in the plant and their role in plant virus infection.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/virologia , Família Multigênica , Doenças das Plantas/genética , Doenças das Plantas/virologia , Potyvirus/fisiologia , Regulação da Expressão Gênica de Plantas , Filogenia , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Tabaco
10.
PLoS Pathog ; 17(1): e1008770, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33428670

RESUMO

Environments such as light condition influence the spread of infectious diseases by affecting insect vector behavior. However, whether and how light affects the host defense which further affects insect preference and performance, remains unclear, nor has been demonstrated how pathogens co-adapt light condition to facilitate vector transmission. We previously showed that begomoviral ßC1 inhibits MYC2-mediated jasmonate signaling to establish plant-dependent mutualism with its insect vector. Here we show red-light as an environmental catalyzer to promote mutualism of whitefly-begomovirus by stabilizing ßC1, which interacts with PHYTOCHROME-INTERACTING FACTORS (PIFs) transcription factors. PIFs positively control plant defenses against whitefly by directly binding to the promoter of terpene synthase genes and promoting their transcription. Moreover, PIFs interact with MYC2 to integrate light and jasmonate signaling and regulate the transcription of terpene synthase genes. However, begomovirus encoded ßC1 inhibits PIFs' and MYC2' transcriptional activity via disturbing their dimerization, thereby impairing plant defenses against whitefly-transmitted begomoviruses. Our results thus describe how a viral pathogen hijacks host external and internal signaling to enhance the mutualistic relationship with its insect vector.


Assuntos
Begomovirus/fisiologia , Hemípteros/virologia , Insetos Vetores/virologia , Doenças das Plantas/virologia , Simbiose , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Arabidopsis/metabolismo , Arabidopsis/virologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Luz , Fitocromo , Tabaco/metabolismo , Tabaco/virologia , Proteínas Virais/genética , Fatores de Virulência/genética
11.
Mol Plant Pathol ; 22(3): 334-347, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33377260

RESUMO

The translation initiation factors 4E are a small family of major susceptibility factors to potyviruses. It has been suggested that knocking out these genes could provide genetic resistance in crops when natural resistance alleles, which encode functional eIF4E proteins, are not available. Here, using the well-characterized Arabidopsis thaliana-potyvirus pathosystem, we evaluate the resistance spectrum of plants knocked out for eIF4E1, the susceptibility factor to clover yellow vein virus (ClYVV). We show that besides resistance to ClYVV, the eIF4E1 loss of function is associated with hypersusceptibility to turnip mosaic virus (TuMV), a potyvirus known to rely on the paralog host factor eIFiso4E. On TuMV infection, plants knocked out for eIF4E1 display striking developmental defects such as early senescence and primordia development stoppage. This phenotype is coupled with a strong TuMV overaccumulation throughout the plant, while remarkably the levels of the viral target eIFiso4E remain uninfluenced. Our data suggest that this hypersusceptibility cannot be explained by virus evolution leading to a gain of TuMV aggressiveness. Furthermore, we report that a functional eIF4E1 resistance allele engineered by CRISPR/Cas9 base-editing technology successfully circumvents the increase of TuMV susceptibility conditioned by eIF4E1 disruption. These findings in Arabidopsis add to several previous findings in crops suggesting that resistance based on knocking out eIF4E factors should be avoided in plant breeding, as it could also expose the plant to the severe threat of potyviruses able to recruit alternative eIF4E copies. At the same time, it provides a simple model that can help understanding of the homeostasis among eIF4E proteins in the plant cell and what makes them available to potyviruses.


Assuntos
Arabidopsis/genética , Resistência à Doença/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Doenças das Plantas/imunologia , Potyvirus/patogenicidade , Alelos , Arabidopsis/imunologia , Arabidopsis/virologia , Fator de Iniciação 4E em Eucariotos/genética , Técnicas de Inativação de Genes , Mutação com Perda de Função , Modelos Biológicos , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas
12.
PLoS Pathog ; 16(12): e1009125, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33270799

RESUMO

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas Argonauta/metabolismo , Cucumovirus/patogenicidade , Metiltransferases/metabolismo , Proteínas Virais/metabolismo , Cucumovirus/metabolismo , Doenças das Plantas/virologia
13.
PLoS Pathog ; 16(12): e1009120, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370420

RESUMO

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Nexinas de Classificação/metabolismo , Tombusvirus/fisiologia , Proteínas do Complexo da Replicase Viral/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virologia , Células Cultivadas , Interações Hospedeiro-Patógeno/genética , Organismos Geneticamente Modificados , Fosfatidilinositóis/metabolismo , Domínios e Motivos de Interação entre Proteínas , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/fisiologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Nexinas de Classificação/química , Nexinas de Classificação/fisiologia , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/genética , Tombusvirus/metabolismo , Proteínas do Complexo da Replicase Viral/fisiologia , Replicação Viral/genética
14.
Int J Mol Sci ; 21(22)2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198167

RESUMO

Turnip mosaic virus (TuMV) is one of the most important plant viruses worldwide. It has a very wide host range infecting at least 318 species in over 43 families, such as Brassicaceae, Fabaceae, Asteraceae, or Chenopodiaceae from dicotyledons. Plant NADPH oxidases, the respiratory burst oxidase homologues (RBOHs), are a major source of reactive oxygen species (ROS) during plant-microbe interactions. The functions of RBOHs in different plant-pathogen interactions have been analyzed using knockout mutants, but little focus has been given to plant-virus responses. Therefore, in this work we tested the response after mechanical inoculation with TuMV in ArabidopsisrbohD and rbohF transposon knockout mutants and analyzed ultrastructural changes after TuMV inoculation. The development of the TuMV infection cycle was promoted in rbohD plants, suggesting that RbohD plays a role in the Arabidopsis resistance response to TuMV. rbohF and rbohD/F mutants display less TuMV accumulation and a lack of virus cytoplasmic inclusions were observed; these observations suggest that RbohF promotes viral replication and increases susceptibility to TuMV. rbohD/F displayed a reduction in H2O2 but enhanced resistance similarly to rbohF. This dominant effect of the rbohF mutation could indicate that RbohF acts as a susceptibility factor. Induction of hydrogen peroxide by TuMV was partially compromised in rbohD mutants whereas it was almost completely abolished in rbohD/F, indicating that these oxidases are responsible for most of the ROS produced in this interaction. The pattern of in situ H2O2 deposition after infection of the more resistant rbohF and rbohD/F genotypes suggests a putative role of these species on systemic signal transport. The ultrastructural localization and quantification of pathogenesis-related protein 1 (PR1) indicate that ROS produced by these oxidases also influence PR1 distribution in the TuMV-A.thaliana pathosystem. Our results revealed the highest activation of PR1 in rbohD and Col-0. Thus, our findings indicate a correlation between PR1 accumulation and susceptibility to TuMV. The specific localization of PR1 in the most resistant genotypes after TuMV inoculation may indicate a connection of PR1 induction with susceptibility, which may be characteristic for this pathosystem. Our results clearly indicate the importance of NADPH oxidases RbohD and RbohF in the regulation of the TuMV infection cycle in Arabidopsis. These findings may help provide a better understanding of the mechanisms modulating A.thaliana-TuMV interactions.


Assuntos
Proteínas de Arabidopsis/metabolismo , NADPH Oxidases/metabolismo , Doenças das Plantas/virologia , Potyvirus/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virologia , Regulação da Expressão Gênica de Plantas/fisiologia , Genótipo , Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
Viruses ; 12(10)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33023227

RESUMO

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant-microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant-virus interactions in complement to Saccharomyces cerevisiae.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Ribonuclease III/metabolismo , Tombusvirus/isolamento & purificação , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Cadeia Dupla , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Saccharomyces cerevisiae/genética , Tabaco/virologia , Replicação Viral
16.
Science ; 370(6513): 227-231, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33033220

RESUMO

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/imunologia , Arabidopsis/virologia , Cucumovirus , Proteínas de Homeodomínio/fisiologia , Imunidade Inata , Doenças das Plantas/virologia , Imunidade Vegetal , Proteínas de Arabidopsis/genética , Proteínas de Homeodomínio/genética , Meristema/citologia , Meristema/imunologia , Meristema/virologia , Metiltransferases/metabolismo , RNA Ribossômico/metabolismo , Células-Tronco/imunologia , Células-Tronco/virologia
17.
Virology ; 551: 64-74, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33038689

RESUMO

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Capsídeo/metabolismo , Carmovirus/metabolismo , Doenças das Plantas/virologia , Sulfito Oxidase/metabolismo , Arabidopsis/enzimologia , Arabidopsis/virologia , Proteínas Argonauta/metabolismo , Interações entre Hospedeiro e Microrganismos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
18.
Plant Cell ; 32(10): 3256-3272, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32769133

RESUMO

Flowering plants and mammals contain imprinted genes that are primarily expressed in the endosperm and placenta in a parent-of-origin manner. In this study, we show that early activation of the geminivirus genes C2 and C3 in Arabidopsis (Arabidopsis thaliana) plants, encoding a viral suppressor of RNA interference and a replication enhancer protein, respectively, is correlated with the transient vegetative expression of VARIANT IN METHYLATION5 (VIM5), an endosperm imprinted gene that is conserved in diverse plant species. VIM5 is a ubiquitin E3 ligase that directly targets the DNA methyltransferases MET1 and CMT3 for degradation by the ubiquitin-26S proteasome proteolytic pathway. Infection with Beet severe curly top virus induced VIM5 expression in rosette leaf tissues, possibly via the expression of the viral replication initiator protein, leading to the early activation of C2 and C3 coupled with reduced symmetric methylation in the C2-3 promoter and the onset of disease symptoms. These findings demonstrate how this small DNA virus recruits a host imprinted gene for the epigenetic activation of viral gene transcription. Our findings reveal a distinct strategy used by plant pathogens to exploit the host machinery in order to inhibit methylation-mediated defense responses when establishing infection.


Assuntos
Arabidopsis/genética , Arabidopsis/virologia , Geminiviridae/patogenicidade , Doenças das Plantas/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA-Citosina Metilases/genética , DNA-Citosina Metilases/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Impressão Genômica , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Folhas de Planta/genética , Folhas de Planta/virologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética
19.
Plant Mol Biol ; 104(4-5): 467-481, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32813230

RESUMO

KEY MESSAGE: The crop yield losses induced by phytoviruses are mainly associated with the symptoms of the disease. DNA modifications as methylation can modulate the information coded by the sequence, process named epigenetics. Viral infection can change the expression patterns of different genes linked to defenses and symptoms. This work represents the initial step to expose the role of epigenetic process, in the production of symptoms associated with plants-virus interactions. Small RNAs (sRNAs) are important molecules for gene regulation in plants and play an essential role in plant-pathogen interactions. Researchers have evaluated the relationship between viral infections as well as the endogenous accumulation of sRNAs and the transcriptional changes associated with the production of symptoms, but little is known about a possible direct role of epigenetics, mediated by 24-nt sRNAs, in the induction of these symptoms. Using different RNA directed DNA methylation (RdDM) pathway mutants and a triple demethylase mutant; here we demonstrate that the disruption of RdDM pathway during viral infection produce alterations in the plant transcriptome and in consequence changes in plant symptoms. This study represents the initial step in exposing that DNA methylation directed by endogenous sRNAs has an important role, uncoupled to defense, in the production of symptoms associated with plant-virus interactions.


Assuntos
Arabidopsis/genética , Arabidopsis/virologia , Metilação de DNA , Interações Hospedeiro-Patógeno/fisiologia , Doenças das Plantas/virologia , Tobamovirus/patogenicidade , Regulação da Expressão Gênica de Plantas , Mutação , RNA de Plantas
20.
Mol Plant Pathol ; 21(10): 1271-1286, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32737952

RESUMO

Virus infections affect plant developmental traits but this aspect of the interaction has not been extensively studied so far. Two strains of Turnip mosaic virus differentially affect Arabidopsis development, especially flower stalk elongation, which allowed phenotypical, cellular, and molecular characterization of the viral determinant, the P3 protein. Transiently expressed wild-type green fluorescent protein-tagged P3 proteins of both strains and selected mutants of them revealed important differences in their behaviour as endoplasmic reticulum (ER)-associated peripheral proteins flowing along the reticulum, forming punctate accumulations. Three-dimensional (3D) model structures of all expressed P3 proteins were computationally constructed through I-TASSER protein structure predictions, which were used to compute protein surfaces and map electrostatic potentials to characterize the effect of amino acid changes on features related to protein interactions and to phenotypical and subcellular results. The amino acid at position 279 was the main determinant affecting stalk development. It also determined the speed of ER-flow of the expressed proteins and their final location. A marked change in the protein surface electrostatic potential correlated with changes in subcellular location. One single amino acid in the P3 viral protein determines all the analysed differential characteristics between strains differentially affecting flower stalk development. A model proposing a role of the protein in the intracellular movement of the viral replication complex, in association with the viral 6K2 protein, is proposed. The type of association between both viral proteins could differ between the strains.


Assuntos
Arabidopsis , Flores , Interações Hospedeiro-Patógeno , Potyvirus/metabolismo , Proteínas não Estruturais Virais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , Retículo Endoplasmático/virologia , Flores/crescimento & desenvolvimento , Flores/virologia , Estrutura Molecular , Mutação Puntual , Potyvirus/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
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