versão On-line ISSN 1678-4677
Fitopatol. bras. v.26 n.1 Brasília mar. 2001
ALICE K. INOUE-NAGATA1*, M. ESTHER N. FONSECA1, TATIANA O.T.A. LOBO1, ANTÔNIO C. DE ÁVILA2 & DAMARES C. MONTE1
1Embrapa Recursos Genéticos e Biotecnologia, Cx. Postal 02372, CEP 70770-900, Brasília, DF, fax: (61) 340-3624, e-mail: email@example.com; 2Embrapa Hortaliças, Cx. Postal 0218, CEP 70359-970, Brasília, DF
(Accepted for publication on 05/12/2000)
Corresponding author: Alice Kazuko Inoue-Nagata
Two Brazilian Potato virus Y (PVY) isolates were biologically characterized as necrotic (PVY-NBR) and common (PVY-OBR) based upon symptoms on test plants. Additional characterization was performed by sequencing a cDNA corresponding to the 3' terminal region of the viral genome. The sequence consisted of 195 nucleotides (nt) coding part of the nuclear inclusion body b (NIb) gene, 804 nt of the coat protein (CP) gene, and 328 nt (PVY-OBR) or 326 nt (PVY-NBR) of the 3'-untranslated region (UTR). Translation of the sequence resulted in one single open reading frame with part of the NIb and a CP of 267 amino acids. The two isolates shared 95.1% similarity in the CP amino acid sequence. The CP and the 3'-UTR sequence of the Brazilian isolates were compared to those of other PVY isolates previously reported and unrooted phylogenetic trees were constructed. The trees revealed a separation of two distinct clusters, one comprising most of the common strains and the other comprising the necrotic strains. PVY-OBR was clustered in the common group and PVY-NBR in the necrotic one.
Key words: PVY, Potyvirus, phylogenetic analysis, taxonomy, coat protein.
Análise da seqüência de nucleotídeos da capa proteica e da região 3' não codificadora de dois isolados brasileiros de Potato virus Y
Dois isolados do vírus Y da batata (Potato virus Y, PVY) foram caracterizados biologicamente como pertencentes às estirpes necrótica (PVY-NBR ) e comum (PVY-OBR) com base nos sintomas induzidos em plantas-teste. Caracterização adicional foi realizada com a determinação da seqüência de nucleotídeos do cDNA correspondente à extremidade 3' do genoma viral. A seqüência obtida continha 195 nucleotídeos (nt) que codificam uma parte do gene da inclusão nuclear b (NIb), 804 nt da capa proteíca (CP) e 328 nt em PVY-OBR e 326 nt em PVY-NBR da região 3' não traduzida (UTR). A análise da seqüência mostrou ser composta por uma única fase aberta de leitura contendo parte do NIb e a CP consistindo de 267 aminoácidos. Os dois isolados apresentaram uma similaridade de 95,1% na seqüência de aminoácidos da CP. A CP e a seqüência 3'-UTR dos isolados brasileiros foram comparados com as seqüências de outros isolados de PVY previamente relatados e árvores filogenéticas foram construídas. As árvores mostraram a delimitação de dois grupos distintos, um contendo a maioria das estirpes comuns e o outro com as estirpes necróticas. PVY-OBR foi agrupado no grupo comum e PVY-NBR no necrótico.
Potato virus Y (PVY) is the type species of the genus Potyvirus from the family Potyviridae. This virus causes serious losses in many important crops worldwide, especially in solanaceous plants such as potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), and tobacco (Nicotiana tabacum L.). Three PVY strains are recognized by their distinct host responses: the common strain (PVYO), which causes mosaic and mottling symptoms in N. tabacum plants; the tobacco veinal necrosis strain (PVYN), causing necrotic symptoms in N. tabacum; and the stipple streak strain (PVYC), characterized by the hypersensitive reaction caused in many potato cultivars (De Bokx & Huttinga, 1981). Recently, a new PVY isolate causing the potato tuber necrotic ring disease (PTNRD) was reported as caused by a new variant of PVY, PVYNTN (Beczner et al., 1984; Le Romancer et al., 1994). Under Brazilian conditions, the occurrence of PVYO and PVYN strains (based upon reaction on potato and tobacco plants) are widely reported (Silberschmidt et al., 1954; Montenegro et al., 1968; Alexandre & Barradas, 1982).
The nucleotide or amino acid sequence of the viral genome has been especially important to classify the species and strains of the Potyvirus genus (Van der Vlugt et al., 1993). The availability of this information is also a prerequisite for the success of new control measures, e.g. methods based on transgenic resistant plants expressing viral genes (Tennant et al., 1994). Many of the transgenic approaches are based upon specific interactions between the transgene and the predominant virus in a given environment, which underlines the need for sequencing local virus strains.
Here, two Brazilian PVY isolates from potato were studied and the nucleotide sequence of part of the inclusion body b (NIb) gene, the coat protein (CP) gene and the 3'-untranslated region (UTR) were determined. The taxonomical position of these Brazilian PVY isolates are discussed in this report.
MATERIALS AND METHODS
Thy PVY isolates, PVY-OBR and PVY-NBR, used in this study were originally collected from potato in the Federal District region more than 15 years ago. Since their collection, they were maintained in the greenhouse of Embrapa Vegetables in N. tabacum TNN plants by mechanical inoculation using 0.01M potassium phosphate buffer, pH 7.0, with 0.01M sodium sulfite as inoculation buffer.
The PVY isolates were mechanically inoculated to 14 test plants (Table 1). Inoculated plants were incubated under greenhouse conditions for symptom development up to 30 days after inoculation.
cDNA Cloning and sequencing of the viral 3' terminal region
Total RNA was extracted from infected N. tabacum TNN plants using Tri Reagent RNA extraction solution (Sigma). The total RNA was then used for cDNA synthesis primed with oligo d(T) (T-primed first-strand kit, Pharmacia). PCR was performed using Taq polymerase (GIBCO-BRL) and the primers PY1 and PY2 (Table 2). The amplified DNA fragments amplified of ca. 1.330 bases were cloned in pGEM-T vector (Promega) following recommendation of the manufacturer. The nucleotide sequence was determined by automated sequencing using vector (T7 and SP6) and internal (PY3, PY4, and PY5) primers (Table 2).
Nucleotide sequence analysis
The nucleotide sequences were compiled and analyzed using programs of the University of Wisconsin Genetics Computer group (GCG, Madison, USA) (Devereux et al., 1984). PVY sequences were fetched from the GenBank and referred to as the locus name (Table 3), except for PVYNNL (Van der Vlugt et al., 1989), PVYOH (Ohshima et al., 1991) and PVYTH (Hataya et al., 1990). The sequences were aligned and Clustal W (http://www2.ebi.ac.uk/clustalw) was used for phylogenetic tree construction, which was viewed by the program Treeview (http://taxonomy.zoology.gla.ac.uk/rod/treeview).
Host response to PVY inoculation
PVY-OBR and PVY-NBR were originally isolated from potato fields in Brazil and maintained in N. tabacum plants. When inoculated into test plants, they showed typical symptoms of the common and necrotic strains, respectively (Table 1). Datura stramonium and pepper plants were not infected with either isolates and only PVY-OBR caused local lesions in C. amaranticolor and C. quinoa. Nicotiana tabacum TNN showed distinct symptoms. PVY-NBR caused necrotic spots and veinal necrosis, while PVY-OBR caused vein clearing, mottling, and pearl spots. Physalis floridana infected with PVY-OBR showed more severe symptoms than when inoculated with PVY-NBR. PVY-OBR appeared to be more severe in S. tuberosum, with systemic necrosis in cv. Achat and mosaic in cv. Bintje. The infection of the test plants was confirmed by DAS-ELISA using a polyclonal antibody (Laboratory of Virology, Embrapa Vegetables) against PVY .
Nucleotide sequence analysis of the 3'end of the PVY genome
PCR amplification was directed to the conserved region in the nuclear inclusion body b (NIb) gene approximately 200 bases upstream of the CP gene, and to the last 15 bases of the 3'-UTR, which were completely conserved among all the PVY sequences analyzed. Amplified PCR products were cloned and the sequence of approximately 1.330 nucleotides was determined for both strains using two clones from different PCR amplifications. The comparison of the nucleotide sequence of both isolates, revealed 87.6% of identity to each other including the NIb, CP and the 3'-UTR (Figure 1). The amplified region of the NIb gene and the CP gene of both isolates were of the same size (nucleotides 1 to 195 for NIb and 196 to 999 for CP). However, the PVY-OBR 3'-UTR (nucleotides 1000 to 1327) was two nucleotides longer than of PVY-NBR (nucleotides 1000 to 1325). The PVY-OBR and PVY-NBR nucleotide sequence were deposited in the GenBank under the accession numbers AF255659 and AF255660, respectively.
Translation of the CP gene resulted in 267 amino acids for both isolates sharing 95.1% similarity. The amino acid sequences of 35 different PVY isolates were aligned and compared to the Brazilian isolates (data not shown). Most of the heterogeneity was found in the amino terminal region. The similarity between PVY-OBR and the other isolates varied from 94.4 to 99.2%. The highest similarity was found with PVY223594 (isolate 0803) and PVY223595 (isolate 0854), both common strains from Switzerland. PVY-NBR showed amino acid similarity from 93.3 to 99.7% when compared to other isolates. The highest degree of similarity with PVY-NBR was found with PVYCPNYSA (a necrotic isolate from Poland) and PVYU91747 (a necrotic isolate from North America).
A phylogenetic tree was constructed based on the alignment and displayed as an unrooted tree (Figure 2). PVY-OBR was clearly clustered with other common isolates, with the exception of two necrotic isolates (PVYNXX and PVYCPWILG). PVY-NBR was clustered with typical necrotic isolates and the two PVYNTN isolates (PVYGCP and PVYPOLYP). A potato isolate from Russia (PVYCAPSI) was isolated in a separate branch close to the PVYN cluster, as well as the common isolate from Argentina (PVYCOAT) and a tobacco isolate from Chile (PVYCAPA), both closer to the PVYO cluster. Pepper and tobacco isolates formed a distinct branch including two tobacco isolates from the USA (PVYCAPD and PVYCAPE) and the causal agent of pepper veinal necrosis (PVYU10378).
The 3'-UTR nucleotide sequence of the Brazilian isolates was aligned with 18 available PVY sequences and used for construction of an unrooted phylogenetic tree (Figure 3). The consensus sequence found throughout the alignment (data not shown) formed two well defined groups, which were visualized in the phylogenetic tree (Figure 3). The first group comprised all PVYO-like isolates, except for PVYNXX, the necrotic strain from France (Robaglia et al., 1989), and included PVYNTN (PVYPOLYP), PVY from pepper (PVYU10378), a PVYN from Poland (PVYCPWILG), an unclassified PVY (PVYPOLYPR) and the PVYN from Russia (PVYCAPSI). The second group comprised the PVYN isolates and PVYNXX. The sequence of PVYNXX showed high identity with the PVYO group in the first 90 nucleotides and with PVYN for the remaining sequence (data not shown). PVY-OBR showed 98.2 to 99.4 % identity within the PVYO group. The highest values were found with PVYPOLYP, PVYU10378, and PVYCPWILG. The identity ranged from 85.6 to 86.5% with the PVYN group and was 89.0% with PVYNXX. The identity of PVY-NBR within the PVYN group ranged from 97.3 to 98.8% and was 94.4% with PVYNXX. The highest values were found with the necrotic isolates from Switzerland (PVYGEN) and New Zealand (PVYCPA). The identity ranged from 85.2 to 86.5% with the PVYO group. The two groups showed a high degree of homogeneity within the group and the variability between the two groups was found in stretches of one to three nucleotides scattered throughout the sequence (data not shown).
The host range and nucleotide sequence of the Brazilian PVY-OBR and PVY-NBR isolates confirmed their inclusion in the PVYO and PVYN group, respectively. A comparison of the amino acid sequence of the CP with other PVY isolates showed that PVY-OBR was closely related to European isolates than the PVYO from Angentina (Bravo-Almonacid & Mentaberry, 1989). Regarding the 3'-UTR, PVY-OBR was closer to necrotic or pepper isolates from Central Europe. PVY-NBR, on the other hand, showed a higher correlation with the necrotic isolates from North America by CP comparison and with the necrotic isolates from Switzerland by 3'-UTR comparison. The lower similarity of the CP sequence from the Brazilian isolates with those from South American, PVYCOAT from Argentina (95.9% with PVY-OBR and 93.3% with PVY-NBR) and PVYCAPA from Chile (96.3% with PVY-OBR and 94.0% with PVY-NBR), is well justified by the high rate of seed potato importation from Canada and Europe (SECEX/DECEX, 1999). This result confirmed that seed potato importation constitutes an easy entrance of new viruses or viral isolates into the country (Figueira et al., 1998), which may quickly spread throughout potato production areas.
The analysis of the phylogenetic trees from the CP and 3'-UTR sequence revealed two distinct clusters, one comprising most of the PVYO sequences and the other with PVYN ones. In the CP analysis, the two clusters showed a good correlation with the biological properties of the isolates, with the exception of PVYNXX and PVYCPWILG, two necrotic isolates grouped in the PVYO cluster. This tree separated the potato strain from Russia (PVYCAPSI), the pepper strain (PVYU10378), the tobacco isolates (PVYCAPD, PVYCAPE and PVYCAPA) and the PVYO from Argentina (PVYCOAT) from the two clusters. The separation of the isolates from Russia, Argentina and Chile (PVYCAPA) may indicate isolated geographical evolution of these viruses. Pepper and tobacco isolates are still poorly studied, but they formed a separate group suggesting a possible correlation among them. This result confirms the grouping of PVY isolates in PVYO, PVYN and non-potato isolates according to RFLP analysis of the CP (Blanco-Urgoiti et al., 1996).
Thirty seven PVY sequences were analyzed for the CP alignment, but only 20 were available for the 3'-UTR analysis. The 3'-UTR tree showed a well defined grouping of PVYO and PVYN and a separate and isolated sequence of PVYNXX, a PVYN isolate from France. Although this isolate was necrotic to N. tabacum (Robaglia et al., 1989), it was consistently clustered with the PVYO isolates when the CP gene was analyzed (Figure 2). This result confirms the previous alignment of 21 CP sequences, which showed the grouping of PVYNXX in the PVYO group (Van der Vlugt et al., 1993). The analysis of the PVYNXX 3'-UTR region showed a mixture of a PVYOlike 5'-region followed by a highly divergent stretch of nucleotides and a PVYN-like sequence at its 3' termini (data not shown). The PVYCPWILG was also a necrotic isolate but which was clustered in both trees with PVYO-like isolates. The PVYNTN isolates (PVYPOLYP and PVYGCP) were grouped in PVYN cluster by the CP analysis, but the 3'-UTR of PVYPOLYP was closer to PVYO. Therefore, the nucleotide sequence of CP and 3'-UTR was not useful to distinguish PVYNTN from other PVY isolates. PVYNTN is also indistinguishable from PVYN by serology and host response (Weilguny & Singh, 1998). These results confirm the report that the necrotic reaction determinants in PVY are not localized in the 3' half portion of the genome (Chachulska et al., 1997).
The nucleotide sequence determination of the CP is postulated as the basis for identification and classification in the Potyvirus genus (Shukla & Ward, 1988). The comparison of two PVY strains with several PVY isolates confirmed the overall clustering into PVYO and PVYN groups. However, the existence of many variants with distinct biological responses and nucleotide sequences showed that characterization studies of new isolates must include a description of their biological properties.
We thank Dr. René Van der Vlugt and Dr. Tatsuya Nagata for critically reading the manuscript.
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* Present address: Embrapa Hortaliças, Caixa Postal 0218, CEP 70359-970, Brasília, DF