Nicotiana benthamiana Class 1 Reversibly Glycosylated Polypeptides Suppress Tobacco Mosaic Virus Infection

Abstract

Reversibly glycosylated polypeptides (RGPs) have been identified in many plant species and play an important role in cell wall formation, intercellular transport regulation, and plant–virus interactions. Most plants have several RGP genes with different expression patterns depending on the organ and developmental stage. Here, we report on four members of the RGP family in N. benthamiana. Based on a homology search, NbRGP1-3 and NbRGP5 were assigned to the class 1 and class 2 RGPs, respectively. We demonstrated that NbRGP1–3 and 5 mRNA accumulation increases significantly in response to tobacco mosaic virus (TMV) infection. Moreover, all identified class 1 NbRGPs (as distinct from NbRGP5) suppress TMV intercellular transport and replication in N. benthamiana. Elevated expression of NbRGP1–2 led to the stimulation of callose deposition at plasmodesmata, indicating that RGP-mediated TMV local spread could be affected via a callose-dependent mechanism. It was also demonstrated that NbRGP1 interacts with TMV movement protein (MP) in vitro and in vivo. Therefore, class 1 NbRGP1–2 play an antiviral role by impeding intercellular transport of the virus by affecting plasmodesmata callose and directly interacting with TMV MP, resulting in the reduced viral spread and replication.

Nicotiana benthamiana Methanol-Inducible Gene (MIG) 21 Encodes a Nucleolus-Localized Protein That Stimulates Viral Intercellular Transport and Downregulates Nuclear Import

“…To obtain a set of constructs encoding NbMIG21 fusions with various fluorescent proteins (35S-NbMIG21:GFP, 35S-NbMIG21:RFP, 35S-NbMIG21:YC, and NbMIG21:YN), a fragment encoding NbMIG21 without a stop codon and flanked by Acc65I/BamHI recognition sites was synthesized using the N-NbMIG21-Acc65I_f/N-NbMIG21-BamHI_r pair of primers. The NbMIG21 fragment was digested with Acc65I/BamHI and inserted into the pCambia-35S vector containing either a GFP-, RFP-, YN-, or YC-encoding sequence without a start codon [ 37 ], using the same sites. The NbMIG21 NoLSmut :GFP fragment was obtained as a product of overlap PCR, and mutations were inserted using NbMIG21-NoLS-mut_f and NbMIG21-NoLS-mut_r primers.…”

“…For NbCoil:YN and NbMIG21:YC pair, YFP fluorescence was distributed in multiple subnuclear structures that corresponded to Cajal bodies (Figure 8, right). An antiviral protein, N. benthamiana reversibly glycosylated polypeptide 1 (NbRGP1) [37], was used as a negative control for the BiFC assay. We concluded that NbMIG21p is distributed between the nucleolus and Cajal bodies.…”

Nicotiana benthamiana Methanol‐Inducible Gene (MIG) 21 Encodes a Nucleolus‐Localized Protein that Stimulates Viral Intercellular Transport and Downregulates Nuclear Import

“…For NbCoil:YC and NbMIG21:YN pair YFP fluorescence was distributed in multiple subnuclear structures that correspond to Cajal bodies (Figure 5B). An antiviral protein, N. benthamiana reversibly glycosylated protein 1 (NbRGP1) [37], was used as a negative control for BiFC assay. We concluded that NbMIG21p is distributed between nucleolus and Cajal bodies.…”

“…To obtain a set of constructs encoding NbMIG21 fusions with various fluorescent protein: 35S-NbMIG21:GFP, 35S-NbMIG21:RFP, 35S-NbMIG21:YC and NbMIG21:YN, a fragment encoding NbMIG21 without a stop codon and flanked by Acc65I/BamHI recognition sites was synthesized using "N-NbMIG21-Acc65I_f"/"N-NbMIG21-BamHI_r" pair of primers. This fragment was digested with Acc65I/BamHI and inserted into pCambia-35S vector containing either GFP-, RFP-, YN-or YC-encoding sequence without start codon [37] using the same sites.…”

Nanoplatforms for the Delivery of Nucleic Acids into Plant Cells

“…Nucleic acid delivery into plant cells could be applied to searching for host resistance [32][33][34][35] or susceptibility factors [36][37][38]. Solving fundamental problems in the field of plant-pathogen interactions paves the way to the transfer of the developed technologies to the applied biotechnology for crop defense and improvement, especially using such instruments as RNAi [12].…”