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The Colorado potato beetle (CPB), Leptinotarsa decemlineata is an important economic pest of potato worldwide. Resistance to organophosphates and carbamates in CPB has been associated in some cases to point mutations in the acetylcholinesterase (AChE) gene Ldace2, an orthologue of Drosophila melanogaster Dmace2. In this paper we report cloning and sequencing of Ldace1, an orthologue of Anopheles gambiae Agace1 that was previously unknown in CPB. The Ldace1 coding enzyme contains all residues conserved in a functionally active AChE. Ldace1 is expressed at higher levels (between 2- and 11-fold) than Ldace2 in embryos, in the four larval instars and in adults. Specific interference of Ldace1 by means of dsRNA injection resulted in a reduction of AChE activity to an approximate 50% compared to control, whilst interference of Ldace2 reduced AChE activity to an approximate 85%. Analysis of zymograms of AChE activity after interference indicates that LdAChE1 is the enzyme predominantly responsible for the activity visualised. Interference of Ldace1 in CPB adults caused a significant increase in mortality (43%) as early as three days post-injection (p.i.), suggesting the essential role of Ldace1. Interference of Ldace2 also caused a significant increase in mortality (29%) compared to control, although at seven days p.i. The effect of the interference of Ldace1 on susceptibility to the organophosphate chlorpyrifos points out that LdAChE1 could be a main target for this insecticide. In the light of our results, studies associating resistance in CPB to mutations in Ldace2 should be reviewed, taking into consideration analysis of the Ldace1 gene., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cyclorrhapha insect genomes contain a single acetylcholinesterase (AChE) gene while other insects contain at least two ace genes (ace1 and ace2). In this study we tested the hypothesis that the two ace paralogous from Blattella germanica have different contributions to AChE activity, using RNA interference (RNAi) to knockdown each one individually. Paralogous-specific depletion of Bgace transcripts was evident in ganglia of injected cockroaches, although the effects at the protein level were less pronounced. Using spectrophotometric and zymogram measurements, we obtained evidence that BgAChE1 represents 65-75% of the total AChE activity in nerve tissue demonstrating that ace1 encodes a predominant AChE. A significant increase in sensitivity of Bgace1-interfered cockroaches was observed after 48 h of exposure to chlorpyrifos. In contrast, Bgace2 knockdown had a negligible effect on mortality to this organophosphate. These results point out a key role, qualitative and/or quantitative, of AChE1 as target of organophosphate insecticides in this species. Silencing the expression of Bgace1 but not Bgace2 also produced an increased mortality in insects when synergized with lambda-cyhalothrin, a situation which resembles the synergistic effects observed between organophosphates and pyrethroids. Gene silencing of ace genes by RNAi offers an exciting approach for examining a possible functional differentiation in ace paralogous., (Copyright 2009 Elsevier Ltd. All rights reserved.)

SUMMARY A comparative analysis of the synergistic interaction between PVX and either PVY or TEV potyviruses was performed in Nicotiana benthamiana and N. tabacum plants. In each PVX/potyvirus combination, doubly infected plants developed much more severe symptoms than singly infected ones. However, while PVX accumulation increased in doubly infected N. tabacum plants compared with singly infected plants, the accumulation of PVX did not vary drastically in doubly infected N. benthamiana plants with respect to single infected ones. These findings suggest that the relationship between viral titre enhancement and synergism in PVX/potyvirus infections is host dependent. Since PVX and potyviruses contain suppressors of a plant antiviral defence system mediated by gene silencing, differences observed in the response of these two related hosts to PVX/potyvirus interactions might reflect the effect of these viruses on host specific antiviral defences.

Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms. Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacterium-mediated transient-expression assay. We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach. We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals. The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not single-stranded RNA. Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation.

We previously reported that Nicotiana benthamiana plants transformed with the wild-type 54-kDa region of the pepper mild mottle tobamovirus, S strain (PMMoV-S), displayed two different resistance responses against PMMoV infection. Some of the transgenic plants exhibited a complete and highly resistant phenotype while the remaining plants showed a delayed resistance (Tenllado et al., 1995, Virology 211, 170--183). Here we show that some of the N. benthamiana plants transformed with a construct expressing a PMMoV-S truncated 54-kDa protein coding sequence also displayed a complete and highly resistant phenotype similar to that shown by the wild-type 54-kDa transgenic plants. This result indicates that the wild-type, full-length 54-kDa protein is not required in mediating the complete resistance phenotype against PMMoV. The remaining truncated 54-kDa transgenic plants were susceptible to PMMoV infection but showed a variable delay in the appearance of symptoms. Unlike the wild-type 54-kDa transgenic plants, which were initially susceptible to the infection but recovered later, the truncated 54-kDa transgenic plants never exhibited this delayed resistance phenotype. However, they displayed a new type of altered symptomatic phenotype. The truncated 54-kDa transgenic lines also exhibited a lower level of transgenic transcripts compared to the wild-type 54-kDa transgenic lines which could account for the absence of the delayed resistance phenotype.

Two Spanish plum pox virus (PPV) isolates, 5.15 and 3.3, were used in transmission experiments involving the aphid vector Myzus persicae, with woody and herbaceous host plants. These isolates differ in the size of their coat protein (CP) and sequence analysis revealed that isolate 3.3 has a 15 amino acid deletion near the N terminus of the CP, affecting the same positions as in a previously reported non-aphid-transmissible PPV isolate from Germany. Aphid transmission experiments showed that isolate 5.15 was transmitted from infected plants whereas isolate 3.3 was not. In contrast, both isolates were readily aphid-transmitted when acquired through artificial membranes from purified virus preparations supplemented with purified helper component (HC) obtained from potato virus Y-infected plants. This indicates that non-transmissibility of isolate 3.3 may be due to a defect in the HC rather than in the CP.

The L3 gene is responsible for the hypersensitive response in Capsicum plants against infection by tobamoviruses. The resistance conferred by this gene is one of the most effective so far described against tobamoviruses. Certain isolates of pepper mild mottle virus (PMMV) are the only tobamoviruses able to overcome the L3 resistance. Chimeric viral genomes between PMMV-S (to which L3 plants are hypersensitive) and PMMV-I (an L3 resistance-breaking isolate) led us to conclude that sequence variation within the coat protein gene of both isolates determines their different virulence in L3L3 plants. Furthermore, the results indicate that a single amino acid substitution, Asn to Met, at position 138 of the PMMV-I coat protein is sufficient to induce the hypersensitive response and localization of viral infection in C. chinense plants. Finally, the use of a mutant coding for a truncated coat protein (maintaining the Met138 coding sequence at the RNA level) demonstrates that a functional coat protein is required for elicitation of the L3 gene-mediated resistance.

The detection for the first time of a plant virus in a single aphid by the high sensitivity polymerase chain reaction (PCR) technology is reported. The application of PCR for the detection of viruses in their vectors will aid the understanding of the complex virus-vector relationship and therefore allow the development of new approaches for control of spread of plant virus diseases.

The entire genomic RNA of a Spanish isolate of pepper mild mottle virus (PMMV-S), a resistance-breaking virus in pepper, was cloned and sequenced and shown to be similar to other tobamoviruses in its genomic organization. It consisted of 6357 nucleotides (nt) and contained four open reading frames (ORFs) which encode a 126K protein and a readthrough 183K protein (nt 70 to 4908), a 28K protein (nt 4909 to 5682) and a 17.5K coat protein (nt 5685 to 6158). This is the first tobamovirus in which none of the ORFs overlap. Both its nucleic acid and predicted protein sequences were compared with the previously determined sequences of other tobamoviruses. The variations and similarities found and their relationship with the pathogenicity of this virus are discussed.

The nucleotide sequences of the 5' and 3' non-coding regions of pepper mild mottle virus strain S (PMMV-S) RNA were determined; they are more like corresponding sequences of tomato mosaic virus (ToMV) RNA than those of any other tobamovirus reported so far. The 5' leader contains a 68 nucleotide guanosine-free sequence which differs in several nucleotides from the corresponding sequences in genomic RNA of tobacco mosaic virus (TMV) and ToMV. The messenger activity of PMMV-S RNA in vitro and the polypeptide translation products made were similar to those of TMV RNA. It therefore seems unlikely that qualitative or quantitative differences in translation in vivo account for the milder symptoms induced by PMMV-S, and its lesser replication, than TMV. The 3' non-coding region of PMMV-S RNA is 199 nucleotides long and can be folded into the same secondary structure as the RNA of other tobamoviruses.