Insights into RNAi-based antiviral immunity in Lepidoptera: acute and persistent infections in Bombyx mori and Trichoplusia ni cell lines.

The control of viral infections in insects is a current issue of major concern and RNA interference (RNAi) is considered the main antiviral immune response in this group of animals. Here we demonstrate that overexpression of key RNAi factors can help to protect insect cells against viral infections. In particular, we show that overexpression of Dicer2 and Argonaute2 in lepidopteran cells leads to improved defense against the acute infection of the Cricket Paralysis Virus (CrPV). We also demonstrate an important role of RNAi in the control of persistent viral infections, as the one caused by the Macula-like Latent Virus (MLV). Specifically, a direct interaction between Argonaute2 and virus-specific small RNAs is shown. Yet, while knocking down Dicer2 and Argonaute2 resulted in higher transcript levels of the persistently infecting MLV in the lepidopteran cells under investigation, overexpression of these proteins could not further reduce these levels. Taken together, our data provide deep insight into the RNAi-based interactions between insects and their viruses. In addition, our results suggest the potential use of an RNAi gain-of-function approach as an alternative strategy to obtain reduced viral-induced mortality in Lepidoptera, an insect order that encompasses multiple species of relevant economic value.

High population densities of Macrolophus pygmaeus on tomato plants can cause economic fruit damage: interaction with Pepino mosaic virus?

BACKGROUND: The zoophytophagous predator Macrolophus pygmaeus Rambur (Hemiptera: Miridae) is a successful biocontrol agent against several pest species in protected tomato crops. This predator is considered to be harmless for the crop. However, in recent years, Heteroptera feeding punctures on tomato fruit in Belgian and Dutch greenhouses have been misinterpreted as Pepino mosaic virus (PepMV) symptoms. In this study, three hypotheses were tested: (1) M. pygmaeus causes fruit damage that increases with population density and surpasses economic thresholds; (2) the presence of prey or alternative prey reduces the damage; (3) an infection of the tomato plants by PepMV triggers or aggravates M. pygmaeus fruit damage. RESULTS: At increasing M. pygmaeus densities, the severity of fruit damage increased from a few dimples towards yellowish discoloration and deformed fruits. A correlation with an infection with PepMV was found. The severity of the symptoms was independent of the presence of prey. A minimum economic density threshold was estimated at 0.32 M. pygmaeus per leaf. CONCLUSION: M. pygmaeus can cause economic damage to tomato fruits at densities common in practice. An infection of the plants with PepMV enhances fruit symptoms significantly. Interacting plant defence responses are most likely the key to explaining this, although confirmation is required. © 2015 Society of Chemical Industry.

Inactivation of Bombyx mori macula-like virus under physical conditions.

The Bombyx mori macula-like virus (BmMLV) is a member of the genus Maculavirus, family Tymoviridae, and contains a positive-sense single-stranded RNA genome. Previously, we reported that almost all B. mori-derived cell lines have already been contaminated with BmMLV via an unknown infection route. Since B. mori-derived cell lines are used for the baculovirus expression vector system, the invasion of BmMLV will cause a serious safety risk in the production of recombinant proteins. In this study, to determine the inactivation effectiveness of BmMLV, viruses were treated with various temperatures as well as gamma and ultraviolet (UV) light radiation. After these treatments, the virus solutions were inoculated into BmMLV-free BmVF cells. At 7 days postinoculation, the amount of virus in cells was evaluated by real-time reverse transcription PCR. Regarding heat treatment, conditions under 56°C for 3 h were tolerated, whereas infectivity disappeared after treatment at 75°C for 1 h. Regarding gamma radiation treatment, viruses were relatively stable at 1 kGy; however, their infectivity was entirely eliminated at a dose of 10 kGy. With 254 nm UV-C treatment, viruses were still active at less than 120 mJ/cm(2); however, their infectivity was completely lost at greater than 140 mJ/cm(2) UV-C radiation. These results provide quantitative evidence of the potential for BmMLV inactivation under a variety of physical conditions.

First infectious clone of the propagatively transmitted Oat blue dwarf virus.

Oat blue dwarf virus (OBDV) is a small, phloem-limited marafivirus that replicates in its leafhopper vector. We have developed complete cDNA clones of OBDV from which infectious transcripts may be derived--the first such clones for any propagatively transmitted plant virus. Prior to clone construction, the reported sequences of the 5' and 3' ends were confirmed using 5' RACE, primer extension, and ligation-anchored PCR. Using vascular puncture of maize seeds with capped transcripts, multiple clones were shown to be infectious at an average rate of 24.3% (range 14-36%). Aster leafhoppers successfully transmitted OBDV to oats and barley after feeding on detached, infected maize leaves. Proteins and RNAs consistent in size with those expected in OBDV infection were detected in young leaves via western and northern blotting, respectively. One construct, pOBDV-2r, was designated as the reference clone. An infectious clone of OBDV will be valuable in examining the interaction of this virus with both its insect and plant hosts.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease.

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus.

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.

Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease.

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.