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.

The size of viral genomes is limited, thus the majority of encoded proteins possess multiple functions. The main function of tobamoviral movement protein (MP) is to perform plasmodesmata gating and mediate intercellular transport of the viral RNA. MP is a remarkable example of a protein that, in addition to the initially discovered and most obvious function, carries out numerous activities that are important both for the manifestation of its key function and for successful and productive infection in general. Briefly, MP binds the viral genome, delivers it to the plasmodesmata (PD) and mediates its intercellular transfer. To implement the transport function, MP interacts with diverse cellular factors. Each of these cellular proteins has its own function, which could be different under normal conditions and upon viral infection. Here, we summarize the data available at present on the plethora of cellular factors that were identified as tobamoviral MP partners and analyze the role of these interactions in infection development.

The biological functions of the multiple members of the xyloglucan endotransglycosylase/hydrolase (XTH) protein family are rather diverse: XTHs are cell wall remodeling enzymes that participate in plant growth and development, are involved in responses to various environmental stresses and interactions with pathogenic and symbiotic microorganisms. However, XTHs’ role upon viral infection remains poorly understood. Here we identified and characterized Nicotiana benthamiana XTH (NbXTH) which is involved in responses to viral infection. We demonstrated that NbXTH is a positive regulator of intercellular transport. NbXTH suppression leads to the inhibition of tobacco mosaic virus (TMV) local spread, resulting in the increased tolerance of N. benthamiana plants to TMV. Therefore, NbXTH could be regarded as a susceptibility factor.

The mechanical damage of plant tissues leads to the activation of methanol production and its release into the atmosphere. The gaseous methanol or vapors emitted by the damaged plant induce resistance in neighboring intact plants to bacterial pathogens but create favorable conditions for viral infection spread. Among the Nicotiana benthamiana methanol-inducible genes (MIGs), most are associated with plant defense and intercellular transport. Here, we characterize NbMIG21, which encodes a 209 aa protein (NbMIG21p) that does not share any homology with annotated proteins. NbMIG21p was demonstrated to contain a nucleolus localization signal (NoLS). Colocalization studies with fibrillarin and coilin, nucleolus and Cajal body marker proteins, revealed that NbMIG21p is distributed among these subnuclear structures. Our results show that recombinant NbMIG21 possesses DNA-binding properties. Similar to a gaseous methanol effect, an increased NbMIG21 expression leads to downregulation of the nuclear import of proteins with nuclear localization signals (NLSs), as was demonstrated with the GFP-NLS model protein. Moreover, upregulated NbMIG21 expression facilitates tobacco mosaic virus (TMV) intercellular transport and reproduction. We identified an NbMIG21 promoter (PrMIG21) and showed that it is methanol sensitive; thus, the induction of NbMIG21 mRNA accumulation occurs at the level of transcription. Our findings suggest that methanol-activated NbMIG21 might participate in creating favorable conditions for viral reproduction and spread.

Mechanical damage of plant tissues leads to the activation of methanol production and its release in the atmosphere. The gaseous methanol or vapors emitted by the damaged plant induce resistance in neighboring intact plants to bacterial pathogens but create favorable conditions for the viral infection spread. Among Nicotiana benthamiana methanol-inducible genes (MIGs) most are associated with plant defense and intercellular transport. Here we characterize NbMIG21 that encodes a 209-aa protein (NbMIG21p) which does not share any homology with annotated proteins. NbMIG21p was demonstrated to contain nucleolus localization signals (NoLS). Co-localization studies with fibrillarin and coilin, nucleolus and Cajal bodies marker proteins, revealed that NbMIG21p is distributed between these subnuclear structures. Our results show that recombinant NbMIG21 possess DNA-binding properties . Similar to gaseous methanol effect an increased NbMIG21 expression leads to the downregulation of the nuclear import of NLS-containing proteins as was demonstrated on the model GFP-NLS protein. Moreover, upregulated NbMIG21 facilitates tobacco mosaic virus (TMV) intercellular transport. We identified NbMIG21 promoter (PrMIG21) and showed that it is methanol-sensitive thus induction of NbMIG21 expression occurs at the level of transcription. Our findings suggest that methanol-activated NbMIG21 might participate in creating favorable conditions for the viral reproduction and spread.

A coordinated and generalized plant response to adverse environmental factors largely depends on the proper and finely-tuned regulation of intercellular transport via plasmodesmata (PD). However, the knowledge of the whole network of PD-controlling mechanisms is far from complete. Earlier, a cellular factor, Kunitz peptidase inhibitor-like protein (KPILP), that affects PD gating and plays a proviral role, was identified in Nicotiana benthamiana plants. Here we characterized its homolog from N. tabacum, NtKPILP, which is hardly detectable in leaves of intact plants, in contrast to roots, flowers and seeds where NtKPILP is highly expressed. However, its mRNA accumulation in leaves increases in response to various stresses, including viral infection. NtKPILP was demonstrated to affect chloroplast functioning. Using the virus-induced gene silencing approach, we have shown that NtKPILP downregulation negatively affects intercellular transport of macromolecules, inducing callose deposition at PD and suppressing beta-1,3-glucanase mRNA accumulation. Together, the obtained results indicate that NtKPILP is a viral infection-responsive cellular factor that is involved in PD permeability regulation, sharing thus the features of KPILPs from other Nicotiana species.