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Viruses infect all cellular life forms and cause various diseases and significant economic losses worldwide. The majority of viruses are positive-sense RNA viruses. A common feature of infection by diverse RNA viruses is to induce the formation of altered membrane structures in infected host cells. Indeed, upon entry into host cells, plant-infecting RNA viruses target preferred organelles of the cellular endomembrane system and remodel organellar membranes to form organelle-like structures for virus genome replication, termed as the viral replication organelle (VRO) or the viral replication complex (VRC). Different viruses may recruit different host factors for membrane modifications. These membrane-enclosed virus-induced replication factories provide an optimum, protective microenvironment to concentrate viral and host components for robust viral replication. Although different viruses prefer specific organelles to build VROs, at least some of them have the ability to exploit alternative organellar membranes for replication. Besides being responsible for viral replication, VROs of some viruses can be mobile to reach plasmodesmata (PD) via the endomembrane system, as well as the cytoskeleton machinery. Viral movement protein (MP) and/or MP-associated viral movement complexes also exploit the endomembrane-cytoskeleton network for trafficking to PD where progeny viruses pass through the cell-wall barrier to enter neighboring cells.

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor-mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin-related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co-opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV-infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1-dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2β) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2β impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2β are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection., (© 2019 Her Majesty the Queen in Right of Canada The Plant Journal © 2019 John Wiley & Sons Ltd and Society for Experimental Biology.)

In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas.

A successful infection by a plant virus results from the complex molecular interplay between the host plant and the invading virus. Thus, dissecting the molecular network of virus-host interactions advances the understanding of the viral infection process and may assist in the development of novel antiviral strategies. In the past decade, molecular identification and functional characterization of host factors in the virus life cycle, particularly single-stranded, positive-sense RNA viruses, have been a research focus in plant virology. As a result, a number of host factors have been identified. These host factors are implicated in all the major steps of the infection process. Some host factors are diverted for the viral genome translation, some are recruited to improvise the viral replicase complexes for genome multiplication, and others are components of transport complexes for cell-to-cell spread via plasmodesmata and systemic movement through the phloem. This review summarizes current knowledge about host factors and discusses future research directions.

NONEXPRESSER OF PATHOGENESIS-RELATED GENES 1 (NPR1) is the master regulator of salicylic acid-mediated basal and systemic acquired resistance in plants. Here, we report that NPR1 plays a pivotal role in restricting compatible infection by turnip mosaic virus, a member of the largest plant RNA virus genus Potyvirus, and that such resistance is counteracted by NUCLEAR INCLUSION B (NIb), the viral RNA-dependent RNA polymerase. We demonstrate that NIb binds to the SUMO-interacting motif 3 (SIM3) of NPR1 to prevent SUMO3 interaction and sumoylation, while sumoylation of NIb by SUMO3 is not essential but can intensify the NIb–NPR1 interaction. We discover that the interaction also impedes the phosphorylation of NPR1 at Ser11/Ser15. Moreover, we show that targeting NPR1 SIM3 is a conserved ability of NIb from diverse potyviruses. These data reveal a molecular "arms race" by which potyviruses deploy NIb to suppress NPR1-mediated resistance through disrupting NPR1 sumoylation. Salicylic acid (SA) signaling pathway restricts the compatible infection of potyviruses. Here, Liu et al. show that potyviral NIb interacts with NPR1, the SA receptor in plants, preventing its sumoylation by SUMO3 and subsequent phosphorylation at Ser11/Ser15. This way, NPR1-mediated immunity is suppressed to promote virus infection. [ABSTRACT FROM AUTHOR]

Mitophagy that selectively eliminates damaged mitochondria is an essential mitochondrial quality control mechanism. Recently, mitophagy has been shown to be induced in host cells infected by a few animal viruses. Here, we report that southern rice black-streaked dwarf virus (SRBSDV), a plant nonenveloped double-stranded RNA virus, can also trigger mitophagy in its planthopper vector to prevent mitochondria-dependent apoptosis and promote persistent viral propagation. We find that the fibrillar structures constructed by the nonstructural protein P7-1 of SRBSDV directly target mitochondria via interaction with the mitophagy receptor BNIP3 (BCL2 interacting protein 3), and these mitochondria are then sequestered within autophagosomes to form mitophagosomes. Moreover, SRBSDV infection or P7-1 expression alone can promote BNIP3 dimerization on the mitochondria, and induce autophagy via the P7-1-ATG8 interaction. Furthermore, SRBSDV infection stimulates the phosphorylation of AMP-activated protein kinase (AMPK), resulting in BNIP3 phosphorylation via the AMPKα-BNIP3 interaction. Together, P7-1 induces BNIP3-mediated mitophagy by promoting the formation of phosphorylated BNIP3 dimers on the mitochondria. Silencing of ATG8, BNIP3, or AMPKα significantly reduces virus-induced mitophagy and viral propagation in insect vectors. These data suggest that in planthopper, SRBSDV-induced mitophagosomes are modified to accommodate virions and facilitate persistent viral propagation. In summary, our results demonstrate a previously unappreciated role of a viral protein in the induction of BNIP3-mediated mitophagy by bridging autophagosomes and mitochondria and reveal the functional importance of virus-induced mitophagy in maintaining persistent viral infection in insect vectors.Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; CASP3: caspase 3; dsRNA: double strand RNA; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GFP: green fluorescent protein; GST: glutathione S-transferase; padp: post-first access to diseased plants; Phos-tag: Phosphate-binding tag; PINK1: PTEN induced kinase 1; Sf9: Spodoptera frugiperda; SQSTM1: sequestosome 1; SRBSDV: southern rice black-streaked dwarf virus; STK11/LKB1: serine/threonine kinase 11; TOMM20: translocase of outer mitochondrial membrane 20; RBSDV: rice black-streaked dwarf virus; TUNEL: terminal deoxynucleotidyl dUTP nick end labeling; ULK1: unc-51 like autophagy activating kinase 1; VDAC1: voltage dependent anion channel 1. [ABSTRACT FROM AUTHOR]

Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus causes severe crop losses and threatens tomato production worldwide. ToBRFV was discovered in greenhouse tomato plants grown in Jordan in spring 2015 and its first outbreak was traced back to 2014 in Israel. To date, the virus has been reported in at least 35 countries across four continents in the world. ToBRFV is transmitted mainly via contaminated seeds and mechanical contact (such as through standard horticultural practices). Given the global nature of the seed production and distribution chain, and ToBRFV's seed transmissibility, the extent of its spread is probably more severe than has been disclosed. ToBRFV can break down genetic resistance to tobamoviruses conferred by R genes Tm‐1, Tm‐2, and Tm‐22 in tomato and L1 and L2 alleles in pepper. Currently, no commercial ToBRFV‐resistant tomato cultivars are available. Integrated pest management‐based measures such as rotation, eradication of infected plants, disinfection of seeds, and chemical treatment of contaminated greenhouses have achieved very limited success. The generation and application of attenuated variants may be a fast and effective approach to protect greenhouse tomato against ToBRFV. Long‐term sustainable control will rely on the development of novel genetic resistance and resistant cultivars, which represents the most effective and environment‐friendly strategy for pathogen control. Taxonomy: Tomato brown rugose fruit virus belongs to the genus Tobamovirus, in the family Virgaviridae. The genus also includes several economically important viruses such as Tobacco mosaic virus and Tomato mosaic virus. Genome and virion: The ToBRFV genome is a single‐stranded, positive‐sense RNA of approximately 6.4 kb, encoding four open reading frames. The viral genomic RNA is encapsidated into virions that are rod‐shaped and about 300 nm long and 18 nm in diameter. Tobamovirus virions are considered extremely stable and can survive in plant debris or on seed surfaces for long periods of time. Disease symptoms: Leaves, particularly young leaves, of tomato plants infected by ToBRFV exhibit mild to severe mosaic symptoms with dark green bulges, narrowness, and deformation. The peduncles and calyces often become necrotic and fail to produce fruit. Yellow blotches, brown or black spots, and rugose wrinkles appear on tomato fruits. In pepper plants, ToBRFV infection results in puckering and yellow mottling on leaves with stunted growth of young seedlings and small yellow to brown rugose dots and necrotic blotches on fruits. [ABSTRACT FROM AUTHOR]

Summary: Autophagy is an evolutionarily conserved pathway in eukaryotes that delivers unwanted cytoplasmic materials to the lysosome/vacuole for degradation/recycling. Stimulated autophagy emerges as an integral part of plant immunity against intracellular pathogens.In this study, we used turnip mosaic virus (TuMV) as a model to investigate the involvement of autophagy in plant RNA virus infection.The small integral membrane protein 6K2 of TuMV, known as a marker of the virus replication site and an elicitor of the unfolded protein response (UPR), upregulates the selective autophagy receptor gene NBR1 in a UPR‐dependent manner. NBR1 interacts with TuMV NIb, the RNA‐dependent RNA polymerase of the virus replication complex (VRC), and the autophagy cargo receptor/adaptor protein ATG8f. The NIb/NBR1/ATG8f interaction complexes colocalise with the 6K2‐stained VRC. Overexpression of NBR1 or ATG8f enhances TuMV replication, and deficiency of NBR1 or ATG8f inhibits virus infection. In addition, ATG8f interacts with the tonoplast‐specific protein TIP1 and the NBR1/ATG8f‐containing VRC is enclosed by the TIP1‐labelled tonoplast. In TuMV‐infected cells, numerous membrane‐bound viral particles are evident in the vacuole.Altogether these results suggest that TuMV activates and manipulates UPR‐dependent NBR1‐ATG8f autophagy to target the VRC to the tonoplast to promote viral replication and virion accumulation. [ABSTRACT FROM AUTHOR]

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant‐infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double‐fluorescent (green and mCherry) protein‐tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N‐terminus (amino acids 1–97), the core (amino acids 98–245), and the C‐terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N‐terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell‐to‐cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans‐expression of the wild‐type CP either by TuMV or through genetic transformation‐based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis‐expressed CP is functional. [ABSTRACT FROM AUTHOR]

Exonuclease-mediated RNA decay in plants is known to be involved primarily in endogenous RNA degradation, and several RNA decay components have been suggested to attenuate RNA silencing possibly through competing for RNA substrates. In this paper, we report that overexpression of key cytoplasmic 5’–3’ RNA decay pathway gene-encoded proteins (5’RDGs) such as decapping protein 2 (DCP2) and exoribonuclease 4 (XRN4) in Nicotiana benthamiana fails to suppress sense transgene-induced post-transcriptional gene silencing (S-PTGS). On the contrary, knock-down of these 5’RDGs attenuates S-PTGS and supresses the generation of small interfering RNAs (siRNAs). We show that 5’RDGs degrade transgene transcripts via the RNA decay pathway when the S-PTGS pathway is disabled. Thus, RNA silencing and RNA decay degrade exogenous gene transcripts in a hierarchical and coordinated manner. Moreover, we present evidence that infection by turnip mosaic virus (TuMV) activates RNA decay and 5’RDGs also negatively regulate TuMV RNA accumulation. We reveal that RNA silencing and RNA decay can mediate degradation of TuMV RNA in the same way that they target transgene transcripts. Furthermore, we demonstrate that VPg and HC-Pro, the two known viral suppressors of RNA silencing (VSRs) of potyviruses, bind to DCP2 and XRN4, respectively, and the interactions compromise their antiviral function. Taken together, our data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and concerted actions against exogenous and viral RNA, and VSRs not only counteract RNA silencing but also subvert RNA decay to promote viral infection. [ABSTRACT FROM AUTHOR]

All positive-strand RNA viruses induce the biogenesis of cytoplasmic membrane-bound virus factories for viral genome multiplication. We have previously demonstrated that upon plant potyvirus infection, the potyviral 6K2 integral membrane protein induces the formation of ER-derived replication vesicles that subsequently target chloroplasts for robust genome replication. Here, we report that following the trafficking of the Turnip mosaic potyvirus (TuMV) 6K2 vesicles to chloroplasts, 6K2 vesicles accumulate at the chloroplasts to form chloroplast-bound elongated tubular structures followed by chloroplast aggregation. A functional actomyosin motility system is required for this process. As vesicle trafficking and fusion in planta are facilitated by a superfamily of proteins known as SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors), we screened ER-localized SNARES or SNARE-like proteins for their possible involvement in TuMV infection. We identified Syp71 and Vap27-1 that colocalize with the chloroplast-bound 6K2 complex. Knockdown of their expression using a Tobacco rattle virus (TRV)-based virus-induced gene silencing vector showed that Syp71 but not Vap27-1 is essential for TuMV infection. In Syp71-downregulated plant cells, the formation of 6K2-induced chloroplast-bound elongated tubular structures and chloroplast aggregates is inhibited and virus accumulation is significantly reduced, but the trafficking of the 6K2 vesicles from the ER to chloroplast is not affected. Taken together, these data suggest that Syp71 is a host factor essential for successful virus infection by mediating the fusion of the virus-induced vesicles with chloroplasts during TuMV infection. [ABSTRACT FROM AUTHOR]

Sumoylation is a transient, reversible dynamic posttranslational modification that regulates diverse cellular processes including plant-pathogen interactions. Sumoylation of NPR1, a master regulator of basal and systemic acquired resistance to a broad spectrum of plant pathogens, activates the defense response. Here, we report that NIb, the only RNA-dependent RNA polymerase of Turnip mosaic virus (TuMV) that targets the nucleus upon translation, interacts exclusively with and is sumoylated by SUMO3 (SMALL UBIQUITIN-LIKE MODIFIER3), but not the three other Arabidopsis thaliana SUMO paralogs. TuMV infection upregulates SUMO3 expression, and the sumoylation of NIb by SUMO3 regulates the nuclear-cytoplasmic partitioning of NIb. We identified the SUMO-interacting motif in NIb that is essential for its sumoylation and found that knockout or overexpression of SUMO3 suppresses TuMV replication and attenuates viral symptoms, suggesting that SUMO3 plays dual roles as a host factor of TuMV and as an antiviral defender. Sumoylation of NIb by SUMO3 is crucial for its role in suppressing the host immune response. Taken together, our findings reveal that sumoylation of NIb promotes TuMV infection by retargeting NIb from the nucleus to the cytoplasm where viral replication takes place and by suppressing host antiviral responses through counteracting the TuMV infection-induced, SUMO3-activated, NPR1-mediated resistance pathway. [ABSTRACT FROM AUTHOR]

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo . The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. [ABSTRACT FROM AUTHOR]

Abstract: Plant potyviruses encode two membrane proteins, 6K and P3. The 6K protein has been shown to induce virus replication vesicles. However, the function of P3 remains unclear. In this study, subcellular localization of the Tobacco etch virus (TEV) P3 protein was investigated in Nicotiana benthamiana leaf cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway. The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments and colocalize with the 6K-containing replication vesicles. Taken together, these data support previous suggestions that P3 may play dual roles in virus movement and replication. [Copyright &y& Elsevier]