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.

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.

One 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.

Plasma membrane-associated cation-binding protein 1-like protein negatively regulates intercellular movement of BaMV.

To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants. Further analysis indicated no significant difference in the accumulation of BaMV in NbPCaP1L knockdown and control protoplasts, suggesting NbPCaP1L may affect cell-to-cell movement of BaMV. Using a viral vector expressing green fluorescent protein in the knockdown plants, the mean area of viral focus, as determined by fluorescence, was found to be larger in NbPCaP1L knockdown plants. Orange fluorescence protein (OFP)-fused NbPCaP1L, NbPCaP1L-OFP, was expressed in N. benthamiana and reduced the accumulation of BaMV to 46%. To reveal the possible interaction of viral protein with NbPCaP1L, we performed yeast two-hybrid and co-immunoprecipitation experiments. The results indicated that NbPCaP1L interacted with BaMV replicase. The results also suggested that NbPCaP1L could trap the BaMV movement RNP complex via interaction with the viral replicase in the complex and so restricted viral cell-to-cell movement.

A putative Rab-GTPase activation protein from Nicotiana benthamiana is important for Bamboo mosaic virus intercellular movement.

The cDNA-amplified fragment length polymorphism technique was applied to isolate the differentially expressed genes during Bamboo mosaic virus (BaMV) infection on Nicotiana benthamiana plants. One of the upregulated genes was cloned and predicted to contain a TBC domain designated as NbRabGAP1 (Rab GTPase activation protein 1). No significant difference was observed in BaMV accumulation in the NbRabGAP1-knockdown and the control protoplasts. However, BaMV accumulation was 50% and 2% in the inoculated and systemic leaves, respectively, of the knockdown plants to those of the control plants. By measuring the spreading area of BaMV infection foci in the inoculated leaves, we found that BaMV moved less efficiently in the NbRabGAP1-knockdown plants than in the control plants. Transient expression of the wild type NbRabGAP1 significantly increases BaMV accumulation in N. benthamiana. These results suggest that NbRabGAP1 with a functional Rab-GAP activity is involved in virus movement.