Abstract
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.
Highlights
Bamboo mosaic virus (BaMV), belonging to the Potexvirus genus of family Alphaflexiviridae (Lin et al, 1994), has one single-stranded positive-sense RNA genome of approximately 6.4 kb long [excluding the poly(A) tail]
In carbonic anhydrase (CA)-silenced N. benthamiana plants, the growth of Phytophthora infestans was considerably increased, probably due to suppression of the hypersensitive reaction (HR) (Restrepo et al, 2005). This earlier research mostly documented that the host CA is necessary for plant defense, in this study, we found that CA could, by contrast, help BaMV accumulation
The accumulation of plus- and minus-strand BaMV RNAs were affected by the reduction in NbCA levels (Figure 3)
Summary
Bamboo mosaic virus (BaMV), belonging to the Potexvirus genus of family Alphaflexiviridae (Lin et al, 1994), has one single-stranded positive-sense RNA genome of approximately 6.4 kb long [excluding the poly(A) tail]. The genome comprises a 5 cap structure, 3 poly(A) tail, and five open reading frames (ORFs 1-5) (Lin et al, 1994). ORFs 2-4, encoding 28-, 13-, and 6-kDa polypeptides, respectively, overlap and are called triplegene-block (TGB), designated TGBp1, -2, and -3, respectively. The movement of BaMV requires these three TGB proteins (Lin et al, 2004, 2006; Chou et al, 2013). ORF5, encoding a 25-kDa polypeptide viral capsid protein (CP) is required for cell-to-cell movement, symptom development, and virion assembly (Lan et al, 2010; Hung et al, 2014a,b). The 3 untranslated region (UTR) plays roles in minus-strand RNA initiation, polyadenylation, and long-distance movement (Chen et al, 2017)
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