Contribution of Ldace1 gene to acetylcholinesterase activity in Colorado potato beetle.

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.

RNAi of ace1 and ace2 in Blattella germanica reveals their differential contribution to acetylcholinesterase activity and sensitivity to insecticides.

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.

Characterization of the recombinant forms arising from a Potato virus X chimeric virus infection under RNA silencing pressure.

Recombination is a frequent phenomenon in RNA viruses whose net result is largely influenced by selective pressures. RNA silencing in plants acts as a defense mechanism against viruses and can be used to engineer virus resistance. Here, we have investigated the influence of RNA silencing as a selective pressure to favor recombinants of PVX-HCT, a chimeric Potato virus X (PVX) vector carrying the helper-component proteinase (HC-Pro) gene from Plum pox virus (PPV). All the plants from two lines expressing a silenced HC-Pro transgene were completely resistant to PPV. However a significant proportion became infected with PVX-HCT. Analysis of viral RNAs accumulating in silenced plants revealed that PVX-HCT escaped silencing-based resistance by removal of the HC-Pro sequences that represented preferential targets for transgene-promoted silencing. The virus vector also tended to lose the HC-Pro insert when infecting transgenic plants containing a nonsilenced HC-Pro transgene or wild-type (wt) Nicotiana benthamiana plants. Nevertheless, loss of HC-Pro sequences was faster in nonsilenced transgenic plants than in wt plants, suggesting the transgene plays a role in promoting a higher selective pressure in favor of recombinant virus versions. These results indicate that the outcome of recombination processes depends on the strength of selection pressures applied to the virus.

Host-dependent differences during synergistic infection by Potyviruses with potato virus X.

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.

Transient expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing.

Specific post-transcriptional gene silencing (PTGS) of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA (dsRNA) molecules. In plants, PTGS is part of a defense mechanism against virus infection. We have previously shown and patented that direct delivery to nontransgenic plants of dsRNA derived from viral sequences specifically interfere with virus infection. Here, we show that transient expression of constructs encoding hairpin RNA homologous to a rapidly replicating plant tobamovirus also interferes with virus multiplication in a sequence-dependent manner. A three-day lag period between delivery of hairpin RNA and virus into the same tissues completely block virus infectivity. Several hallmarks characteristic of PTGS were associated with viral interference mediated by hairpin RNA: high level of sequence identity between the hairpin RNA and the target RNA, presence of siRNAs in extracts derived from leaves infiltrated with hairpin RNA, and helper component-proteinase (HC-Pro) of potyviruses, a suppressor of PTGS, overcame interference. No evidence for a mobile silencing suppression signal induced by transient expression of HC-Pro was observed. The approach described here has the potential to be used as a versatile tool for studying the onset of PTGS in cases involving virus infection, in opposition to dsRNA-transgenic plants, which allow primarily for the study of PTGS maintenance.

Amino acid substitutions within the Cys-rich domain of the tobacco etch potyvirus HC-Pro result in loss of transmissibility by aphids.

We examined the role of several amino acid residues located at the N-terminus of the tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro) in virus transmissibility by aphids. Site-directed mutagenesis resulted in changes affecting amino acids that appear highly conserved among a number of potyviruses. The TEV HC-Pro amino acid residues Gly343, Val345, Ala346, Ile348, Pro355, Lys358, and Ile359 were arranged within a Cys-rich domain in a region dispensable for TEV infectivity. Two HC-Pro mutants (TEV-P355R and -K358N) exhibited a drastically reduced rate of aphid transmission whereas other mutants (TEV-G343D, -V345E, -A346H, -I348D, and -P355L) were completely unable to be aphid transmitted. In contrast, the I359M mutation had no effect on aphid transmissibility of TEV. This lack of transmissibility did not appear to be due to large differences in the amounts of both coat protein (CP) and HC-Pro in infected tobacco plants. Our results indicated that these amino acid residues likely play a highly conserved role in aphid transmission among potyviruses.

Double-stranded RNA-mediated interference with plant virus infection.

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.

Helper component mutations in nonconserved residues associated with aphid transmission efficiency of a pepper isolate of potato virus y.

ABSTRACT The aphid transmission properties of a pepper isolate of potato virus Y belonging to the pathotype 1-2 (PVY 1-2) have been characterized. PVY 1-2 was not transmitted in plant-to-plant experiments, although purified virus particles were efficiently transmitted when supplemented with heterologous helper component (HC) of the transmissible isolate PVY 0 AT through membrane acquisition assays, indicating that its coat protein was functional in transmission. Additionally, virions of PVY 1-2 were able to bind to different HCs in in vitro binding assays. Analysis of the sequence of the PVY 1-2 HC gene and comparison with that of PVY 0 AT revealed 19 nucleotide differences, but only 2 resulted in amino acid changes, one of which induced a change of charge. Neither of these two amino acid changes occurred within the cysteine-rich domain, nor did they coincide with conserved motifs of the HC protein known to be involved in aphid transmission and which are present in all known potyvi-ruses. However, both changes are located in positions highly conserved among PVY strains. The possible role of both mutations on the activity of the PVY 1-2 HC in aphid transmission is discussed.

Serological characterization of Hungarian plum pox virus isolates.

Three Hungarian (no. 2, 4 and 9), and a Moldavian (K) plum pox virus isolates were compared with a characterized Spanish isolate (5.15) by RT-PCR, ELISA, dot-blot and Western blot analysis. Monoclonal antibodies prepared against the external, intermediate and internal sequences of the coat protein of the Spanish isolate were able to differentiate the four isolates. Hungarian isolate No. 2 proved to be serologically identical to the Spanish isolate, while No. 4 showed appreciable differences and No. 9 could be recognized only by the monoclonal antibodies representing the intermedial and internal parts of the coat protein. K isolate showed a more distant relationship to other isolates. Our experiment provided the first demonstration of the presence of D type isolates in Hungary.

The coat protein is required for the elicitation of the Capsicum L2 gene-mediated resistance against the tobamoviruses.

In Capsicum, the resistance against tobamoviruses conferred by the L2 gene is effective against all but one of the known tobamoviruses. Pepper mild mottle virus (PMMoV) is the only virus which escapes its action. To identify the viral factors affecting induction of the hypersensitive reaction (HR) mediated by the Capsicum spp. L2 resistance gene, we have constructed chimeric viral genomes between paprika mild mottle virus (PaMMV) (a virus able to induce the HR) and PMMoV. A hybrid virus with the PaMMV coat protein gene substituted in the PMMoV-S sequences was able to elicit the HR in Capsicum frutescens (L2L2) plants. These data indicate that the sequences that affect induction of the HR mediated by the L2 resistance gene reside in the coat protein gene. Furthermore, a mutant that codes for a truncated coat protein was able to systemically spread in these plants. Thus, the elicitation of the host response requires the coat protein and not the RNA.

Resistance to pepper mild mottle tobamovirus conferred by the 54-kDa gene sequence in transgenic plants does not require expression of the wild-type 54-kDa protein.

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.

Transmission by aphids of a naturally non-transmissible plum pox virus isolate with the aid of potato virus Y helper component.

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.

Nicotiana benthamiana plants transformed with the 54-kDa region of the pepper mild mottle tobamovirus replicase gene exhibit two types of resistance responses against viral infection.

Nicotiana benthamiana plants transformed with the 54-kDa region of the pepper mild mottle tobamovirus (PMMV) replicase gene were generated and six independently transformed plant lines were analyzed for resistance to PMMV. Two different resistance responses were obtained. Some of the transgenic plants from only two lines showed a preestablished, complete, and highly resistant phenotype since no viral symptoms were observed, although a low level of virus replication occurred. The remaining plants from these two lines and all of the plants from the other four lines tested showed a delayed, induced, and also highly resistant phenotype since they were susceptible early, but were able to recover from the systemic PMMV infection. Recovered, symptomless leaves were resistant to the PMMV strain from which the 54-kDa gene was derived and to a closely related strain but not to tobacco mosaic virus. Such a delayed resistance phenotype has not been previously described for any plant expressing viral replicase sequences. The transgenic plants within the lines displaying complete or delayed resistance phenotypes were analyzed for transgene expression before and after PMMV inoculation and the two types of resistance responses were shown to be independent of the transgene transcript level.

The Capsicum L3 gene-mediated resistance against the tobamoviruses is elicited by the coat protein.

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.

Rapid detection and differentiation of tobamoviruses infecting L-resistant genotypes of pepper by RT-PCR and restriction analysis.

A procedure involving reverse transcription followed by polymerase chain reaction (RT-PCR) was developed for typing pathotypes of the tobamoviruses infecting the L-resistant genotypes of pepper. The method provides a much simpler alternative to the bioassay tests for the different Capsicum spp. genotypes previously used. Discrimination between the two pathotypes, P1,2 and P1,2,3, which cannot be differentiated by serological means, was achieved by restriction enzyme analysis of the PCR products. The assay also detects and distinguishes both pathotypes in a single mixed-infected plant. The procedure should be useful for the diagnosis and control of the disease and helpful to breeders and biotechnologists when producing and evaluating resistance in pepper plants.

Tomato necrosis and the 369 nucleotide Y satellite of cucumber mosaic virus: factors affecting satellite biological expression.

To determine which factors can affect biological expression of the Y satellite RNA of cucumber mosaic virus (CMV) in tomato, three laboratories collaboratively exchanged their natural satellite variants, the corresponding recombinant DNA clones and helper virus strains, as well as tomato varieties, on which different observations previously reported were based. The effects of these materials and the influence of temperature on symptom expression were systematically studied. The results show that in a standardized tomato bioassay at 24 degrees C, the Y satellite, when supported by either CMV-1 or CMV-Y, did not induce tomato necrosis in the Rutgers variety but elicited a slower necrotic response in the Best of All variety that was variably lethal, as compared to the faster inevitably lethal response induced by a prototype necrogenic D satellite variant in both tomato varieties. At higher temperatures (26.5 to 32 degrees C) an extremely fast-killing necrosis caused by CMV-Y itself was observed. The study demonstrates that in experiments on virus symptom modulation induced by CMV satellites, the nature of the helper virus, host plant varieties, as well as the environmental conditions should be precisely defined, and the effects of each parameter change determined separately.

The nucleotide sequence of the coat protein genes and 3' non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses.

The nucleotide sequence of the coat protein genes and 3' non-coding regions of two different resistance-breaking tobamoviruses in pepper have been determined. The deduced coat protein of an Italian isolate of pepper mild mottle virus (PMMV-I) consists of 156 amino acids and its 3' non-coding region is 198 nucleotides long. They have been found to be very similar in sequence and structure to those previously reported for a Spanish isolate (PMMV-S). In contrast, a Dutch isolate termed P 11 codes for a coat protein of 160 amino acids and its 3' non-coding region is 291 nucleotides long, which may have arisen by duplication. The nucleotide and the predicted coat protein amino acid sequence analysis show that this isolate should be considered as a new virus within the tobamovirus group. The term paprika mild mottle virus (PaMMV) is proposed.

Detection of cauliflower mosaic virus (CaMV) in single aphids by the polymerase chain reaction (PCR).

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.

Morphogenesis of African swine fever virus in monkey kidney cells after reversible inhibition of replication by cycloheximide.

The late cytoplasmic phases of African swine fever virus (ASFV) morphogenesis in monkey kidney cells have been studied by transmission electron microscopy, focusing attention on the synthesis of viral envelopes. Morphogenesis was studied after reversible cycloheximide blockage of monkey kidney cells infected with ASFV. ASFV appears to synthesize its external and internal envelopes within the cellular cytoplasm, at the same time as the capsid is formed, with intracellular and extracellular virions showing similar structure and polypeptide composition.

Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper.

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.