A conserved motif in three viral movement proteins from different genera is required for host factor recruitment and cell-to-cell movement.

Due to their minimal genomes, plant viruses are forced to hijack specific cellular pathways to ensure host colonization, a condition that most frequently involves physical interaction between viral and host proteins. Among putative viral interactors are the movement proteins, responsible for plasmodesma gating and genome binding during viral transport. Two of them, DGBp1 and DGBp2, are required for alpha-, beta- and gammacarmovirus cell-to-cell movement, but the number of DGBp-host interactors identified at present is limited. By using two different approaches, yeast two-hybrid and bimolecular fluorescence complementation assays, we found three Arabidopsis factors, eIF3g1, RPP3A and WRKY36, interacting with DGBp1s from each genus mentioned above. eIF3g1 and RPP3A are mainly involved in protein translation initiation and elongation phases, respectively, while WRKY36 belongs to WRKY transcription factor family, important regulators of many defence responses. These host proteins are not expected to be associated with viral movement, but knocking out WRKY36 or silencing either RPP3A or eIF3g1 negatively affected Arabidopsis infection by Turnip crinkle virus. A highly conserved FNF motif at DGBp1 C-terminus was required for protein-protein interaction and cell-to-cell movement, suggesting an important biological role.

Hexanoic Acid Treatment Prevents Systemic MNSV Movement in Cucumis melo Plants by Priming Callose Deposition Correlating SA and OPDA Accumulation.

Unlike fungal and bacterial diseases, no direct method is available to control viral diseases. The use of resistance-inducing compounds can be an alternative strategy for plant viruses. Here we studied the basal response of melon to Melon necrotic spot virus (MNSV) and demonstrated the efficacy of hexanoic acid (Hx) priming, which prevents the virus from systemically spreading. We analysed callose deposition and the hormonal profile and gene expression at the whole plant level. This allowed us to determine hormonal homeostasis in the melon roots, cotyledons, hypocotyls, stems and leaves involved in basal and hexanoic acid-induced resistance (Hx-IR) to MNSV. Our data indicate important roles of salicylic acid (SA), 12-oxo-phytodienoic acid (OPDA), jasmonic-isoleucine, and ferulic acid in both responses to MNSV. The hormonal and metabolites balance, depending on the time and location associated with basal and Hx-IR, demonstrated the reprogramming of plant metabolism in MNSV-inoculated plants. The treatment with both SA and OPDA prior to virus infection significantly reduced MNSV systemic movement by inducing callose deposition. This demonstrates their relevance in Hx-IR against MNSV and a high correlation with callose deposition. Our data also provide valuable evidence to unravel priming mechanisms by natural compounds.

Dissecting the multifunctional role of the N-terminal domain of the Melon necrotic spot virus coat protein in RNA packaging, viral movement and interference with antiviral plant defence.

The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S-domain builds a robust protein shell around the viral genome, whereas the C-terminal protruding domain, or P-domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N-terminal domain, or R-domain, and the arm region, which connects the R-domain and S-domain, are involved in different key steps of the viral cycle, such as cell-to-cell movement and the suppression of RNA silencing and pathogenesis through their RNA-binding capabilities. Deletion mutants revealed that the CP RNA-binding ability was abolished only after complete, but not partial, deletion of the R-domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA-binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R-domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full-length CPs were required to encapsidate full-length genomes. We also found that the R-domain carboxyl portion and the arm region were essential for efficient cell-to-cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R-domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.

Comparative proteomic analysis of melon phloem exudates in response to viral infection.

Phloem vasculature is the route that most plant viruses use to spread widely around the plant. In addition, phloem sap transports signals that trigger systemic defense responses to infection. We investigated the proteome-level changes that occur in phloem sap during virus infection using the 2D-DIGE technique. Total proteins were extracted from phloem exudates of healthy and Melon necrotic spot virus infected melon plants and analyzed by 2D-DIGE. A total of 1046 spots were detected but only 25 had significant changes in abundance. After mass spectrometry, 19 different proteins corresponding to 22 spots were further identified (13 of them up-accumulated and 9 down-accumulated). Most of them were involved in controlling redox balance and cell death. Only two of the differentially altered proteins had never been described to be present in the phloem before: a carboxylesterase and the fumarylacetoacetate hydrolase 1, both considered negative regulators of cell death. RT-PCR analysis of phloem sap RNAs revealed that the transcripts corresponding to some of the identified protein could be also loaded into the sieve elements. The impact of these proteins in the host response against viral infections and the potential involvement in regulating development, growth and stress response in melon plants is discussed. BIOLOGICAL SIGNIFICANCE: Despite the importance of phloem as an integrative pathway for resource distribution, signaling and plant virus transport little is known about the modifications induced by these pathogens in phloem sap proteome. Only one previous study has actually examined the phloem sap proteome during viral infection using conventional two-dimensional electrophoresis. Since the major limitation of this technique has been its low sensitivity, the authors only identified five phloem proteins with altered abundance. To circumvent this issue we use two-dimensional difference in-gel electrophoresis (2D DIGE) technique, which combined with DeCyder Differential Analysis Software allows a more accurate and sensitive quantitative analysis than with conventional 2D PAGE. We identified 19 different proteins which accumulation in phloem sap was altered during a compatible plant virus infection including redox and hypersensitivity response-related proteins. Therefore, this work would help to understand the basic processes that occur in phloem during plant-virus interaction.

A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements.

Viral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus p7B, a type II transmembrane protein, leaves the endoplasmic reticulum (ER) through the COPII-dependent Golgi pathway to reach the plasmodesmata. Here we investigated the sequence requirements and putative mechanisms governing p7B transport through the early secretory pathway. Deletion of either the cytoplasmic N-terminal region (CR) or the luminal C-terminal region (LR) led to ER retention, suggesting that they are both essential for ER export. Through alanine-scanning mutagenesis, we identified residues in the CR and LR that are critical for both ER export and for viral cell-to-cell movement. Within the CR, alanine substitution of aspartic and proline residues in the DSSP ß-turn motif (D7 AP10 A) led to movement of discrete structures along the cortical ER in an actin-dependent manner. In contrast, alanine substitution of a lysine residue in the LR (K49 A) resulted in a homogenous ER distribution of the movement protein and inhibition of ER-Golgi traffic. Moreover, the ability of p7B to recruit Sar1 to the ER membrane is lost in the D7 AP10 A mutant, but enhanced in the K49 A mutant. In addition, fluorescence recovery after photobleaching revealed that K49 A but not D7 AP10 A dramatically diminished protein lateral mobility. From these data, we propose a model whereby the LR directs actin-dependent mobility toward the cortical ER, where the cytoplasmic DSSP ß-turn favors assembly of COPII vesicles for export of p7B from the ER.