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Competing Interests: The authors have declared that no competing interests exist.

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis -cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability. IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes., (Copyright © 2020 American Society for Microbiology.)

In this study, we demonstrate a novel pro-viral role for the Nicotiana benthamiana ARGONAUTE 1 (AGO1) in potyvirus infection. AGO1 strongly enhanced potato virus A (PVA) particle production and benefited the infection when supplied in excess. We subsequently identified the potyviral silencing suppressor, helper-component protease (HCPro), as the recruiter of host AGO1. After the identification of a conserved AGO1-binding GW/WG motif in potyviral HCPros, we used site-directed mutagenesis to introduce a tryptophan-to-alanine change into the HCPro (HCProAG) of PVA (PVAAG) and turnip mosaic virus (TuMVAG). AGO1 co-localization and co-immunoprecipitation with PVA HCPro was significantly reduced by the mutation suggesting the interaction was compromised. Although the mutation did not interfere with HCPro's complementation or silencing suppression capacity, it nevertheless impaired virus particle accumulation and the systemic spread of both PVA and TuMV. Furthermore, we found that the HCPro-AGO1 interaction was important for AGO1's association with the PVA coat protein. The coat protein was also more stable in wild type PVA infection than in PVAAG infection. Based on these findings we suggest that potyviral HCPro recruits host AGO1 through its WG motif and engages AGO1 in the production of stable virus particles, which are required for an efficient systemic infection., Competing Interests: The authors have declared that no competing interests exist.

P3N-PIPO, the only dedicated movement protein (MP) of potyviruses, directs cylindrical inclusion (CI) protein from the cytoplasm to the plasmodesma (PD), where CI forms conical structures for intercellular movement. To better understand potyviral cell-to-cell movement, we further characterized P3N-PIPO using Turnip mosaic virus (TuMV) as a model virus. We found that P3N-PIPO interacts with P3 via the shared P3N domain and that TuMV mutants lacking the P3N domain of either P3N-PIPO or P3 are defective in cell-to-cell movement. Moreover, we found that the PIPO domain of P3N-PIPO is sufficient to direct CI to the PD, whereas the P3N domain is necessary for localization of P3N-PIPO to 6K2-labeled vesicles or aggregates. Finally, we discovered that the interaction between P3 and P3N-PIPO is essential for the recruitment of CI to cytoplasmic 6K2-containing structures and the association of 6K2-containing structures with PD-located CI inclusions. These data suggest that both P3N and PIPO domains are indispensable for potyviral cell-to-cell movement and that the 6K2 vesicles in proximity to PDs resulting from multipartite interactions among 6K2, P3, P3N-PIPO, and CI may also play an essential role in this process. IMPORTANCE Potyviruses include numerous economically important viruses that represent approximately 30% of known plant viruses. However, there is still limited information about the mechanism of potyviral cell-to-cell movement. Here, we show that P3N-PIPO interacts with and recruits CI to the PD via the PIPO domain and interacts with P3 via the shared P3N domain. We further report that the interaction of P3N-PIPO and P3 is associated with 6K2 vesicles and brings the 6K2 vesicles into proximity with PD-located CI structures. These results support the notion that the replication and cell-to-cell movement of potyviruses are processes coupled by anchoring viral replication complexes at the entrance of PDs, which greatly increase our knowledge of the intercellular movement of potyviruses., (Copyright © 2020 American Society for Microbiology.)

Potyviruses represent the largest group of known plant RNA viruses and include many agriculturally important viruses, such as Plum pox virus , Soybean mosaic virus , Turnip mosaic virus , and Potato virus Y . Potyviruses adopt polyprotein processing as their genome expression strategy. Among the 11 known viral proteins, the nuclear inclusion protein b (NIb) is the RNA-dependent RNA polymerase responsible for viral genome replication. Beyond its principal role as an RNA replicase, NIb has been shown to play key roles in diverse virus-host interactions. NIb recruits several host proteins into the viral replication complexes (VRCs), which are essential for the formation of functional VRCs for virus multiplication, and interacts with the sumoylation pathway proteins to suppress NPR1-mediated immunity response. On the other hand, NIb serves as a target of selective autophagy as well as an elicitor of effector-triggered immunity, resulting in attenuated virus infection. These contrasting roles of NIb provide an excellent example of the complex co-evolutionary arms race between plant hosts and potyviruses. This review highlights the current knowledge about the multifunctional roles of NIb in potyvirus infection, and discusses future research directions.

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.)

Autophagy emerges as an essential immunity defense against intracellular pathogens. Here we report that turnip mosaic virus (TuMV) infection activates autophagy in plants and that Beclin1 (ATG6), a core component of autophagy, inhibits virus replication. Beclin1 interacts with NIb, the RNA-dependent RNA polymerase (RdRp) of TuMV, via the highly conserved GDD motif and the interaction complex is targeted for autophagic degradation likely through the adaptor protein ATG8a. Beclin1-mediated NIb degradation is inhibited by autophagy inhibitors. Deficiency of Beclin1 or ATG8a enhances NIb accumulation and promotes viral infection and vice versa. These data suggest that Beclin1 may be a selective autophagy receptor. Overexpression of a Beclin1 truncation mutant that binds to NIb but lacks the ability to mediate NIb degradation also inhibits virus replication. The Beclin1-RdRp interaction further extends to several RNA viruses. Thus Beclin1 restricts viral infection through suppression and also likely autophagic degradation of the viral RdRp.

Like other positive-strand RNA viruses, plant potyviruses assemble viral replication complexes (VRCs) on modified cellular membranes. Potyviruses encode two membrane proteins, 6K2 and P3. The former is known to play pivotal roles in the formation of membrane-associated VRCs. However, P3 remains to be one of the least characterized potyviral proteins. The P3 cistron codes for P3 as well as P3N-PIPO which results from RNA polymerase slippage. In this study, we show that the P3N-PIPO of Turnip mosaic virus (TuMV) is required for viral cell-to-cell movement but not for viral replication. We demonstrate that the C-terminal region of P3 (P3C) is indispensable for P3 to form cytoplasmic punctate inclusions and target VRCs. We reveal that TuMV mutants that lack P3C are replication-defective. Taken together, these data suggest that the P3 cistron has two distinct functions: P3N-PIPO as a dedicated movement protein and P3 as an essential component of the VRC., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

RNA silencing is an innate antiviral immunity response of plants and animals. To counteract this host immune response, viruses have evolved an effective strategy to protect themselves by the expression of viral suppressors of RNA silencing (VSRs). Most potyviruses encode two VSRs, helper component-proteinase (HC-Pro) and viral genome-linked protein (VPg). The molecular biology of the former has been well characterized, whereas how VPg exerts its function in the suppression of RNA silencing is yet to be understood. In this study, we show that infection by Turnip mosaic virus (TuMV) causes reduced levels of suppressor of gene silencing 3 (SGS3), a key component of the RNA silencing pathway that functions in double-stranded RNA synthesis for virus-derived small interfering RNA (vsiRNA) production. We also demonstrate that among 11 TuMV-encoded viral proteins, VPg is the only one that interacts with SGS3. We furthermore present evidence that the expression of VPg alone, independent of viral infection, is sufficient to induce the degradation of SGS3 and its intimate partner RNA-dependent RNA polymerase 6 (RDR6). Moreover, we discover that the VPg-mediated degradation of SGS3 occurs via both the 20S ubiquitin-proteasome and autophagy pathways. Taken together, our data suggest a role for VPg-mediated degradation of SGS3 in suppression of silencing by VPg., Importance: Potyviruses represent the largest group of known plant viruses and cause significant losses of many agriculturally important crops in the world. In order to establish infection, potyviruses must overcome the host antiviral silencing response. A viral protein called VPg has been shown to play a role in this process, but how it works is unclear. In this paper, we found that the VPg protein of Turnip mosaic virus (TuMV), which is a potyvirus, interacts with a host protein named SGS3, a key protein in the RNA silencing pathway. Moreover, this interaction leads to the degradation of SGS3 and its interacting and functional partner RDR6, which is another essential component of the RNA silencing pathway. We also identified the cellular pathways that are recruited for the VPg-mediated degradation of SGS3. Therefore, this work reveals a possible mechanism by which VPg sabotages host antiviral RNA silencing to promote virus infection., (Copyright © 2016 American Society for Microbiology.)

Unlabelled: The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage., Importance: Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: The multifunctional cylindrical inclusion (CI) protein of potyviruses contains ATP binding and RNA helicase activities. As part of the viral replication complex, it assists viral genome replication, possibly by binding to RNA and unwinding the RNA duplex. It also functions in viral cell-to-cell movement, likely via the formation of conical structures at plasmodesmata (PD) and the interaction with coat protein (CP)., Methods: To further understand the role of CI in the viral infection process, we employed the alanine-scanning mutagenesis approach to mutate CI in the infectious full-length cDNA clone of Turnip mosaic virus (TuMV) tagged by green fluorescent protein. A total of 40 double-substitutions were made at the clustered charged residues. The effect of these mutations on viral genome amplification was determined using a protoplast inoculation assay. All the mutants were also introduced into Nicotiana benthamiana plants to assess their cell-to-cell and long-distance movement. Three cell-to-cell movement-abolished mutants were randomly selected to determine if their mutated CI protein targets PD and interacts with CP by confocal microscopy., Results: Twenty CI mutants were replication-defective (5 abolished and 15 reduced), one produced an elevated level of viral genome in comparison with the parental virus, and the remaining 19 retained the same replication level as the parental virus. The replication-defective mutations were predominately located in the helicase domains and C-terminal region. All 15 replication-reduced mutants showed delayed or abolished cell-to-cell movement. Nine of 20 replication-competent mutants contained infection within single cells. Five of them distributed mutations within the N-terminal 100 amino acids. Most of replication-defective or cell-to-cell movement-abolished mutants failed to infect plants systemically. Analysis of three randomly selected replication-competent yet cell-to-cell movement-abolished mutants revealed that the mutated CI failed to form regular punctate structures at PD and/or to interact with CP., Conclusions: The helicase domain and C-terminal region of TuMV CI are essential for viral genome replication, and the N-terminal sequence modulates viral cell-to-cell movement. TuMV CI plays both interlinked and distinct roles in replication and intercellular movement. The ability of CI to target PD and interact with CP is associated with its functional role in viral cell-to-cell movement.

SUMOylation, which is catalyzed by small ubiquitin-like modifier (SUMO) enzymes, is a transient, reversible posttranslational protein modification that regulates diverse cellular processes. Potyviruses, the largest group of known plant viruses, comprise many agriculturally important viruses, such as Turnip mosaic virus (TuMV). The potyviral genome encodes 11 mature proteins. To investigate if SUMOylation plays a role in potyvirus infection, a yeast two-hybrid screen was performed to examine possible interactions of each of the 11 viral proteins of TuMV with AtSCE1, the only SUMO-conjugating enzyme in Arabidopsis thaliana homologous to the key SUMO-conjugating enzyme E2 in mammalian cells or Ubc9 in yeast. A positive reaction was found between AtSCE1 and NIb, the potyviral RNA-dependent RNA polymerase. Further bimolecular fluorescence complementation (BiFC) and fluorescence resonance energy transfer (FRET) assays revealed that the NIb and AtSCE1 interaction occurred in both the cytoplasm and nuclei of epidermal cells of Nicotiana benthamiana. The interaction motif was mapped to a region encompassing NIb amino acids 171 to 300 which contains a potential negatively charged amino acid-dependent SUMOylation motif (NDSM). An Escherichia coli SUMOylation assay showed that NIb can be SUMOylated and that the lysine residue (K172) in the motif is a potent SUMOylation site. A TuMV infectious clone with an arginine (R) substitution mutation at K172 compromised TuMV infectivity in plants. In comparison with wild-type Arabidopsis plants, sce1 knockdown plants exhibited increased resistance to TuMV as well as a nonrelated RNA virus. To the best of our knowledge, this is the first report showing that the host SUMO modification system plays an essential role in infection by plant RNA viruses.

Background: Genetic resistance is the most effective and sustainable approach to the control of plant pathogens that are a major constraint to agriculture worldwide. In soybean, three dominant R genes, i.e., Rsv1, Rsv3 and Rsv4, have been identified and deployed against Soybean mosaic virus (SMV) with strain-specificities. Molecular identification of virulent determinants of SMV on these resistance genes will provide essential information for the proper utilization of these resistance genes to protect soybean against SMV, and advance knowledge of virus-host interactions in general., Methodology/principal Findings: To study the gain and loss of SMV virulence on all the three resistance loci, SMV strains G7 and two G2 isolates L and LRB were used as parental viruses. SMV chimeras and mutants were created by partial genome swapping and point mutagenesis and then assessed for virulence on soybean cultivars PI96983 (Rsv1), L-29 (Rsv3), V94-5152 (Rsv4) and Williams 82 (rsv). It was found that P3 played an essential role in virulence determination on all three resistance loci and CI was required for virulence on Rsv1- and Rsv3-genotype soybeans. In addition, essential mutations in HC-Pro were also required for the gain of virulence on Rsv1-genotype soybean. To our best knowledge, this is the first report that CI and P3 are involved in virulence on Rsv1- and Rsv3-mediated resistance, respectively., Conclusions/significance: Multiple viral proteins, i.e., HC-Pro, P3 and CI, are involved in virulence on the three resistance loci and simultaneous mutations at essential positions of different viral proteins are required for an avirulent SMV strain to gain virulence on all three resistance loci. The likelihood of such mutations occurring naturally and concurrently on multiple viral proteins is low. Thus, incorporation of all three resistance genes in a soybean cultivar through gene pyramiding may provide durable resistance to SMV.

Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.

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., (Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.)

Replication of Tomato ringspot virus (ToRSV) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleotide triphosphate-binding protein (NTB) of ToRSV is an ER-targeted protein and that an intermediate polyprotein containing the domains for NTB and for the genome-linked viral protein (VPg) is associated with the replication complex. We now report the detection of a 95-kDa polyprotein that contains the domains for the RNA-dependent RNA polymerase (Pol), the proteinase (Pro) and the VPg. This polyprotein appears to be a truncated version of the full-length 111-kDa VPg-Pro-Pol polyprotein and was termed VPg-Pro-Pol'. A subpopulation of VPg-Pro-Pol' was peripherally associated with ER-derived membranes active in viral replication. However, the VPg, Pro and Pol domains did not target to membranes in the absence of viral infection. We propose a model in which VPg-Pro-Pol' is brought to the site of replication through interaction with a viral membrane protein.

Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q(423)/G and Q(620)/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.

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. [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]