Interaction between Hantavirus Nucleocapsid Protein (N) and RNA-Dependent RNA Polymerase (RdRp) Mutants Reveals the Requirement of an N-RdRp Interaction for Viral RNA Synthesis

Viral ribonucleocapsids harboring the viral genomic RNA are used as the template for viral mRNA synthesis and replication of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Here we show that hantavirus nucleocapsid protein (N protein) interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain at the N terminus of N protein. Similarly, the N protein binding pocket is located at the C terminus of RdRp. We demonstrate that an N protein-RdRp interaction is required for RdRp function during the course of virus infection in the host cell.     

Influence of cytoplasmic heat shock protein 70 on viral infection of Nicotiana benthamiana

The accumulation of heat shock protein 70 (Hsp70) generally occurs in plants infected with viruses. However, the effect of Hsp70 accumulation on plant viral infection and pathogenesis remains elusive. In this study, the expression of six Hsp70 genes was found to be induced by the four diverse RNA viruses, Tobacco mosaic virus, Potato virus X (PVX), Cucumber mosaic virus and Watermelon mosaic virus, in Nicotiana benthamiana. Heat treatment enhanced the accumulation and systemic infection of these viruses. Similar results were obtained for viral infection in plants heterologously expressing an Arabidopsis cytoplasmic Hsp70 through either a PVX vector or Agrobacterium infiltration. In contrast, viral infection was compromised in cytoplasmic NbHsp70c‐1 gene‐silenced plants. These data demonstrate that the cytoplasmic Hsp70s can enhance the infection of N. benthamiana by diverse viruses.     

The ORF5 of Grapevine virus A is involved in symptoms expression in Nicotiana benthamiana plants

Grapevine virus A (GVA), a member of the genus Vitivirus which belongs to the family Flexiviridae, has a single‐stranded RNA genome of about 7.4 kb that comprises five open reading frames (ORFs). ORF5 encodes a small 10‐kDa protein (p10), which is believed to interact with nucleic acids and to suppress the plant's RNA‐ silencing response. We obtained molecular and biological data indicating that ORF5‐encoded product, specifically its N‐terminus, affects the appearance of symptoms in Nicotiana benthamiana plants. The ORF5‐encoded products of the severe GR5 and the mild GTR1‐1 isolates were found to affect RNA silencing similarly in mesophyll cells of N. benthamiana, despite being involved in different expressions of symptoms on this host.     

The unfolded protein response plays dual roles in rice stripe virus infection through fine-tuning the movement protein accumulation

The movement of plant viruses is a complex process that requires support by the virus-encoded movement protein and multiple host factors. The unfolded protein response (UPR) plays important roles in plant virus infection, while how UPR regulates viral infection remains to be elucidated. Here, we show that rice stripe virus (RSV) elicits the UPR in Nicotiana benthamiana. The RSV-induced UPR activates the host autophagy pathway by which the RSV-encoded movement protein, NSvc4, is targeted for autophagic degradation. As a counteract, we revealed that NSvc4 hijacks UPR-activated type-I J-domain proteins, NbMIP1s, to protect itself from autophagic degradation. Unexpectedly, we found NbMIP1 stabilizes NSvc4 in a non-canonical HSP70-independent manner. Silencing NbMIP1 family genes in N. benthamiana, delays RSV infection, while over-expressing NbMIP1.4b promotes viral cell-to-cell movement. Moreover, OsDjA5, the homologue of NbMIP1 family in rice, behaves in a similar manner toward facilitating RSV infection. This study exemplifies an arms race between RSV and the host plant, and reveals the dual roles of the UPR in RSV infection though fine-tuning the accumulation of viral movement protein.     

Protein composition of 6K2‐induced membrane structures formed during Potato virus A infection

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein‐induced membranous structures from Potato virus A (PVA)‐infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N‐terminal Twin‐Strep‐tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non‐tagged Cerulean‐6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep‐tag‐based affinity chromatography was developed. Both (+)‐ and (–)‐strand PVA RNA and viral protein VPg were co‐purified specifically with the affinity tagged PVA‐SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA‐SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2‐induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co‐purified with PVA‐derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.     

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.     

Overexpression of a soybean nuclear localized type–III DnaJ domain‐containing HSP40 reveals its roles in cell death and disease resistance

Heat‐shock proteins such as HSP70 and HSP90 are important molecular chaperones that play critical roles in biotic and abiotic stress responses; however, the involvement of their co‐chaperones in stress biology remains largely uninvestigated. In a screen for candidate genes stimulating cell death in Glycine max (soybean), we transiently overexpressed full‐length cDNAs of soybean genes that are highly induced during soybean rust infection in Nicotiana benthamiana leaves. Overexpression of a type‐III DnaJ domain‐containing HSP40 (GmHSP40.1), a co‐chaperone of HSP70, caused hypersensitive response (HR)‐like cell death. The HR‐like cell death was dependent on MAPKKKα and WIPK, because silencing each of these genes suppressed the HR. Consistent with the presence of a nuclear localization signal (NLS) motif within the GmHSP40.1 coding sequence, GFP‐GmHSP40.1 was exclusively present in nuclear bodies or speckles. Nuclear localization of GmHSP40.1 was necessary for its function, because deletion of the NLS or addition of a nuclear export signal abolished its HR‐inducing ability. GmHSP40.1 co‐localized with HcRed‐SE, a protein involved in pri‐miRNA processing, which has been shown to be co‐localized with SR33‐YFP, a protein involved in pre‐mRNA splicing, suggesting a possible role for GmHSP40.1 in mRNA splicing or miRNA processing, and a link between these processes and cell death. Silencing GmHSP40.1 enhanced the susceptibility of soybean plants to Soybean mosaic virus, confirming its positive role in pathogen defense. Together, the results demonstrate a critical role of a nuclear‐localized DnaJ domain‐containing GmHSP40.1 in cell death and disease resistance in soybean.     

NbEXPA1, an α‐expansin,is plasmodesmata‐specific and a novel host factor for potyviral infection

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD‐enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2‐fold increase) and 48 (≥2‐fold reduction) are significantly differentially accumulated in the PD‐enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α‐expansin designated NbEXPA1, a cell wall loosening protein, is PD‐specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA‐dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD‐proteome dataset that is useful in future studies to expound PD biology and PD‐mediated virus–host interactions but also characterizes NbEXPA1 as the first PD‐specific cell wall loosening protein and its essential role in potyviral infection.     

A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus

An up‐regulated gene derived from Bamboo mosaic virus (BaMV)‐infected Nicotiana benthamiana plants was cloned and characterized in this study. BaMV is a single‐stranded, positive‐sense RNA virus. This gene product, designated as NbTRXh2, was matched with sequences of thioredoxin h proteins, a group of small proteins with a conserved active‐site motif WCXPC conferring disulfide reductase activity. To examine how NbTRXh2 is involved in the infection cycle of BaMV, we used the virus‐induced gene silencing technique to knock down NbTRXh2 expression in N. benthamiana and inoculated the plants with BaMV. We observed that, compared with control plants, BaMV coat protein accumulation increased in knockdown plants at 5 days post‐inoculation (dpi). Furthermore, BaMV coat protein accumulation did not differ significantly between NbTRXh2‐knockdown and control protoplasts at 24 hpi. The BaMV infection foci in NbTRXh2‐knockdown plants were larger than those in control plants. In addition, BaMV coat protein accumulation decreased when NbTRXh2 was transiently expressed in plants. These results suggest that NbTRXh2 plays a role in restricting BaMV accumulation. Moreover, confocal microscopy results showed that NbTRXh2‐OFP (NbTRXh2 fused with orange fluorescent protein) localized at the plasma membrane, similar to AtTRXh9, a homologue in Arabidopsis. The expression of the mutant that did not target the substrates failed to reduce BaMV accumulation. Co‐immunoprecipitation experiments revealed that the viral movement protein TGBp2 could be the target of NbTRXh2. Overall, the functional role of NbTRXh2 in reducing the disulfide bonds of targeting factors, encoded either by the host or virus (TGBp2), is crucial in restricting BaMV movement.     

Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.     

Rice stripe virus activates the bZIP17/28 branch of the unfolded protein response signalling pathway to promote viral infection

The unfolded protein response (UPR) plays important roles in plant virus infection. Our previous study has proved that rice stripe virus (RSV) infection elicits host UPR. However, the mechanism on how the UPR is triggered upon RSV infection remains obscure. Here, we show that the bZIP17/28 branch of the UPR signalling pathway is activated upon RSV infection in Nicotiana benthamiana. We found that membrane-associated proteins NSvc2 and NSvc4 encoded by RSV are responsible for the activation of the bZIP17/28 branch. Ectopic expression of NSvc2 or NSvc4 in plant leaves induced the proteolytic processing of NbbZIP17/28 and up-regulated the expression of UPR-related genes. Silencing NbbZIP17/28 significantly inhibited RSV infection. We show that RSV can specifically elicit the UPR through the bZIP17/28 branch, thus promoting virus infection of N. benthamiana plants.     

The hypersensitive induced reaction 3 (HIR3) gene contributes to plant basal resistance via an EDS1 and salicylic acid‐dependent pathway

The hypersensitive‐induced reaction (HIR) gene family is associated with the hypersensitive response (HR) that is a part of the plant defense system against bacterial and fungal pathogens. The involvement of HIR genes in response to viral pathogens has not yet been studied. We now report that the HIR3 genes of Nicotiana benthamiana and Oryza sativa (rice) were upregulated following rice stripe virus (RSV) infection. Silencing of HIR3s in N. benthamiana resulted in an increased accumulation of RSV RNAs, whereas overexpression of HIR3s in N. benthamiana or rice reduced the expression of RSV RNAs and decreased symptom severity, while also conferring resistance to Turnip mosaic virus, Potato virus X, and the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. Silencing of HIR3 genes in N. benthamiana reduced the content of salicylic acid (SA) and was accompanied by the downregulated expression of genes in the SA pathway. Transient expression of the two HIR3 gene homologs from N. benthamiana or the rice HIR3 gene in N. benthamiana leaves caused cell death and an accumulation of SA, but did not do so in EDS1‐silenced plants or in plants expressing NahG. The results indicate that HIR3 contributes to plant basal resistance via an EDS1‐ and SA‐dependent pathway.     

The C2 protein of tomato leaf curl Taiwan virus is a pathogenicity determinant that interferes with expression of host genes encoding chromomethylases

Tomato (Solanum lycopersicum) is one of the most important crops worldwide and is severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus‐encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato cv. Micro‐Tom and Nicotiana benthamiana. Severe stunting of tomato and N. benthamiana plants infected with ToLCTWV was observed. Expression of ToLCTWV C2‐green fluorescent protein (GFP) fusion protein was predominately located in the nucleus and contributed to activation of a coat protein promoter. Notably, the C2‐GFP fluorescence was distributed in nuclear aggregates. Tomato and N. benthamiana plants inoculated with potato virus X (PVX)‐C2 displayed chlorotic lesions and stunted growth. PVX‐C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential recognition target to induce host‐defense responses. In tomato and N. benthamiana, ToLCTWV C2 was found to interfere with expression of genes encoding chromomethylases. N. benthamiana plants with suppressed NbCMT3–2 expression were more susceptible to ToLCTWV infection. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the NbCMT3–2 gene and pNbCMT3–2::GUS (β‐glucuronidase) promoter activity. C2 protein is an important pathogenicity determinant of ToLCTWV and interferes with host components involved in DNA methylation.