The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus.

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection. In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation. The results show a significant decrease in BaMV RNA accumulation when the expression level of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3' untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis. The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis.

Perception of volatiles produced by UVC-irradiated plants alters the response to viral infection in naïve neighboring plants.

Interplant communication of stress via volatile signals is a well-known phenomenon. It has been shown that plants undergoing stress caused by pathogenic bacteria or insects generate volatile signals that elicit defense response in neighboring naïve plants. Similarly, we have recently shown that naïve plants sharing the same gaseous environment with UVC-exposed plants exhibit similar changes in genome instability as UVC-exposed plants. We found that methyl salicylate (MeSA) and methyl jasmonate (MeJA) serve as volatile signals communicating genome instability (as measured by an increase in the homologous recombination frequency). UVC-exposed plants produce high levels of MeSA and MeJA, a response that is missing in an npr1 mutant. Concomitantly, npr1 mutants are impaired in communicating the signal leading to genome instability, presumably because this mutant does not develop new necrotic lesion after UVC irradiation as observed in wt plants. To analyze the potential biological significance of such plant-plant communication, we have now determined whether bystander plants that receive volatile signals from UVC-irradiated plants, become more resistant to UVC irradiation or infection with oilseed rape mosaic virus (ORMV). Specifically, we analyzed the number of UVC-elicited necrotic lesions, the level of anthocyanin pigments, and the mRNA levels corresponding to ORMV coat protein and the NPR1-regulated pathogenesis-related protein PR1 in the irradiated or virus-infected bystander plants that have been previously exposed to volatiles produced by UVC-irradiated plants. These experiments showed that the bystander plants responded similarly to control plants following UVC irradiation. Interestingly, however, the bystander plants appeared to be more susceptible to ORMV infection, even though PR1 mRNA levels in systemic tissue were significantly higher than in the control plants, which indicates that bystander plants could be primed to strongly respond to bacterial infection.

The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis.

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.