A plant RNA virus inhibits NPR1 sumoylation and subverts NPR1-mediated plant immunity.

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.

Sumoylation of Turnip mosaic virus RNA Polymerase Promotes Viral Infection by Counteracting the Host NPR1-Mediated Immune Response.

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.

Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication.

Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb.

SCE1, the SUMO-conjugating enzyme in plants that interacts with NIb, the RNA-dependent RNA polymerase of Turnip mosaic virus, is required for viral infection.

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.

Five proteins of Laodelphax striatellus are potentially involved in the interactions between rice stripe virus and vector.

Rice stripe virus (RSV) is the type member of the genus Tenuivirus, which relies on the small brown planthopper (Laodelphax striatellus Fallén) for its transmission in a persistent, circulative-propagative manner. To be transmitted, virus must cross the midgut and salivary glands epithelial barriers in a transcytosis mechanism where vector receptors interact with virions, and as propagative virus, RSV need utilize host components to complete viral propagation in vector cells. At present, these mechanisms remain unknown. In this paper, we screened L. striatellus proteins, separated by two-dimensional electrophoresis (2-DE), as potential RSV binding molecules using a virus overlay assay of protein blots. The results, five L. striatellus proteins that bound to purified RSV particles in vitro were resolved and identified using mass spectrometry. The virus-binding capacities of five proteins were further elucidated in yeast two-hybrid screen (YTHS) and virus-binding experiments of expressed proteins. Among five proteins, the receptor for activated protein kinase C (RACK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH3) did not interact with RSV nucleocapsid protein (NCP) in YTHS and in far-Western blot, and three ribosomal proteins (RPL5, RPL7a and RPL8) had specific interactions with RSV. In dot immunobinding assay (DIBA), all five proteins were able to bind to RSV particles. The five proteins' potential contributions to the interactions between RSV and L. striatellus were discussed. We proposed that RACK and GAPDH3 might be involved in the epithelial transcytosis of virus particles, and three ribosomal proteins probably played potential crucial roles in the infection and propagation of RSV in vector cells.

Formation of complexes at plasmodesmata for potyvirus intercellular movement is mediated by the viral protein P3N-PIPO.

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.

Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus.

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.

Identification of a movement protein of the tenuivirus rice stripe virus.

Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.