Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid– and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement. In plants, spreading virus infection occurs via small pores in the cell wall named plasmodesmata that connect adjacent cells. Two decades have passed since the first discovery of specific viral proteins (movement proteins; MP) that assist this process. However, the manner by which these proteins adapt plasmodesmata to allow the movement of relatively large viral structures remains largely unknown. Here, we show that a family of plasmodesmata-located proteins, called PDLPs, which are conserved amongst higher plants, specifically mediate this process. PDLPs bind classes of MP that assemble into tubules within plasmodesmata to promote the movement of entire virions. This class of MP occurs for a diverse range of plant virus genera and we show that representatives of these viruses have MPs that bind PDLPs. The importance of PDLPs in this process was shown when reduction in accumulation led to reduced tubule formation, delayed infection and attenuated symptoms. Altogether, our study supports a scenario whereby the PDLPs work together to support virus infection of plants and as such provide important mechanistic insights into the movement mechanism of plant viruses within their hosts.

Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear “viral compartment.” We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells. To establish infection, plant viruses spread cell-to-cell via narrow channels in the cell wall, the plasmodesmata (PD). Movement proteins (MP) are virus-encoded proteins essential for virus intercellular transport through PD. Plasmodesmata located plant proteins (PDLPs), are specifically recognised by the MPs of tubule-forming viruses. Here we show that PDLP targeting to PD depends on the molecular motors myosin XI-K and XI-2. Consistently, and in support of a function of PDLP as PD receptor for MP, overexpression of dominant negative myosin mutants inhibits tubule formation by Grapevine fanleaf virus (GFLV) MP and dramatically reduces virus movement.

The 5′-terminal cistron of beet necrotic yellow vein furovirus RNA 2 encodes the 21 kDa major viral coat protein and terminates with an amber stop codon which can undergo suppression to give rise to a 75 kDa readthrough (RT) protein referred to as P75. P75 is a minor component of virions and the 54 kDa RT domain following the coat protein sequence is important both for virus assembly and transmission by the fungal vector Polymyxa betae. To better define the regions of the RT domain involved in these two steps, RNA 2 transcripts encoding different in-frame RT domain deletion mutants were tested for their ability to form virions when inoculated to plants with the other viral RNAs and to be fungus-transmitted. All deletions in the N-terminal half of the RT domain interfered with virus assembly and partially or completely inhibited fungus transmission. A 411 nucleotide deletion within the C-terminal half of the RT domain did not inhibit assembly but blocked fungus transmission of the virus. Alanine scanning mutagenesis within the aforesaid 411 nucleotide subdomain identified a peptide motif (KTER) which is important for the fungus transmission process.