1 INRA/ENSA, Unité Mixte de Recherche Biologie des Organismes et des Populations appliquée à la Protection des Plantes (BiO3P), Domaine de la Motte BP 35327, F-35653 Le Rheu cedex, France
2 FNPPPT (Fédération Nationale des Producteurs de Plants de Pomme de Terre), 9 rue d'Athènes, F-75009 Paris, France
Correspondence
Emmanuel Jacquot
Emmanuel.Jacquot{at}rennes.inra.fr
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ABSTRACT |
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Analysis of the PVYN-605 sequence (GenBank accession no. X97895) allows the identification of unique restriction sites in the P1/HC-Pro/P3 region. Of them, the BssHII, NruI, BstZ17I and BglII sites, located at nucleotide positions 421, 2086, 2591 and 2763, respectively, were used to create PVYN/OBsBg, PVYN/ONrBg, PVYN/ONrBz and PVYN/OBzBg (Fig. 2). These genetically modified PVY genomes were produced in two successive cloning steps. Firstly, the PVYN sequence to be replaced, bordered by two unique restriction sites, was deleted by a digestion/ligation process from the original N-605 infectious clone and replaced by a short linker sequence to produce a deleted version of the PVYN-605 clone. Secondly, the homologous sequence from PVYO-139 was amplified by RT-PCR using viral RNA extracted from O-139-infected N. tabacum and a degenerate primer pair. The latter contained restriction sites allowing the cloning of the PCR fragment into the deleted N-605 genome. This sequential cloning procedure provides full-length recombinant plasmids corresponding to PVYN/O chimeric constructions. These constructions were sequenced to check the precise exchange between PVYN and PVYO sequences at the selected restriction sites and the absence of mutations in the PVYO sequence, which could potentially be introduced during the PCR steps. The PVYN/ONrBg, PVYN/ONrBz and PVYN/OBzBg constructs were inoculated biolistically onto N. clevelandii (Fig. 2
). Considerable variation in the efficiency of the biolistic-mediated inoculations of the PVYN/O constructs (from 1/13 to 13/14) was observed in our experiments. Similar results were obtained with the wild-type infectious N-605 clone. Indeed, the mean of N-605-infected N. clevelandii (34·65 %; 35/101) results from six independent inoculation experiments that were associated with infection efficiencies ranging from 15 to 75 % (2/13 to 9/12). Surprisingly, the PVYN/OBsBg genome was not infectious on N. clevelandii, as denoted by the negative ELISA results obtained with the 77 biolistically inoculated plants that resulted from seven independent experiments, each performed with eight to 13 test plants. As N. clevelandii was used only to establish infection from PVY infectious clones, a single infected N. clevelandii for each chimera was sufficient to produce the required amount of infectious viral particles. Crude sap from infected N. clevelandii was used in mechanical inoculations of young N. tabacum cv. Xanthi plants (three- to four-leaf stage). Three weeks post-inoculation, all plants were tested successfully for virus infection by ELISA assays and monitored for leaf symptoms. For each tested construct, the HC-Pro/P3 nucleotide sequence of the viral progeny produced in infected N. tabacum cv. Xanthi was determined and compared to the PVY sequence in the inoculated plasmid. No differences were found. As expected, PVYN-605 and PVYO-139 induce TVN and mosaic symptoms on Xanthi, respectively. From the three chimeric PVY tested, only PVYN/OBzBg induced TVN. The inability of PVYN/ONrBg and PVYN/ONrBz to induce necrosis on N. tabacum cv. Xanthi illustrates the significance of the PVYN-605 region between nt 2087 and 2591 in the induction of the TVN symptom. Sequence comparison between N-605 and O-139 revealed that this region possesses 83·65 and 94·67 % similarity at the nucleotide (not illustrated) and encoded amino acid (Fig. 3
) levels, respectively. The ability/inability to induce TVN should be supported by at least one of nine PVYN-605/PVYO-139 differential residues identified. However, when the protein sequence comparison was completed by using data collected from five PVYN isolates [sc (GenBank accession no. AY691550), 607 (AY691551), B203 (AY691552), Wi-P (AF248500) and N242 (AF248499), from Scotland, the Netherlands, Spain, Poland and France, respectively] and three PVYO isolates [sc (GenBank accession no. AY691546), 702 (AY691547) and B4 (accession no. AY691548), from Scotland, the Netherlands and France, respectively], only the HC-Pro residues at locations 400 (nt 22122214) and 419 (nt 22692271) were restricted to necrotic (K400 and E/G419) or non-necrotic (R400 and D419) isolates. The SpeI restriction site (position 2257) located between K400 and E419 codons in the N-605 sequence was used to produce the chimeric PVYN/ONrSp and PVYN/OSpBz constructs (Fig. 2
). These two mutants were infectious in N. clevelandii (Fig. 2
); however, neither of them was able to induce necrosis symptoms on N. tabacum cv. Xanthi. These results denote the requirement of at least one residue in both the NruISpeI and SpeIBstZ17I regions in the TVN process. Taken together with the sequence alignment (Fig. 3
), this work suggests that K400 and E419 in the PVY HC-Pro sequence support the TVN property of the PVYN-605 isolate. Point mutations corresponding to A/G2213 and A/C2271 were introduced in the N-605 infectious clone sequence by using degenerate primers and the megaprimer cloning strategy (Ke & Madison, 1997
) to produce the PVYN/O-KE/RD clone (Fig. 2
). When tested on N. tabacum, PVYN/O-KE/RD induced mosaic symptoms, indicating that these specific amino acid changes in the N-605 HC-Pro protein are sufficient to change the phenotype of PVYN-605.
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ACKNOWLEDGEMENTS |
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Received 26 January 2005;
accepted 13 April 2005.
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