Production of plants of Nicotiana benthamiana and Prunus domestica transgenic for plum pox potyvirus coat protein,and demonstration of PPV resistance in transformed N. benthamiana1

Leaf discs of Nicotiana benthamiana plants were transformed with Agrobacterium tumefaciens and transgenic plants expressing plum pox potyvirus (PPV) coat protein (CP) were generated. Homozygous R₂ progeny from these plants were inoculated with PPV. Plants were scored for the appearance of symptoms and tested for infection by DAS-ELISA. Various levels of resistance were obtained after an initial stage in which PPV was able to multiply in all the transgenic plants. Within 2–3 weeks after inoculation, the transgenic resistant plants fully recovered from virus infection. Conversely, control and susceptible transgenic lines developed severe symptoms and high virus titres. Prunus domestica (plum) was transformed by inoculating hypocotyl slices with A. tumefaciens containing a binary plasmid which included the NPTII, GUS, and PPV CP genes within its T-DNA region. Transgenic shoots were rooted and established in the glasshouse. Analysis of selected transformants by PCR showed that the engineered foreign genes had been integrated, including that for PPV CP. Histological assays on young leaves of these putative transformants gave a positive reaction. This suggests that all genes transferred are expressed in these transformed plums.     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

Approaches to pathogen-mediated resistance breeding against plum pox potyvirus in stone-fruit trees1

In a programme for developing systems which allow the transfer of foreign genes into apricot cultivars, we have tested cotyledons of immature embryos, somatic embryos and leaf discs. Apricot plants have been transformed, and then regenerated, with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids: pBinGUSint, carrying the marker gene β-glucuronidase (GUS), and pBinPPVm, carrying the coat-protein gene of plum pox potyvirus (PPV). The marker gene GUS was used for visual evaluation of the efficiency of the transformation system. The coat-protein gene was used in the hope of introducing coat protein-mediated resistance to one of the most important stone-fruit pathogens in Europe and the Mediterranean area.     

A new aubergine disease caused by a whitefly‐borne strain of Tomato mild mottle virus (TomMMoV)

The aims of the present study were to further characterize the causal agent of a new viral disease of aubergines in Israel, first observed in 2003 and tentatively named eggplant mild leaf mottle virus (EMLMV) in a previous work, and to identify the vector responsible for its spread. The disease could be transmitted mechanically from infected source plants to healthy aubergines or laboratory test plants. Transmission electron microscopy (TEM) analysis of purified virus preparations indicated the presence of viral particles with a flexible filamentous morphology (approximately 720 nm long). TEM analysis of ultrathin sections prepared from infected leaf tissue revealed the presence of cytoplasmic inclusion bodies with pinwheel and crystalline structures, typical of those induced by potyviral infection. The viral coat protein subunit was shown to have a molecular weight of 37·5 kDa by SDS‐PAGE analysis. The viral particles reacted positively in western blot analysis with an antiserum against Tomato mild mottle virus (TomMMoV) from Yemen, described as a potyvirus, vectored by the aphid Myzus persicae. The current study describes some biological properties of EMLMV and presents evidence for its transmission by the whitefly Bemisia tabaci, but not by three aphid species. The taxonomic relationship between EMLMV and TomMMoV is discussed based on their biological characteristics and sequence analysis of their genomes. It is suggested that the Israeli EMLMV should be considered a distant strain of TomMMoV, designated TomMMoV‐IL, according to the present rules of Potyviridae molecular taxonomy.     

Cross reactions of an antiserum to plum pox potyvirus1

In the early spring of 1992, plum pox-like viruses (PPLVs) were detected by standard ELISA in some Prunus species. The isolates reacted positively with plum pox potyvirus (PPV) antisera in immunosorbent electron microscopy and Western blot analysis. In Western blot analyses, bands associated with the coat protein subunits of the PPLVs were 48–56 kDa, whereas bands associated with the coat protein subunits of known PPV isolates were 32–37 kDa in size. Also, the PPLVs differed from known PPV isolates in their symptoms on woody and herbaceous indicators, and in their herbaceous host range. None of these PPLVs appears to be an isolate of PPV.     

A potyvirus isolated from solanaceous hosts

A potyvirus was isolated from Datura stramonium, Lycopersicon esculentum (tomato) and Solanum nigrum in the Yemen. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. Its flexuous rod-shaped particles had a mean length of 719 nm and some of its pinwheel inclusion bodies in infected Nicotiana clevelandii leaves were unusual in that they were dichotomously branched. The virus infected various solanaceous species, but the symptoms it induced were distinct from those of pepper veinal mottle (PVMV) and potato Y viruses. Its particles were purified from N. glutinosa and their coat protein had an atypically high molecular mass a potyvirus of 41·5 kDa. They showed a distant serological relationship to those of PVMV and potato virus V in ISEM decoration tests, but did not react with antisera to particles of any other potyvirus tested. The virus has been tentatively named tomato mild mottle virus.     

Serological characterization of Italian isolates of plum pox potyvirus1

An Italian isolate of plum pox potyvirus (PPV) from apricot, Ispave 17, was used as antigen for production of monoclonal antibodies. Six clones secreting specific antibodies to PPV were obtained. All these monoclonal antibodies were used to test a collection of different Italian PPV isolates, collected from plum, apricot and peach orchards, and other European isolates (including PPV-D and PPV-M serotypes), using DAS-ELISA, SDS-PAGE, western blot and GIEM. In western blot analysis, the PPV-M and PPV-D coat protein, detected directly from crude peach GF305 extracts, showed different electrophoretic mobility, the coat protein of PPV-M being slightly larger than that of PPV-D. ELISA tests, performed with fixed dilutions of antibodies and limiting dilutions of clarified samples, showed with some monoclonal antibodies a marked difference between PPV-M and PPV-D strains, at ratios greater than 1:40 (w/v). Also in GIEM some monoclonal antibodies gave a good labelling reaction only with PPV-D serotype. With the help of this differentiation, it was found that all Italian isolates tested were of the D serotype and none of the severe M strain of PPV, which has not been reported in Italy.     

Isolation and characterization of a novel potyvirus tentatively named Ornithogalum virus 2

A novel potyvirus, tentatively named Ornithogalum virus 2 (OV-2) because only its nucleotide sequence of the coat protein gene has been revealed, was isolated for the first time from Ornithogalum thyrsoides. OV-2 had a flexuous particle (700–740 nm in length) and was sap and aphid transmissible. The virus had a narrow host range; of 36 test plants in 12 families, only O. thyrsoides and O. dubium were infected. Because the virus caused characteristic stripe mosaic on O. thyrsoides, we propose Ornithogalum stripe mosaic virus (OrSMV), instead of OV-2 for the proper name of the virus. The nucleotide sequence data reported is available in the DDBJ/EMBL/GenBank databases under accession number AB271783.     

Transmission of plum pox potyvirus in Spain1

Transmission tests were conducted in the laboratory to determine which are the aphid species responsible for the great natural spread of plum pox potyvirus (PPV) observed in the field in Spain. Woody hosts were used in these tests and different transmission techniques were compared. The aphid species tested were Myzus persicae, Aphis gossypii, A. spiraecola, A. fabae, Hyalopterus pruni and Brachycaudus prunicola. Although the transmission rates obtained were, in general, quite low, it can be stated that, except for B. prunicola (pending confirmation of results), all species tested transmitted PPV under the conditions of the trial.     

A potyvirus isolated from Senna occidentalis

A potyvirus causing severe mosaic symptoms was isolated from Senna occidentalis (syn. Cassia occidentalis ) in the Yemen Republic and Ethiopia. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. The flexuous, rod-shaped particles had a mean length of 830 nm, and pinwheels and scrolls were observed by electron microscopy of thin sections of infected Nicotiana clevelandii leaves. Its host range was narrow with only a few legume species, Nicotiana clevelandii and N. benthamiana susceptible to experimental infection. This virus was purified from N. clevelandii and the coat protein had a molecular mass of 34-5 kDa. It reacted positively in ELISA with monoclonal antibody 197 that is specific for potyviruses, but was not decorated by antibodies to any other potyvirus tested when examined by electron microscopy. The virus has been tentatively named cassia severe mosaic potyvirus.     

New potyvirus isolated from <Emphasis Type="Italic">Cryptotaenia japonica</Emphasis>

 A potyvirus, for which the name Japanese hornwort mosaic virus (JHMV) is proposed, was isolated from Japanese hornwort plants (Cryptotaenia japonica) with mosaic disease symptoms. The virus was used to inoculate mechanically 34 plants belonging to 33 species of 10 families. Of these species seven from two families were infected. Faint chlorotic spots appeared on the inoculated leaves of Chenopodium quinoa and C. amaranticolor, but no systemic infection occurred in these plants. JHMV systemically infected only Umbelliferae plants; they did not infect 26 other species in eight families. JHMV was transmitted in a nonpersistent manner by aphids (Myzus persicae). The virus was a flexuous rod-shaped particle about 750 nm in length. Sequencing the nucleotides in the 3′ terminal region of JHMV revealed that the coat protein contains 280 amino acids with a molecular mass of 32.2 kDa. The nucleotide sequence of the coat protein of JHMV had the highest similarity with that of Zantedeschia mosaic virus (83.3%) compared to those of other potyviruses (57.0%–64.9%). An antiserum against JHMV reacted strongly with JHMV and weakly with Potato virus Y. These results indicate that JHMV is a new potyvirus. Received: September 9, 2002 / Accepted: November 7, 2002 RID="*" ID="*" The nucleotide sequence determined in this work appears in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB081518     

Identification and characterization of a potyvirus causing chlorotic spots on <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchids

A putative virus-induced disease showing chlorotic spots on leaves of Phalaenopsis orchids was observed in central Taiwan. A virus culture, phalaenopsis isolate 7-2, was isolated from a diseased Phalaenopsis orchid and established in Chenopodium quinoa and Nicotiana benthamiana. The virus reacted with the monoclonal antibody (POTY) against the potyvirus group. Potyvirus-like long flexuous filament particles around 12–15 × 750–800 nm were observed in the crude sap and purified virus preparations, and pinwheel inclusion bodies were observed in the infected cells. The conserved region of the viral RNA was amplified using the degenerate primers for the potyviruses and sequence analysis of the virus isolate 7-2 showed 56.6–63.1% nucleotide and 44.8–65.1% amino acid identities with those of Bean yellow mosaic virus (BYMV), Beet mosaic virus (BtMV), Turnip mosaic virus (TuMV) and Bean common mosaic virus (BCMV). The coat protein (CP) gene of isolate 7-2 was amplified, sequenced and found to have 280 amino acids. A homology search in GenBank indicated that the virus is a potyvirus but no highly homologous sequence was found. The virus was designated as Phalaenopsis chlorotic spot virus (PhCSV) in early 2006. Subsequently, a potyvirus, named Basella rugose mosaic virus isolated from malabar spinach was reported in December 2006. It was found to share 96.8% amino acid identity with the CP of PhCSV. Back-inoculation with the isolated virus was conducted to confirm that PhCSV is the causal agent of chlorotic spot disease of Phalaenopsis orchids in Taiwan. This is the first report of a potyvirus causing a disease on Phalaenopsis orchids.     

Biological, serological, and molecular differences among isolates of potato a potyvirus

ABSTRACT Sequences of the coat protein (CP) and 3'-end nontranslated region (3'NTR) of 13 isolates and the helper component proteinase (HC) of nine isolates of potato A potyvirus (PVA) were determined and compared with the eight previously determined PVA CP and 3'NTR sequences and one HC sequence. CP amino acid (aa), 3'NTR nucleotide, and HC aa sequence identities were 92.9, 93.4, and 94.8%, respectively. Sequence data, serological tests, and the necrotic local lesions induced in the leaves of the potato hybrid 'A6' confirmed that tamarillo mosaic virus is a strain of PVA. The aa substitutions A6T and G7S in the CP N-terminus were correlated with loss of aphid transmissibility. Development of necrotic lesions or nonnecrotic symptoms in the systemically infected leaves or lack of systemic spread in potato cv. King Edward were used to place the PVA isolates into four strain groups, but this grouping was not correlated with any differences in CP, HC, or 3'NTR. Recognition of CP by three monoclonal antibodies was used to place the PVA isolates into three groups different from the four groups above. The epitopes of two mono-clonal antibodies were mapped by site-directed mutagenesis to the same lysine residue at the CP aa 34.     

Expression of the mature form of plum pox potyvirus helper component1

Polyclonal antibodies raised to the plum pox potyvirus (PPV) helper component (HC) have been produced for the specific detection of HC protein in PPV-infected plants. These experiments suggested that PPV HC is a single soluble protein of 48 kDa. The independent expression of the first two and half cistrons of the PPV genome in transgenic plants suggests that the P1 and P2 (HC-Pro) proteases are involved in the processing of the mature form of HC.     

First report of Colombian datura potyvirus in tomato

Colombian datura potyvirus (CDV) was detected in about 300 plants of tomato Cabrion in one greenhouse in the Netherlands. Virus identification was based on host range and symptomatology, aphid transmission, electron microscopy and serology. Evidence was obtained that the tomato plants were infected by viruliferousMyzus persicae that acquired the virus from a CDV-infectedBrugmansia plant present in the same greenhouse.     

Pfaffia mosaic virus: a new potyvirus found infecting Pfaffia glomerata in Brazil

In Brazil plants of Pfaffia glomerata with mosaic symptoms were found to be infected with a previously undescribed potyvirus, Pfaffia mosaic virus (PfMV). Virus particles were long and flexuous, c.  10 × 700–800 nm, and cylindrical inclusions typical of potyviruses were present in cells of infected tissue. Partial host-range studies revealed that in addition to P. glomerata , PfMV infected only Chenopodium amaranticolor and Chenopodium quinoa . It was efficiently transmitted by the aphids Aphis gossypii and Myzus persicae . Polyclonal antiserum produced against the PfMV coat protein (CP) reacted with Potato virus Y (PVY), but not with four other potyviruses in PTA-ELISA. The similarity of the nucleotide sequence of the PfMV coat-protein gene ( CP ) varied from 7 to 76% when compared with other members of the family Potyviridae . Similarity of the 3' NTR sequence varied from 4 to 23%. In both cases the highest similarity was with PVY. These data indicate that PfMV is a new species in the genus Potyvirus .     

Resistance to Plum pox virus in plants expressing cytosolic and nuclear single‐chain antibodies against the viral RNA NIb replicase

The expression of engineered single‐chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A‐3 (six non‐infected plants out of 10), 6K2‐1 (17 out of 33) and NLS‐11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non‐structural NIb protein in different cell compartments during viral infection.