The 5' noncoding region of grapevine chrome mosaic nepovirus RNA-2 triggers a necrotic response on three Nicotiana spp.

The 5' noncoding region (NCR) of grapevine chrome mosaic nepovirus (GCMV) was cloned in a viral vector derived from potato virus X (PVX). The recombinant virus obtained was inoculated to Nicotiana benthamiana, N. clevelandii, and N. tabacum plants. Infected plants developed necrotic symptoms in place of the vein clearing and mosaic typically observed after inoculation with PVX. Northern (RNA) blot analysis showed that the replication of PVX was not specifically altered by the presence of the GCMV 5' NCR. Inoculation of recombinant PVX harboring deleted forms of the GCMV 5' NCR showed that the three stem-loop structures at the 3' end of the 5' NCR (nucleotides 153 to 206) are dispensable for the induction of necrosis. Further deletion analysis indicated that neither the 5'-most 70 nucleotides of the 5' NCR nor the downstream region (nucleotides 71 to 217) alone is able to induce the necrotic symptoms. In the presence of both the sequence encoding the GCMV coat protein and the GCMV 3' NCR, the GCMV 5' NCR failed to induce necrosis in the PVX background. The mechanisms by which the expression of the 5' NCR might modify PVX symptoms are discussed.

Transgenic tobaccos transformed with a gene encoding a truncated form of the coat protein of tomato black ring nepovirus are resistant to viral infection.

Tomato black ring virus (TBRV) belongs to the nepoviruses, an important genus of phytoviruses characterized by isometric particles and by their transmission by longidorid nematodes. As for all other nepoviruses, the coat protein (CP) of TBRV is encoded by the 3' terminal part of the viral RNA2 (positions 2801-4334). A hybrid gene driving the expression of a truncated form of the TBRV CP was constructed. It contains a frameshift deletion at position T4065 so that in the encoded protein the last 90 amino acids of the wild-type CP are replaced by 52 amino acids encoded by a different reading frame of the viral RNA. This hybrid gene was introduced into the genome of Nicotiana tabacum cv 'Xanthi' plants. When compared to control plants, progeny of some transformants expressing the mutated CP gene (CPm+ plants) showed resistance against TBRV infection. This resistance is characterized by a delay in the appearance of symptoms, a reduction in the number of infected plants and a reduction in virus accumulation.

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy. Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission. However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs. Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT. Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.

Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants.

A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intro-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.

High resistance and control of biological risks in transgenic plants expressing modified plum pox virus coat protein.

Transgenic plums transformed with the plum pox virus coat protein (PPV CP) gene displayed a resistance to the sharka disease (Ravelonandro et al., 1997). However, the expression of PPV CP in transgenic plants may lead to complementation of deficient characteristic of an incoming potyvirus. Indeed, an aphid-intransmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) could be transmitted when encapsidated by the engineered PPV CP (Lecoq et al., 1993). To control such a risk, new PPV CP constructs were designed and introduced into Nicotiana benthamiana genome. In the first construct, the DAG amino acid triplet involved in the potyvirus aphid-transmission was deleted. The second construct encoded a truncated PPV CP lacking its first 140 amino acids. In the last construct, the nucleotides encoding the charged amino-acids R220, Q221 and D264 localized in the core of the PPV CP were removed. A bacterial expression system was developed to show that these deletions prevent the assembly of the PPV CP subunits. For each construct, several transgenic lines were produced and first challenged with several strains of PPV. Two phenotypes of resistance were observed: recovery and immunity. Their biochemical characterization showed that the resistance was RNA-mediated and therefore can be classified as homology-dependent (Jacquet et al., 1998a). Resistant lines producing high level of wild type or modified PPV CP were then inoculated with ZYMV-NAT to perform an aphid-transmission assay. Results of these experiments demonstrated that the use of modified forms of PPV CP genes in transgenic plants provide a good way to control the biological risks associated with heteroencapsidation (Jacquet et al., 1998b).

Characterization of phenotype resistance to plum pox of transgenic plums expressing plum pox virus capsid gene.

Resistance to plum pox virus (PPV) infection can be obtained in transgenic plants that express the virus capsid gene. An Agrobacterium-mediated transformation was used to introduce the PPV capsid gene into Prunus domestica plants. Over 11 regenerated plants (clones) were observed for the development of the disease symptoms and analysed for the presence of PPV by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) through 4 dormancy cycles. The level of protection against PPV was determined in the transformed plants, non-transformed plants, and a control transgenic plant "transformed" with the plasmid vector alone. One clone, C-5, appeared fully protected, while PT-6 and C-4 clones accumulated a low concentration of virus and the rest of the clones was entirely susceptible. Little is known about the mechanisms of resistance to virus infection in transgenic woody plants. To investigate this aspect, comparative studies based on the characteristics of resistant and susceptible clones have been started. A question, whether the phenotype resistance of clone C-5 is similar to that observed in transgenic herbaceous plants or not, has been addressed. Recent progress in this investigation is presented.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus.

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

Biological and molecular variability of lettuce mosaic virus isolates.

ABSTRACT Lettuce mosaic potyvirus (LMV) causes severe disease of commercial lettuce crops. LMV isolates show wide biological variability, particularly in their ability to overcome the resistance genes described in Lactuca sativa. For a better understanding of the molecular interaction between lettuce and LMV, biological and molecular characterization of a collection of 10 LMV isolates known to differ in virulence or aggressiveness was performed. The ability of these isolates to overcome the resistance genes was reevaluated under standardized conditions. To study the molecular variability of LMV, an immunocapture-reverse transcription-poly-merase chain reaction technique, coupled with direct sequencing, was used to obtain nucleotide sequence data from three short regions of the LMV genome. Clustering analysis was performed and compared to the biological properties of the 10 isolates. Three groups of LMV isolates were discriminated based on the molecular data. These groups appear to correlate with the geographic origin of the isolates rather than with their pathogenicity. Sequence comparison with California isolates clearly showed that the California isolates are related to the western European isolates, raising the possibility of past exchanges of LMV between western Europe and California.

Comparison of the complete nucleotide sequences of two isolates of lettuce mosaic virus differing in their biological properties.

The complete nucleotide sequences of the genomic RNAs of the 0 and E isolates of lettuce mosaic potyvirus (LMV) have been determined. These two isolates differ by their behavior towards two lettuce resistance genes and by their seed transmission properties. LMV-0 is unable to induce disease in lettuce carrying either one of the mol1 and mol2 recessive resistance genes, whereas LMV-E is able to induce disease in the same plants. The genomes of these two isolates are 10080 nucleotides (nt) in length, excluding the poly(A) tract, and encode polyproteins of 3255 amino acids (aa). The open reading frame is flanked by a 5' non-coding region of 103 nt and a 3' non-coding region of 212 nucleotides. Ten proteins were predicted. The P3 protein, with 377 aa, is the longest potyviral P3 protein characterized to date while the P1 protein, with 437 aa, is among the longest P1 proteins reported. Sequence comparisons between the two isolates demonstrated only limited sequence difference. The overall nucleotide and amino acid sequence identities between LMV-0 and LMV-E are 94 and 97% respectively. The greatest variability occurs in the P1 and in the variable N-terminal region of the coat protein, while the NIa protease domain, the NIb protein, the C-terminus of the helper component protease and the 3' non-coding region are extensively conserved. While this sequence analysis does not allow direct identification of determinants involved in the resistance breaking or in the seed transmissibility properties, these data are a first step towards the characterization of these determinants.

An RNA-dependent-RNA-polymerase activity associated with grapevine chrome mosaic nepovirus infection.

A virus-induced, viral RNA-specific, RNA-dependent-RNA-polymerase activity has been observed in vitro associated with membrane extracts of plants of three different species after infection with grapevine chrome mosaic nepovirus (GCMV). The products of this activity are full-length, positive sense GCMV RNAs present in double-stranded structures. As has been demonstrated for a relative to nepoviruses, cowpea mosaic comovirus, this activity probably corresponds to the nepoviral replication complex.

Complete nucleotide sequence of the genome of a severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV).

The genome of the Balaton 1 severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV-Bal1) has been cloned and sequenced. The genomic RNA is 7549 nucleotide long, excluding the poly A tail. The genomic organization, with three overlapping open reading frames (ORF), is similar to that of the other sequenced ACLSV isolates. Sequence comparisons indicate a high variability between ACLSV isolates, with overall nucleotide sequence homology levels between 76 and 82%. The coat protein, encoded internally inside a larger ORF, is the most conserved protein (identity levels between 87 and 93%) while the central ORF, encoding the putative movement protein, is the most divergent (77 to 85% identity).

Resistance of Transgenic Prunus domestica to Plum Pox Virus Infection.

Transgenic plum trees (Prunus domestica) containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV by aphid feeding or chip budding. Infection was monitored by evaluation of virus symptoms, DAS-ELISA, and immunoblot assays. Based on observations and analyses over 3 years including two dormancy cycles, one out of five transgenic clones (C-5), was found to be resistant to infection whether inoculated by aphids or by chip budding. PPV could not be detected in any inoculated plants of the C-5 clone by immunoblot or immunocap-ture-reverse transcriptase-polymerase chain reaction assays. To our knowledge, this is the first P. domestica clone resistant to PPV infection produced by genetic engineering.

Frequent occurrence of recombinant potyvirus isolates.

We have performed a systematic search for recombination in the region encoding coat protein and the 3' non-translated region in natural isolates of potyviruses, the largest group of plant RNA viruses. The presence of recombination, and the localization of the cross-over points, were confirmed statistically, by three different methods. Recombination was detected or suspected in 18 out of 109 potyvirus isolates tested, belonging to four out of eight virus species, and was most prevalent in potato virus Y, clear in bean common mosaic virus, and possible in bean yellow mosaic and zucchini yellow mosaic viruses. Recombination was not detected in the four other potyvirus species tested, including plum pox virus, despite the availability of numerous sequences for this last species. Though it was not specifically researched, no evidence for inter-specific recombination was found. For several reasons, including the fact that only a minor portion of the genome was analysed, the above figures certainly represent an underestimate of the extent of recombination among isolates of potyviruses, which might thus be a common phenomenon.

In vitro translation of apple chlorotic leaf spot virus (ACLSV) RNA.

The genomic RNA of apple chlorotic leaf spot virus was translated in a rabbit reticulocyte lysate system, yielding a large, 190 K product as well as several other polypeptides of smaller size (60, 56, 22 and 15 kDa). The 22 kDa product was immunoprecipitated by an anti-ACLSV serum and comigrated with purified ACLSV coat protein. In vitro translation of RNA transcripts prepared from cloned ACLSV cDNAs demonstrates that the coat protein is synthesised by initiation on the second in frame AUG codon of the 28 kDa open reading frame located at the 3' end of the genome. In the in vitro translation system used, the ability of various ACLSV-derived RNAs to direct the synthesis of the coat protein appears to be the result of initiation on the internal AUG codon.

The nucleotide sequence of the RNA-2 of an isolate of the English serotype of tomato black ring virus: RNA recombination in the history of nepoviruses.

The RNA-2 of a carrot isolate from the English serotype of tomato black ring nepovirus (TBRV-ED) has been sequenced. It is 4618 nucleotides long and contains one open reading frame encoding a polypeptide of 1344 amino acids. The 5' non-coding region contains three repetitions of a stem-loop structure also conserved in TBRV-Scottish and grapevine chrome mosaic nepovirus (GCMV). The coat protein domain was mapped to the carboxy-terminal one-third of the polyprotein. Sequence comparisons indicate that TBRV-ED RNA-2 probably arose by an RNA recombination event that resulted in the exchange of the putative movement protein gene between TBRV and GCMV.

Transfer of the 3' non-translated region of grapevine chrome mosaic virus RNA-1 by recombination to tomato black ring virus RNA-2 in pseudorecombinant isolates.

In grapevine chrome mosaic and tomato black ring viruses (GCMV and TBRV), as in many other nepoviruses, the 3' non-translated regions (3'NTR) are identical between the two genomic RNAs. We have investigated the structure of the 3'NTR of two recombinant isolates which contain GCMV RNA-1 and TBRV RNA-2. In these isolates, the 3'NTR of RNA-1 was transferred to RNA-2, thus restoring the 3' identity. The transfer occurred within three passages, and probably contributes to the spread of randomly appearing mutations from one genomic RNA to the other. The site of recombination is near the 3' end of the open reading frame.

A multiple alignment of the capsid protein sequences of nepoviruses and comoviruses suggests a common structure.

The amino acid sequences of the regions encoding the structural proteins of eleven nepoviruses and five comoviruses, two genera of the family Comoviridae, have been aligned. The properties predicted by computer analysis (three-dimensional-3D-structure, hydrophobicity) are also correlated along this alignment, and aligned to the experimentally determined 3D structure of two comoviruses. It can thus be assumed that the 3D structure of the unique nepovirus coat protein matches that of the bipartite protomer found in the comovirus particles. In this model, the spatial locations of two amino-acid motifs characteristic of nepoviruses are in close vicinity, at the external surface of the virion. The coat proteins of nepoviruses and comoviruses may thus share a common evolutionary origin. A phylogenetic analysis was made using the multiple alignment, allowing a better understanding of the molecular relationships between these two groups of viruses.

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene.

Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and ß-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.