An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication.

One up-regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. The full length cDNA of this gene was cloned by 5' and 3'-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus-based virus-induced gene silencing (loss-of-function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up-regulated gene, NbUbE3R1 that plays a role in BaMV replication.

A terpenoid phytoalexin plays a role in basal defense of Nicotiana benthamiana against Potato virus X.

Terpenoid phytoalexins function as defense compound against a broad spectrum of pathogens and pests in the plant kingdom. However, the role of phytoalexin in antiviral defense is still elusive. In this study, we identified the biosynthesis pathway of a sesquiterpenoid phytoalexin, capsidiol 3-acetate as an antiviral response against RNA virus Potato Virus X (PVX) in Nicotiana benthamiana. NbTPS1 and NbEAH genes were found strongly induced by PVX-infection. Enzymatic activity and genetic evidence indicated that both genes were involved in the PVX-induced biosynthesis of capsidiol 3-acetate. NbTPS1- or NbEAH-silenced plant was more susceptible to PVX. The accumulation of capsidiol 3-acetate in PVX-infected plant was partially regulated by jasmonic acid signaling receptor COI1. These findings provide an insight into a novel mechanism of how plant uses the basal arsenal machinery to mount a fight against virus attack even in susceptible species.

The abscisic acid pathway has multifaceted effects on the accumulation of Bamboo mosaic virus.

Accepted 29 October 2013. Abscisic acid (ABA) plays a key role in modulating plant responses to different biotic and abiotic stresses. However, the effect of ABA on virus infection is not fully understood. Here, we describe the effects of the ABA pathway on the accumulation of Bamboo mosaic virus (BaMV) and Cucumber mosaic virus (CMV) in two different hosts: Arabidopsis thaliana and Nicotiana benthamiana. We report that ABA2 plays a critical role in the accumulation of BaMV and CMV. Mutants downstream of ABA2 (aao3, abi1-1, abi3-1, and abi4-1) were susceptible to BaMV, indicating that the ABA pathway downstream of ABA2 is essential for BaMV resistance. The aba2-1 mutant decreased the accumulation of BaMV (+)RNA, (-)RNA, and coat protein, with the most dramatic effect being observed for (-)RNA. These findings were further validated by the use of virus-induced gene silencing and enzyme-linked immunosorbent assay in N. benthamiana. In addition, infecting N. benthamiana with BaMV or CMV increased ABA contents and activated the SA and ABA pathways, thereby disrupting the antagonism between these two cascades. Our findings uncover a novel role for ABA2 in supporting BaMV and CMV accumulation, distinct from the opposing role of its downstream genes.

The overexpression of a maize mitogen-activated protein kinase gene (ZmMPK5) confers salt stress tolerance and induces defence responses in tobacco.

As sessile organisms, plants are exposed to potential dangers, including multiple biotic and abiotic stresses. The mitogen-activated protein kinase (MAPK) is a universal signalling pathways involved in these processes. A previous study showed that maize ZmMPK5 is induced by various stimuli at transcriptional and post-translational levels. In this study, ZmMPK5 was overexpressed in tobacco to further analyse its biological functions. Under salt and oxidative stresses, ZmMPK5-overexpressing lines displayed less severe damage and stronger growth phenotypes corresponding to a series of physiological changes. In addition, the transgenic lines accumulated less reactive oxygen species (ROS) and had higher levels of antioxidant enzyme activity and metabolites than wild-type (WT) plants following NaCl treatment. Quantitative RT-PCR revealed that the expression of ROS-related and stress-responsive genes was higher in transgenic plants than in WT plants. Furthermore, transgenic lines exhibited enhanced resistance to viral pathogens, and expressed constitutively higher transcript levels of pathogenesis-related genes, such as PR1a, PR4, PR5 and EREBP. Taken together, these results demonstrated that ZmMPK5 is involved in salt stress, oxidative stress and pathogen defence signalling pathways, and its function may be at least partly devoted to efficiently eliminating ROS accumulation under salt stress.

The silencing suppressor P25 of Potato virus X interacts with Argonaute1 and mediates its degradation through the proteasome pathway.

Previous evidence has indicated that the P25 protein encoded by Potato virus X (PVX) inhibits either the assembly or function of the effector complexes in the RNA silencing-based antiviral defence system (Bayne et al., Cell-to-cell movement of Potato Potexvirus X is dependent on suppression of RNA silencing. Plant J.44, 471-482). This finding prompted us to investigate the possibility that P25 targets the Argonaute (AGO) effector nuclease of RNA silencing. Co-immunoprecipitation and Western blot analysis indicated that there is a strong interaction between P25 and AGO1 of Arabidopsis when these proteins are transiently co-expressed in Nicotiana benthamiana. P25 also interacts with AGO1, AGO2, AGO3 and AGO4, but not with AGO5 and AGO9. As an effective suppressor, the amount of AGO1 accumulated in the presence of P25 was dramatically lower than that infiltrated with HcPro, but was restored when treated with a proteasome inhibitor MG132. These findings are consistent with the idea that RNA silencing is an antiviral defence mechanism and that the counter-defence role of P25 is through the degradation of AGO proteins via the proteasome pathway. Further support for this idea is provided by the observation that plants treated with MG132 are less susceptible to PVX and its relative Bamboo mosaic virus.

[Effect of fucoidan on the ultrastructure of mesophyll cells of Datura stramonium L. and accumulation of potato virus X in them].

Influence of fucoidan from brown alga Fucus evanescens C. Ag. on the development of infection induced by potato virus X (PVX) in Datura stramonium leaves was studied. It as been shown that 24 h after the treatment of the leaves with fucoidan and following infection of them with PVX the accumulation of virus particles in infected cells during early infection period was substantially less than that in untreated control. Using ultrastructure-morphometric analysis, it has been established that fucoidan treatment increases at protein-synthesizing capability of cells (nucleolus dimension, amount of mitochondria and rough endoplasmic reticulum membranes become increased). At the same time, the fucoidan treatment causes some activation of lytic compartment which leads to destruction of virus particles and, therefore, might be considered as one of fucoidan-dependent protective mechanisms limiting virus accumulation in cells. Fucoidan stimulation of the formation of PVX-specific laminated structures capable of virus particles binding is possibly another induced antiviral cell mechanism, preventing from virus reproduction and transposition.

Effect of pH, ions, bovine serum albumin and heterologous antisera on the stability of immunosorbed flexuous potato viruses.

Electron microscopic studies on the stability of immunosorbed (trapped) virions of potato viruses X, S and Y0 (PVX, PVS and PVY0) revealed disintegration and dislodging of PVY0 virions upon incubation with (1) antisera to PVX, PVS, or both diluted in saline, (2) 0.86% NaCl (saline) or 0.1 mol/l CaCl2 but not with 0.1 mol/l CaSO4 or 0.1 mol/l MgSO4. PVX virions, on the other hand, showed partial dislodging upon incubation with an antiserum to PVS diluted in saline, but complete disintegration and dislodging with saline. 0.1 mol/l CaCl2 caused partial dislodging while MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) had no apparent adverse effect. PVS virions were not affected by saline, CaCl2, MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) and were only partially dislodged by antisera to PVX or PVY0. Disintegration and/or dislodging of the PVX and PVY0 virions was prevented when (1) they were fixed with glutaraldehyde prior to incubation or (2) the virus extract contained bovine serum albumin (BSA) or (3) heterologous antisera were diluted in 0.1 mol/l phosphate buffer (PB) before use except the PVS antiserum which still caused disintegration and dislodging of PVY0 virions. Prior fixation of virions prevented their disruption and dislodging by saline only in the case of PVY0 but not PVX. On the other hand, BSA reverted the adverse effect of saline but not that of the PVS antiserum on PVY0 virions. The results presented here suggest (1) a disruptive effect of Cl' on PVX and PVY0 virions particularly when it was associated with Na+ and (2) an interaction between the immunosorbed virions of PVX or PVY0 and the antiserum to PVS.