Bamboo Mosaic Virus Accumulation Research Articles

Importin α2 participates in RNA interference against bamboo mosaic virus accumulation in Nicotiana benthamiana via NbAGO10a-mediated small RNA clearance.

Karyopherins, the nucleocytoplasmic transporters, participate in multiple RNA silencing stages by transporting associated proteins into the nucleus. Importin α is a member of karyopherins and has been reported to facilitate virus infection via nuclear import of viral proteins. Unlike other RNA viruses, silencing of importin α2 (α2i) by virus-induced gene silencing (VIGS) boosted the titre of bamboo mosaic virus (BaMV) in protoplasts, and inoculated and systemic leaves of Nicotiana benthamiana. The enhanced BaMV accumulation in importin α2i plants was linked to reduced levels of RDR6-dependent secondary virus-derived small-interfering RNAs (vsiRNAs). Small RNA-seq revealed importin α2 silencing did not affect the abundance of siRNAs derived from host mRNAs but significantly reduced the 21 and 22 nucleotide vsiRNAs in BaMV-infected plants. Deletion of BaMV TGBp1, an RNA silencing suppressor, compromised importin α2i-mediated BaMV enhancement. Moreover, silencing of importin α2 upregulated NbAGO10a, a proviral protein recruited by TGBp1 for BaMV vsiRNAs clearance, but hindered the nuclear import of NbAGO10a. Taken together, these results indicate that importin α2 acts as a negative regulator of BaMV invasion by controlling the expression and nucleocytoplasmic shuttling of NbAGO10a, which removes vsiRNAs via the TGBp1-NbAGO10a-SDN1 pathway. Our findings reveal the hidden antiviral mechanism of importin α2 in countering BaMV infection in N. benthamiana.

A viral movement protein co-opts endoplasmic reticulum luminal-binding protein and calreticulin to promote intracellular movement.

Intracellular movement is an important step for the initial spread of virus in plants during infection. This process requires virus-encoded movement proteins (MPs) and their interaction with host factors. Despite the large number of known host factors involved in the movement of different viruses, little is known about host proteins that interact with one of the MPs encoded by potexviruses, the triple-gene-block protein 3 (TGBp3). The main obstacle lies in the relatively low expression level of potexviral TGBp3 in hosts and the weak or transient nature of interactions. Here, we used TurboID-based proximity labeling to identify the network of proteins directly or indirectly interacting with the TGBp3 of a potexvirus, Bamboo mosaic virus (BaMV). Endoplasmic reticulum (ER) luminal-binding protein 4 and calreticulin 3 of Nicotiana benthamiana (NbBiP4 and NbCRT3, respectively) associated with the functional TGBp3-containing BaMV movement complexes, but not the movement-defective mutant, TGBp3M. Fluorescent microscopy revealed that TGBp3 colocalizes with NbBiP4 or NbCRT3 and the complexes move together along ER networks to cell periphery in N. benthamiana. Loss- and gain-of-function experiments revealed that NbBiP4 or NbCRT3 is required for the efficient spread and accumulation of BaMV in infected leaves. In addition, overexpression of NbBiP4 or NbCRT3 enhanced the targeting of BaMV TGBp1 to plasmodesmata (PD), indicating that NbBiP4 and NbCRT3 interact with TGBp3 to promote the intracellular transport of virion cargo to PD that facilitates virus cell-to-cell movement. Our findings revealed additional roles for NbBiP4 and NbCRT3 in BaMV intracellular movement through ER networks or ER-derived vesicles to PD, which enhances the spread of BaMV in N. benthamiana.

Voltage-dependent anion channel proteins associate with dynamic Bamboo mosaic virus-induced complexes.

Infection cycles of viruses are highly dependent on membrane-associated host factors. To uncover the infection cycle of Bamboo mosaic virus (BaMV) in detail, we purified the membrane-associated viral complexes from infected Nicotiana benthamiana plants and analyzed the involved host factors. Four isoforms of voltage-dependent anion channel (VDAC) proteins on the outer membrane of mitochondria were identified due to their upregulated expression in the BaMV complex-enriched membranous fraction. Results from loss- and gain-of-function experiments indicated that NbVDAC2, -3, and -4 are essential for efficient BaMV accumulation. During BaMV infection, all NbVDACs concentrated into larger aggregates, which overlapped and trafficked with BaMV virions to the structure designated as the "dynamic BaMV-induced complex." Besides the endoplasmic reticulum and mitochondria, BaMV replicase and double-stranded RNAs were also found in this complex, suggesting the dynamic BaMV-induced complex is a replication complex. Yeast two-hybrid and pull-down assays confirmed that BaMV triple gene block protein 1 (TGBp1) could interact with NbVDACs. Confocal microscopy revealed that TGBp1 is sufficient to induce NbVDAC aggregates, which suggests that TGBp1 may play a pivotal role in the NbVDAC-virion complex. Collectively, these findings indicate that NbVDACs may associate with the dynamic BaMV-induced complex via TGBp1 and NbVDAC2, -3, or -4 and can promote BaMV accumulation. This study reveals the involvement of mitochondrial proteins in a viral complex and virus infection.

MiR398-regulated antioxidants contribute to Bamboo mosaic virus accumulation and symptom manifestation.

Virus infections that cause mosaic or mottling in leaves commonly also induce increased levels of reactive oxygen species (ROS). However, how ROS contributes to symptoms is less well documented. Bamboo mosaic virus (BaMV) causes chlorotic mosaic symptoms in both Brachypodium distachyon and Nicotiana benthamiana. The BaMV △CPN35 mutant with an N-terminal deletion of its coat protein gene exhibits asymptomatic infection independently of virus titer. Histochemical staining of ROS in mock-, BaMV-, and BaMV△CPN35-infected leaves revealed that hydrogen peroxide (H2O2) accumulated solely in BaMV-induced chlorotic spots. Moreover, exogenous H2O2 treatment enhanced yellowish chlorosis in BaMV-infected leaves. Both BaMV and BaMV△CPN35 infection could induce the expression of Cu/Zu superoxide dismutase (CSD) antioxidants at messenger RNA and protein level. However, BaMV triggered the abundant accumulation of full-length NbCSD2 preprotein (prNbCSD2, without transit peptide cleavage), whereas BaMV△CPN35 induced a truncated prNbCSD2. Confocal microscopy showed that majority of NbCSD2-green fluorescent protein (GFP) predominantly localized in the cytosol upon BaMV infection, but BaMV△CPN35 infection tended to cause NbCSD2-GFP to remain in chloroplasts. By 5′-RNA ligase-mediated rapid amplification of cDNA ends, we validated CSDs are the targets of miR398 in vivo. Furthermore, BaMV infection increased the level of miR398, while the level of BaMV titer was regulated positively by miR398 but negatively by CSD2. In contrast, overexpression of cytosolic form NbCSD2, impairing the transport into chloroplasts, greatly enhanced BaMV accumulation. Taken together, our results indicate that induction of miR398 by BaMV infection may facilitate viral titer accumulation, and cytosolic prNbCSD2 induction may contribute to H2O2 accumulation, resulting in the development of BaMV chlorotic symptoms in plants.

NbPsbO1 Interacts Specifically with the Bamboo Mosaic Virus (BaMV) Subgenomic RNA (sgRNA) Promoter and Is Required for Efficient BaMV sgRNA Transcription.

ABSTRACTMany positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in the infection cycle through the combined activities of viral replicase and host proteins. However, knowledge about host proteins involved in direct sgRNA promoter recognition is limited. Here, in the partially purified replicase complexes from Bamboo mosaic virus (BaMV)-infected tissue, we have identified the Nicotiana benthamiana photosystem II oxygen-evolving complex protein, NbPsbO1, which specifically interacted with the promoter of sgRNA but not that of genomic RNA (gRNA). Silencing of NbPsbO1 expression suppressed BaMV accumulation in N. benthamiana protoplasts without affecting viral gRNA replication. Overexpression of wild-type NbPsbO1 stimulated BaMV sgRNA accumulation. Fluorescent microscopy examination revealed that the fluorescence associated with NbPsbO1 was redistributed from chloroplast granal thylakoids to stroma in BaMV-infected cells. Overexpression of a mislocalized mutant of NbPsbO1, dTPPsbO1-T7, inhibited BaMV RNA accumulation in N. benthamiana, whereas overexpression of an NbPsbO1 derivative, sPsbO1-T7, designed to be targeted to chloroplast stroma, upregulated the sgRNA level. Furthermore, depletion of NbPsbO1 in BaMV RdRp preparation significantly inhibited sgRNA synthesis in vitro but exerted no effect on (+) or (−) gRNA synthesis, which indicates that NbPsbO1 is required for efficient sgRNA synthesis. These results reveal a novel role for NbPsbO1 in the selective enhancement of BaMV sgRNA transcription, most likely via direct interaction with the sgRNA promoter.IMPORTANCE Production of subgenomic RNAs (sgRNAs) for efficient translation of downstream viral proteins is one of the major strategies adapted for viruses that contain a multicistronic RNA genome. Both viral genomic RNA (gRNA) replication and sgRNA transcription rely on the combined activities of viral replicase and host proteins, which recognize promoter regions for the initiation of RNA synthesis. However, compared to the cis-acting elements involved in the regulation of sgRNA synthesis, the host factors involved in sgRNA promoter recognition mostly remain to be elucidated. Here, we found a chloroplast protein, NbPsbO1, which specifically interacts with Bamboo mosaic virus (BaMV) sgRNA promoter. We showed that NbPsbO1 is relocated to the BaMV replication site in BaMV-infected cells and demonstrated that NbPsbO1 is required for efficient BaMV sgRNA transcription but exerts no effect on gRNA replication. This study provides a new insight into the regulating mechanism of viral gRNA and sgRNA synthesis.

Dissecting the role of a plant-specific Rab5 small GTPase NbRabF1 in Bamboo mosaic virus infection.

NbRabF1, a small GTPase from Nicotiana benthamiana and a homolog of Arabidopsis thaliana Ara6, plays a key role in regulating Bamboo mosaic virus (BaMV) movement by vesicle transport between endosomal membranes. Reducing the expression of NbRabF1 in N. benthamiana by virus-induced gene silencing decreased the accumulation of BaMV, and with smaller infection foci on inoculated leaves, but had no effect in protoplasts. Furthermore, transient expression of NbRabF1 increased the accumulation of BaMV in inoculated leaves. Thus, NbRabF1 may be involved in the cell-to-cell movement of BaMV. The potential acyl modification sites at the second and third amino acid positions of NbRabF1 were crucial for membrane targeting and BaMV accumulation. The localization of mutant forms of NbRabF1 with the GDP-bound (donor site) and GTP-bound (acceptor site) suggested that NbRabF1 might regulate vesicle trafficking between the Golgi apparatus and plasma membrane. Furthermore, GTPase activity could also be involved in BaMV cell-to-cell movement. Overall, in this study, we identified a small GTPase, NbRabF1, from N. benthamiana that interacts with its activation protein NbRabGAP1 and regulates vesicle transport from the Golgi apparatus to the plasma membrane. We suggest that the BaMV movement complex might move from cell to cell through this vesicle trafficking route.

Disrupting the Homeostasis of High Mobility Group Protein Promotes the Systemic Movement of Bamboo mosaic virus.

Viruses hijack various organelles and machineries for their replication and movement. Ever more lines of evidence indicate that specific nuclear factors are involved in systemic trafficking of several viruses. However, how such factors regulate viral systemic movement remains unclear. Here, we identify a novel role for Nicotiana benthamiana high mobility group nucleoprotein (NbHMG1/2a) in virus movement. Although infection of N. benthamiana with Bamboo mosaic virus (BaMV) decreased NbHMG1/2a expression levels, nuclear-localized NbHMG1/2a protein was shuttled out of the nucleus into cytoplasm upon BaMV infection. NbHMG1/2a knockdown or even overexpression did not affect BaMV accumulation in inoculated leaves, but it did enhance systemic movement of the virus. Interestingly, the positive regulator Rap-GTPase activation protein 1 was highly upregulated upon infection with BaMV, whereas the negative regulator thioredoxin h protein was greatly reduced, no matter if NbHMG1a/2a was silenced or overexpressed. Our findings indicate that NbHMG1/2a may have a role in plant defense responses. Once its homeostasis is disrupted, expression of relevant host factors may be perturbed that, in turn, facilitates BaMV systemic movement.

The Lipid Transfer Protein 1 from Nicotiana benthamiana Assists Bamboo mosaic virus Accumulation.

Host factors play a pivotal role in regulating virus infection. Uncovering the mechanism of how host factors are involved in virus infection could pave the way to defeat viral disease. In this study, we characterized a lipid transfer protein, designated NbLTP1 in Nicotiana benthamiana, which was downregulated after Bamboo mosaic virus (BaMV) inoculation. BaMV accumulation significantly decreased in NbLTP1-knockdown leaves and protoplasts compared with the controls. The subcellular localization of the NbLTP1-orange fluorescent protein (OFP) was mainly the extracellular matrix. However, when we removed the signal peptide (NbLTP1/ΔSP-OFP), most of the expressed protein targeted chloroplasts. Both NbLTP1-OFP and NbLTP1/ΔSP-OFP were localized in chloroplasts when we removed the cell wall. These results suggest that NbLTP1 may have a secondary targeting signal. Transient overexpression of NbLTP1 had no effect on BaMV accumulation, but that of NbLTP1/ΔSP significantly increased BaMV expression. NbLTP1 may be a positive regulator of BaMV accumulation especially when its expression is associated with chloroplasts, where BaMV replicates. The mutation was introduced to the predicted phosphorylation site to simulate the phosphorylated status, NbLTP/ΔSP/P(+), which could still assist BaMV accumulation. By contrast, a mutant lacking calmodulin-binding or simulates the phosphorylation-negative status could not support BaMV accumulation. The lipid-binding activity of LTP1 was reported to be associated with calmodulin-binding and phosphorylation, by which the C-terminus functional domain of NbLTP1 may play a critical role in BaMV accumulation.

Nicotiana benthamiana Argonaute10 plays a pro-viral role in Bamboo mosaic virus infection.

RNA silencing is a major defense mechanism against invading viruses in plants. Argonaute proteins (AGOs) are the key players in RNA silencing. The number of AGO family members involved varies depending on the plant species and they play distinct or sometimes redundant roles in antiviral defense. By using a virus-induced gene silencing technique, it was found that Nicotiana benthamiana AGO1 restricted Bamboo mosaic virus (BaMV) accumulation, but NbAGO10, the closest paralog of NbAGO1, positively regulated BaMV accumulation. Immunoprecipitation assay revealed BaMV virus-derived small interfering RNAs (vsiRNAs) in NbAGO10 complexes. Transient overexpression of NbAGO10 increased BaMV RNA accumulation, but with co-expression of NbAGO1, BaMV RNA accumulation was reduced, which suggests that NbAGO10 may have competed with NbAGO1 for sequestering BaMV vsiRNA and prevented the formation of RNA-induced silencing complexes. In addition, overexpression of NbAGO10 decreased BaMV vsiRNA accumulation. A host enzyme, small RNA degrading nuclease 1 (SDN1), also was found to interact with NbAGO10 on in vivo pull-down assay. Silencing of SDN1 elevated BaMV vsiRNA level and decreased BaMV RNA accumulation in N.benthamiana, indicating that NbAGO10 might recruit SDN1 for BaMV vsiRNA degradation. The results herein suggested that NbAGO10 plays a pro-viral role by BaMV vsiRNA sequestration and degradation.

Autophagy is involved in assisting the replication of Bamboo mosaic virus in Nicotiana benthamiana.

Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.

An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication.

SummaryOne up‐regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single‐stranded positive‐sense RNA virus. The full length cDNA of this gene was cloned by 5′ and 3′‐rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus‐based virus‐induced gene silencing (loss‐of‐function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild‐type NbUbE3R1‐orange fluorescent protein (NbUbE3R1‐OFP), NbUbE3R1/△TM‐OFP (removal of the transmembrane domain) and NbUbE3R1/mRING‐OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate‐interacting domain. Yeast two‐hybrid and co‐immunoprecipitation experiments used to determine the possible viral‐encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up‐regulated gene, NbUbE3R1 that plays a role in BaMV replication.

Nuclear-Encoded Plastidal Carbonic Anhydrase Is Involved in Replication of Bamboo mosaic virus RNA in Nicotiana benthamiana.

On inoculation of Nicotiana benthamiana with Bamboo mosaic virus (BaMV), a gene with downregulated expression was found involved in the infection cycle of BaMV. To uncover how this downregulated gene affects the accumulation of BaMV in plants, we used loss- and gain-of-function experiments. Knockdown of this gene decreased the accumulation of BaMV coat protein to approximately 60% in both plants and protoplasts of N. benthamiana but had no effect on Potato virus X and Cucumber mosaic virus infection. The full-length gene was cloned and revealed as an N. benthamiana nuclear-encoded chloroplast carbonic anhydrase (CA) and so designated NbCA. As compared with the accumulation of BaMV RNAs in NbCA-knockdown protoplasts, both plus- and minus-strand RNAs were reduced. We further fused NbCA with Orange fluorescent protein to confirm its localization in chloroplasts on confocal microscopy. However, transiently expressed NbCA in chloroplasts did not considerably increase BaMV accumulation. The addition of exogenous CA may not have any additive effect on BaMV accumulation because of the natural abundance of CA in chloroplasts. In an in vitro replication assay, the addition of Escherichia coli-expressed NbCA enhanced exogenous template level (re-initiation and elongation) but not endogenous template level (only elongation). These results suggest that NbCA is possibly involved in re-initiation step of BaMV RNA replication. Further analysis indicated that proton concentration could influence the endogenous and exogenous template activities. Hence, our results implied that NbCA could be playing a role in harnessing proton concentration and favoring the replicase with the re-initiation template.

Antiviral Roles of Abscisic Acid in Plants.

Abscisic acid (ABA) is a key hormone involved in tuning responses to several abiotic stresses and also has remarkable impacts on plant defense against various pathogens. The roles of ABA in plant defense against bacteria and fungi are multifaceted, inducing or reducing defense responses depending on its time of action. However, ABA induces different resistance mechanisms to viruses regardless of the induction time. Recent studies have linked ABA to the antiviral silencing pathway, which interferes with virus accumulation, and the micro RNA (miRNA) pathway through which ABA affects the maturation and stability of miRNAs. ABA also induces callose deposition at plasmodesmata, a mechanism that limits viral cell-to-cell movement. Bamboo mosaic virus (BaMV) is a member of the potexvirus group and is one of the most studied viruses in terms of the effects of ABA on its accumulation and resistance. In this review, we summarize how ABA interferes with the accumulation and movement of BaMV and other viruses. We also highlight aspects of ABA that may have an effect on other types of resistance and that require further investigation.

Nicotiana benthamiana Elicitor-Inducible Leucine-Rich Repeat Receptor-Like Protein Assists Bamboo Mosaic Virus Cell-to-Cell Movement

For successful infection, a virus requires various host factors at different stages such as translation, targeting, replication, and spreading. One of the host genes upregulated after Nicotiana benthamiana infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA potexvirus, assists in viral movement. To understand how this host protein is involved in BaMV movement, we cloned its full-length cDNA by rapid amplification of cDNA ends. The gene has 3199 nt and encodes a 969-amino acid polypeptide. The sequence of the encoded polypeptide is orthologous to that of N. tabacum elicitor-inducible leucine-rich repeat (LRR) receptor-like protein (NtEILP), a plant viral resistance gene, and is designated NbEILP. To reveal how NbEILP is involved in BaMV movement, we fused green fluorescent protein (GFP) to its C-terminus. Unfortunately, the gene’s expression in N. benthamiana was beyond our detection limit possibly because of its large size (∼135 kDa). However, NbEILP at such low expression could still enhance BaMV accumulation in inoculated leaves. A short version of NbEILP was constructed to remove the LRR domain, NbEILP/ΔLRR-GFP; the expression of this deletion mutant could still enhance BaMV accumulation to 1.7-fold that of the control. Hence, the LRR domain in NbEILP is not an essential element in BaMV movement. We constructed a few deletion mutants — NbEILP/ΔLRRΔTMD (without the transmembrane domain), NbEILP/ΔLRRΔCD (without the cytoplasmic domain), and NbEILP/ΔLRRΔSP (without the signal peptide) — to examine whether these domains are involved in BaMV movement. For BaMV movement, NbEILP requires the signal peptide to target the endoplasmic reticulum and the transmembrane domain to retain on the membrane.

Host Factors in the Infection Cycle of Bamboo mosaic virus.

To complete the infection cycle efficiently, the virus must hijack the host systems in order to benefit for all the steps and has to face all the defense mechanisms from the host. This review involves a discussion of how these positive and negative factors regulate the viral RNA accumulation identified for the Bamboo mosaic virus (BaMV), a single-stranded RNA virus. The genome of BaMV is approximately 6.4 kb in length, encoding five functional polypeptides. To reveal the host factors involved in the infection cycle of BaMV, a few different approaches were taken to screen the candidates. One of the approaches is isolating the viral replicase-associated proteins by co-immunoprecipitation with the transiently expressed tagged viral replicase in plants. Another approach is using the cDNA-amplified fragment length polymorphism technique to screen the differentially expressed genes derived from N. benthamiana plants after infection. The candidates are examined by knocking down the expression in plants using the Tobacco rattle virus-based virus-induced gene silencing technique following BaMV inoculation. The positive or negative regulators could be described as reducing or enhancing the accumulation of BaMV in plants when the expression levels of these proteins are knocked down. The possible roles of these host factors acting on the accumulation of BaMV will be discussed.

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.

Promotion of Bamboo Mosaic Virus Accumulation in Nicotiana benthamiana by 5'→3' Exonuclease NbXRN4.

Bamboo mosaic virus (BaMV) has a 6.4-kb (+) sense RNA genome with a 5′ cap and a 3′ poly(A) tail. ORF1 of this potexvirus encodes a 155-kDa replication protein responsible for the viral RNA replication/transcription and 5′ cap formation. To learn more about the replication complex of BaMV, a protein preparation enriched in the 155-kDa replication protein was obtained from Nicotiana benthamiana by a protocol involving agroinfiltration and immunoprecipitation. Subsequent analysis by SDS-PAGE and mass spectrometry identified a handful of host proteins that may participate in the viral replication. Among them, the cytoplasmic exoribonuclease NbXRN4 particularly caught our attention. NbXRN4 has been shown to have an antiviral activity against Tomato bushy stunt virus and Tomato mosaic virus. In Arabidopsis, the enzyme could reduce RNAi- and miRNA-mediated RNA decay. This study found that downregulation of NbXRN4 greatly decreased BaMV accumulation, while overexpression of NbXRN4 resulted in an opposite effect. Mutations at the catalytically essential residues abolished the function of NbXRN4 in the increase of BaMV accumulation. Nonetheless, NbXRN4 was still able to promote BaMV accumulation in the presence of the RNA silencing suppressor P19. In summary, the replication efficiency of BaMV may be improved by the exoribonuclease activity of NbXRN4.

NbRABG3f, a member of Rab GTPase, is involved in Bamboo mosaic virus infection in Nicotiana benthamiana.

The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up-regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up-regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus-induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane-targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP-NbRABG3f/T22N clearly co-localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane-targeting ability are crucial for BaMV accumulation at the cell level.

The Abscisic Acid Pathway Has Multifaceted Effects on the Accumulation of Bamboo mosaic virus

Accepted 29 October 2013. Abscisic acid (ABA) plays a key role in modulating plant responses to different biotic and abiotic stresses. However, the effect of ABA on virus infection is not fully understood. Here, we describe the effects of the ABA pathway on the accumulation of Bamboo mosaic virus (BaMV) and Cucumber mosaic virus (CMV) in two different hosts: Arabidopsis thaliana and Nicotiana benthamiana. We report that ABA2 plays a critical role in the accumulation of BaMV and CMV. Mutants downstream of ABA2 (aao3, abi1-1, abi3-1, and abi4-1) were susceptible to BaMV, indicating that the ABA pathway downstream of ABA2 is essential for BaMV resistance. The aba2-1 mutant decreased the accumulation of BaMV (+)RNA, (-)RNA, and coat protein, with the most dramatic effect being observed for (-)RNA. These findings were further validated by the use of virus-induced gene silencing and enzyme-linked immunosorbent assay in N. benthamiana. In addition, infecting N. benthamiana with BaMV or CMV increased ABA contents and activated the SA and ABA pathways, thereby disrupting the antagonism between these two cascades. Our findings uncover a novel role for ABA2 in supporting BaMV and CMV accumulation, distinct from the opposing role of its downstream genes.

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N.benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N.benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.