Livia Stavolone

A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells.

Hitching a ride on vesicles: Cauliflower mosaic virus movement protein trafficking in the endomembrane system

A tubule-forming viral movement protein traffics in post-Golgi compartments and requires endocytosis for tubule formation and virus movement. The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) μA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.

Extracellular Matrix in Plants and Animals: Hooks and Locks for Viruses

The extracellular matrix (ECM) of animal and plants cells plays important roles in viral diseases. While in animal cells extracellular matrix components can be exploited by viruses for recognition, attachment and entry, the plant cell wall acts as a physical barrier to viral entry and adds a higher level of difficulty to intercellular movement of viruses. Interestingly, both in plant and animal systems, ECM can be strongly remodeled during virus infection, and the understanding of remodeling mechanisms and molecular players offers new perspectives for therapeutic intervention. This review focuses on the different roles played by the ECM in plant and animal hosts during virus infection with special emphasis on the similarities and differences. Possible biotechnological applications aimed at improving viral resistance are discussed.

Unusual genomic features of a badnavirus infecting mulberry

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95 % sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.

S‐acylation mediates Mungbean yellow mosaic virus AC4 localization to the plasma membrane and in turns gene silencing suppression

RNA silencing plays a critical role in plant resistance against viruses. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that interfere with the cellular silencing machinery through various mechanisms not always well understood. We examined the role of Mungbean yellow mosaic virus (MYMV) AC4 and showed that it is essential for infectivity but not for virus replication. It acts as a determinant of pathogenicity and counteracts virus induced gene silencing by strongly suppressing the systemic phase of silencing whereas it does not interfere with local production of siRNA. We demonstrate the ability of AC4 to bind native 21–25 nt siRNAs in vitro by electrophoretic mobility shift assay. While most of the known VSRs have cytoplasmic localization, we observed that despite its hydrophilic nature and the absence of trans-membrane domain, MYMV AC4 specifically accumulates to the plasma membrane (PM). We show that AC4 binds to PM via S-palmitoylation, a process of post-translational modification regulating membrane–protein interactions, not known for plant viral protein before. When localized to the PM, AC4 strongly suppresses systemic silencing whereas its delocalization impairs VSR activity of the protein. We also show that AC4 interacts with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), a positive regulator of the cell-to-cell movement of RNAi. The absolute requirement of PM localization for direct silencing suppression activity of AC4 is novel and intriguing. We discuss a possible model of action: palmitoylated AC4 anchors to the PM by means of palmitate to acquire the optimal conformation to bind siRNAs, hinder their systemic movement and hence suppress the spread of the PTGS signal in the plant.

Prosystemin overexpression induces transcriptional modifications of defense-related and receptor-like kinase genes and reduces the susceptibility to Cucumber mosaic virus and its satellite RNAs in transgenic tomato plants

Systemin is a plant signal peptide hormone involved in the responses to wounding and insect damage in the Solanaceae family. It works in the same signaling pathway of jasmonic acid (JA) and enhances the expression of proteinase inhibitors. With the aim of studying a role for systemin in plant antiviral responses, a tomato (Solanum lycopersicum) transgenic line overexpressing the prosystemin cDNA, i.e. the systemin precursor, was inoculated with Cucumber mosaic virus (CMV) strain Fny supporting either a necrogenic or a non-necrogenic satellite RNA (satRNA) variant. Transgenic plants showed reduced susceptibility to both CMV/satRNA combinations. While symptoms of the non-necrogenic inoculum were completely suppressed, a delayed onset of lethal disease occurred in about half of plants challenged with the necrogenic inoculum. RT-qPCR analysis showed a correlation between the systemin-mediated reduced susceptibility and the JA biosynthetic and signaling pathways (e.g. transcriptional alteration of lipoxygenase D and proteinase inhibitor II). Moreover, transgenically overexpressed systemin modulated the expression of a selected set of receptor-like protein kinase (RLK) genes, including some playing a known role in plant innate immunity. A significant correlation was found between the expression profiles of some RLKs and the systemin-mediated reduced susceptibility to CMV/satRNA. These results show that systemin can increase plant defenses against CMV/satRNA through transcriptional reprogramming of diverse signaling pathways.

Interference of Brefeldin A in viral movement protein tubules assembly.

Plant virus genomes cross the barrier of the host cell wall and move to neighboring cells either in the form of nucleoprotein complex or encapsidated into virions. Virus transport is facilitated by virus-encoded movement proteins (MP), which are different from one another in number, size, sequence, and in the strategy used to overcome the size exclusion limit of plasmodesmata (PD).1 A group of them forms tubules inside the lumen of highly modified PDs upon removal of the desmotubule. To date the molecular mechanism(s) and the host factors involved in the assembly of MP tubules as well as the mechanistic aspects of virus particle transport throughout them remain substantially unknown. In a recent study, we showed that Cauliflower mosaic virus (CaMV) MP traffics in the endocytic pathway with the help of 3 tyrosine-sorting signals, which are not required to target MP to the plasma membrane but are essential for tubule formation.2 This evidence unravels a previously unknown connection between the plant endosomal system and tubule-mediated virus movement that is here supported by demonstration of hindrance of tubule assembly upon Brefeldin A (BFA) treatment. We discuss the implications of our data on the mechanisms of viral transport through tubules and draw parallels with plant mechanisms of polarized growth.

Evaluation of Constitutive Cestrum Yellow Leaf Curling Virus Promoter in Maize and Tomato

We have cloned and evaluated two versions of a novel, strong and constitutive promoter from Cestrum yellow leaf-curling virus (CmYLCV) called CmpC (short- 346bp) and CmpS (longer- 400bp), which can be used for regulating transgene expression in a wide variety of plant species. CmYLCV belongs to the Caulimoviridae family and was first reported in Cestrum parqui from the Solanaceae by Ragozzino (1974). Recently, CmYLCV was cloned and seven open reading frames were identified in the genomic sequence (Hohn et al., 2001). To evaluate the utility of the CmYLCV promoter to drive expression of heterologous genes in plants, two versions of the full-length transcript promoter were cloned in front of the GUS, CAT and FP reporter genes and tested in transient assays in Nicotiana plumbaginifolia, Orichophragmus violaceus and Oriza sativa protoplasts as well as in stably transformed Zea mays and Lycopersicon esculentum. The transient expression experiments show that, depending on the plant system used, the expression level of CmpC and CmpS promoter fragments are higher than the expression level of the widely used 35S promoter from Cauliflower Mosaic Virus (Hohn et al., 2001) and that the longer promoter fragment is the weaker one. Expression analysis of CmpC and CmpS promoter fragments in stably transformed maize (Figurel) have shown that both fragments are on average ten times greater than the strong constitutive Ubi 1 promoter from Z. mays (Christensen et al., 1992) and that the expression levels in tomato (Figure 2) are comparable with the strong, constitutive SMAS promoter (Ni et al., 1994). Moreover, the spatial expression analysis has shown that both promoters express in various tissue types, except pollen in both maize and tomato and that both promoter fragments retain their high expression levels through at least two generations. We limited our analysis to the single copy events only (Ingham et al., 2001) since it is a widely accepted idea that the copy number of a transgene affects the expression level in transgenic plants.