Biomedical subjects

G P Accotto

Publications and source records attributed to G P Accotto.

At least 19 recordsLinked to original sources

PubMed2004

TYLCSV DNA, but not infectivity, can be transovarially inherited by the progeny of the whitefly vector Bemisia tabaci (Gennadius).

The transovarial transmission of two species of begomovirus, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), through generations of Bemisia tabaci of the B and Q biotypes has been investigated. Different life stages of the progeny of viruliferous female whiteflies have been analysed by PCR detection of viral DNA and infectivity tests. Our results indicate that TYLCSV DNA can be detected in eggs and nymphs, and to a lesser extent adults, of the first-generation progeny. Infectivity tests using a large number of adult progeny of the first, second, and third generation indicate that even when viral DNA is inherited, infectivity is not. For TYLCV, neither viral DNA nor infectivity were associated with the progeny of viruliferous female whiteflies. Because the inherited viral DNA is unable to give rise to infections, the transovarial transmission of TYLCSV DNA appears to have no epidemiological relevance.

Animals↗

PubMed2004

Severe outbreaks of tomato yellow leaf curl Sardinia virus in Calabria, Southern Italy.

During the winter 2003--2004 a serious disease was observed in protected tomato crops in Castrovillari, Reggio Calabria province, Southern Italy. Symptoms consisted in marginal leaf yellowing, leaf curling, plant stunting, flower abortion. The disease was detected in a group of greenhouses (about 10ha) where several tomato cultivars were grown hydroponically. The highest incidence of infection (60-100%) was observed in tomatoes grafted on Beaufort DRS tomato rootstock. Since the symptoms were similar to those described for Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), detection assays for these viruses were used. In DAS-ELISA positive results were obtained with a abroad-spectrums reagent combination (distributed by Bioreba AG) detecting TYLCV, TYLCSV, and other begomoviruses. When DNA probes were used in tissue print assays, positive reactions were obtained for TYLCSV, but not for TYLCV. The two probes consisted of digoxigenin-labelled DNAs representing the coat protein gene of either TYLCSV or TYLCV. Attempts to isolate the viral agent by mechanical inoculation failed, except in few cases where Potato virus Y and Tobacco mosaic virus were identified following transmission from symptomatic plants to herbaceous indicatorpplants. By contrast, grafting onto tomato seedlings always successfully transmitted the disease. In the Castrovillari area TYLCSV was not reported before. The rootstocks that nurseries used for grafting were obtained from Sicily, where the disease is endemic and both TYLCSV and TYLCV are widespread. Probably the grafted plantlets represented the primary source of infection from which subsequent diffusion by way of the vector Bemisia tabaci followed. In fact the vector had previously been detected in both the glasshouse-grown and open field tomato crops in Calabria region. TYLCV was previously reported in a different area of Calabria in 1991, but apparently it was an occasional outbreak, and B. tabaci was not detected. Since in the Castrovillari area surveyed in the present study tomato is grown throughtout the year in protected crops, the whitefly vector of the virus is present, and some natural hosts of the virus are found, it is feared that TYLCSV may become endemic, as already happened in Sicily, Sardinia, and Spain several years ago. In Spain and Sicily TYLCV, together with TYLCSV, was reported as the causal agent of very severe tomato crop losses. Therefore the danger exists that also TYLCV will reach this area, furthermore complicating the management of tomato crops.

Agriculture↗

PubMed2003

The partial sequence of RNA 1 of the ophiovirus Ranunculus white mottle virus indicates its relationship to rhabdoviruses and provides candidate primers for an ophiovirus-specific RT-PCR test.

A 4018 nucleotide sequence was obtained for RNA 1 of Ranunculus white mottle virus (RWMV), genus Ophiovirus, representing an incomplete ORF of 1339 aa. Amino acid sequence analysis revealed significant similarities with RNA polymerases of viruses in the family Rhabdoviridae and a conserved domain of 685 aa, corresponding to the RdRp domain of those in the order Mononegavirales. Phylogenetic analysis indicated that the genus Ophiovirus is not related to the genus Tenuivirus or the family Bunyaviridae, with which it has been linked, and probably deserves a special taxonomic position, within a new family. A pair of degenerate primers was designed from a consensus sequence obtained from a relatively conserved region in the RNA 1 of two members of the genus, Citrus psorosis virus (CPsV) and RWMV. The primers, used in RT-PCR experiments, amplified a 136 bp DNA fragment from all the three recognized members of the genus, i.e. CPsV, RWMV and Tulip mild mottle mosaic virus (TMMMV) and from two tentative ophioviruses from lettuce and freesia. The amplified DNAs were sequenced and compared with the corresponding sequences of CPsV and RWMV and phylogenetic relationships were evaluated. Assays using extracts from plants infected by viruses belonging to the genera Tospovirus, Tenuivirus, Rhabdovirus and Varicosavirus indicated that the primers are genus-specific.

Amino Acid Sequence↗

PubMed2002

Nucleotide sequence, genome organisation and phylogenetic analysis of Indian citrus ringspot virus. Brief report.

The sequence of the single-stranded RNA genome of Indian citrus ringspot virus (ICRSV) consists of 7560 nucleotides. It contains six open reading frames (ORFs) which encode putative proteins of 187.3, 25, 12, 6.4, 34 and 23 kDa respectively. ORF1 encodes a polypeptide that contains all the elements of a replicase; ORFs 2, 3 and 4 compose a triple-gene block; ORF5 encodes the capsid protein; the function of ORF6 is unknown. Phylogenetic analysis of the complete genome and each ORF separately, and database searches indicate that ICRSV, though showing some similarities to potexviruses, is significantly different, as in the presence of ORF6, the genome and CP sizes, and particle morphology. These differences favour its inclusion in a new virus genus.

Base Sequence↗

PubMed2001

Transgenically expressed T-Rep of tomato yellow leaf curl Sardinia virus acts as a trans-dominant-negative mutant, inhibiting viral transcription and replication.

We have previously shown that transgenic expression of a truncated C1 gene of Tomato yellow leaf curl Sardinia virus (TYLCSV), expressing the first 210 amino acids of the replication-associated protein (T-Rep) and potentially coexpressing the C4 protein, confers resistance to the homologous virus in Nicotiana benthamiana plants. In the present study we have investigated the role of T-Rep and C4 proteins in the resistance mechanism, analyzing changes in virus transcription and replication. Transgenic plants and protoplasts were challenged with TYLCSV and the related TYLCSV Murcia strain (TYLCSV-ES[1]). TYLCSV-resistant plants were susceptible to TYLCSV-ES[1]; moreover, TYLCSV but not TYLCSV-ES[1] replication was strongly inhibited in transgenic protoplasts as well as in wild-type (wt) protoplasts transiently expressing T-Rep but not the C4 protein. Viral circular single-stranded DNA (cssDNA) was usually undetectable in transgenically and transiently T-Rep-expressing protoplasts, while viral DNAs migrating more slowly than the cssDNA were observed. Biochemical studies showed that these DNAs were partial duplexes with the minus strand incomplete. Interestingly, similar viral DNA forms were also found at early stages of TYLCSV replication in wt N. benthamiana protoplasts. Transgenically expressed T-Rep repressed the transcription of the GUS reporter gene up to 300-fold when fused to the homologous (TYLCSV) but not to the heterologous (TYLCSV-ES[1]) C1 promoter. Similarly, transiently expressed T-Rep but not C4 protein strongly repressed GUS transcription when fused to the C1 promoter of TYLCSV. A model of T-Rep interference with TYLCSV transcription-replication is proposed.

DNA Helicases↗

PubMed2000

An Ophiovirus isolated from lettuce with big-vein symptoms.

Big-vein is a widespread and damaging disease of lettuce, transmitted through soil by the chytrid fungus Olpidium brassicae, and generally supposed to be caused by Lettuce big-vein virus (LBVV; genus Varicosavirus). This virus is reported to have rigid rod-shaped particles, a divided double-stranded RNA genome, and one capsid protein of 48 kD, but has not been isolated or rigorously shown to cause the disease. We provide evidence that a totally different virus, here named Mirafiori lettuce virus (MiLV), is also very frequently associated with lettuce showing big-vein symptoms. MiLV was mechanically transmissible from lettuce to Chenopodium quinoa and to several other herbaceous test plants. The virus was partially purified, and an antiserum prepared, which did not react with LBVV particles in decoration tests. As reported for LBVV, MiLV was labile, soil-transmitted and had a single capsid protein of 48 kD, but the particles morphologically resembled those of ophioviruses, and like these, MiLV had a genome of three RNA segments approximately 8.5, 1.9 and 1.7 kb in size. MiLV preparations reacted strongly in Western blots and in ISEM with antiserum to Tulip mild mottle mosaic virus, an ophiovirus from Japan also apparently Olpidium-transmitted. They reacted weakly but clearly in Western blots with antiserum to Ranunculus white mottle virus, another ophiovirus. When lettuce seedlings were mechanically inoculated with crude or partially purified extracts from MiLV-infected test plants, many became systemically infected with MiLV and some developed big-vein symptoms. Such plants did not react in ELISA using an LBVV antiserum or an antiserum to tobacco stunt virus, and varicosavirus-like particles were never seen in them in the EM after negative staining. We conclude that MiLV is a hitherto undescribed virus assignable to the genus Ophiovirus. The cause or causes of lettuce big-vein disease and the properties of LBVV may need to be re-evaluated in light of our results.

Blotting, Northern↗

PubMed2000

Indian citrus ringspot virus: a proposed new species with some affinities to potex-, carla-, fovea- and allexiviruses.

An isolate of Indian citrus ringspot virus from Kinnow mandarin in northern India had flexuous particles with evident cross-banding and a modal length of 650 nm. It was mechanically transmitted to five herbaceous hosts including Phaseolus vulgaris cv Saxa, in which it became systemic. In thin sections, virus particles were observed in the cytoplasm of parenchyma cells but no specific inclusions were seen. The virus was purified from infected Saxa bean leaves and an antiserum prepared. There was no serological cross-reaction with representative allexi-, capillo-, potex- and trichoviruses, except a faint one-way reaction with Potato virus X. Purified virus yielded a major band, the presumed coat protein (CP), of about 34 kDa, and a single ssRNA of about 7.5 kb, which was infectious. Two ORFs encoding putative proteins of 34 kDa and 23 kDa were located in the 3' part of the RNA. The product of the 34 kDa ORF was confirmed as the CP by expression in E. coli. The derived amino acid sequence of the CP contained some short motifs similar to those of potex-, fovea-, carla- and allexiviruses but otherwise there was no strong similarity to any of these. The 23 kDa ORF contained a zinc finger-like sequence, as in similar ORFs in carla- and allexiviruses but overall amino acid homology with these was low. The virus does not appear to fall into any known genus. A new species is proposed. Serological and molecular diagnostic reagents were prepared.

3' Untranslated Regions↗

PubMed2000

Natural recombination between Tomato yellow leaf curl virus-is and Tomato leaf curl virus.

The complete genome sequences (2791 and 2793 nt) of isolates of Tomato yellow leaf curl virus-Is (TYLCV-Is) from Spain (SP72/97) and Portugal (Port2/95) were determined. These isolates are closely related to TYLCV-Is isolates reported in Japan (Japan-A and Japan-S) and Israel (Israel/Mild). Comparison of all sequenced isolates of TYLCV-Is showed that part of the genome comprising the intergenic region and the 5'-end of the rep gene of the Iran and Israel isolates was not closely related to that of other isolates. Phylogenetic analyses suggest that the Israel and Iran isolates may have chimeric genomes that have arisen by recombination between TYLCV-Is-like and tomato leaf curl virus (ToLCV)-like ancestors. The TYLCV-Is donors of the Iran and the Israel genomes were closely related to each other and to other known TYLCV-Is isolates. However, the ToLCV donors differed from each other, although both were related to ToLCV isolates from India (Bangalore-2 and Bangalore-4).

Geminiviridae↗

PubMed1998

Amino acids in the capsid protein of tomato yellow leaf curl virus that are crucial for systemic infection, particle formation, and insect transmission.

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sic, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sic, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.

Amino Acid Sequence↗

PubMed1997

Partial characterization of a new virus from ranunculus with a divided RNA genome and circular supercoiled thread-like particles.

An undescribed virus, here named ranunculus white mottle virus, was isolated in Italy from cultivated ranunculus showing mottle and distortion of leaves. The virus was mechanically transmissible to several herbaceous hosts. In negative stain, the particles appeared as circularised supercoiled threads 3 nm in diameter of different contour lengths; in some conditions the circles collapsed to form linear pseudobranched structures 9 nm in diameter. Immunolabeling of thin sections showed that viral antigen was widely distributed in the cytoplasm of parenchyma cells. The virus was not serologically related to the morphologically similar tenuiviruses, citrus psorosis-ringspot virus and tulip mild mottle mosaic virus. A major 43 kDa protein was present in purified preparations and in infected plant tissue, as also was a minor 28 kDa protein, serologically related to the major one. Nucleic acids extracted from purified particles consisted of at least three RNAs, of approximately 7.5, 1.8 and 1.5 kb, which appeared partly in single- and partly in double-stranded form. Purified preparations, but not viral RNAs, when mechanically inoculated, were infectious. Host range, tissue tropism, particle morphology and coat protein size place the virus closest to citrus psorosis-ringspot and tulip mild mottle mosaic viruses. These three viruses in turn show similarities with the Tenuiviruses and Bunyaviridae.

Capsid↗

PubMed1996

Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed with a truncated viral C1 gene.

The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication. A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced into Nicotiana benthamiana using Agrobacterium tumefaciens. The same sequence was also cloned in antisense orientation. When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were further studied. The presence of the transgene was verified and its expression was followed at intervals. All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection. Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed. The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection.

Agrobacterium tumefaciens↗

PubMed1996

DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus.

The in vitro DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus (TYLCV) was studied following its expression in Escherichia coli as a fusion protein with an His tag N-terminal extension (His-C2). Southwestern blotting experiments demonstrated that the C2 protein is able to bind both single-stranded and double-stranded DNA probes. In electrophoretic mobility shift assays performed using purified protein and single-stranded DNA probes several shifted complexes were formed. The presence of NaCl (up to 800 mM) did not substantially affect binding profiles, demonstrating a stable interaction. His-C2 appeared to bind single-stranded DNA in a sequence-nonspecific manner, with a preference for single-stranded compared to double-stranded DNA. Deletion mutants demonstrated that the central core of C2 (amino acids 33 to 104), which contains a Cys-His rich region, is sufficient for conferring binding activity. The potential significance of this DNA-binding activity with respect to possible biological functions of TYLCV C2 protein is discussed.

Base Sequence↗

PubMed1996

A polyclonal antiserum against a recombinant viral protein combines specificity with versatility.

A polyclonal rabbit antiserum was obtained to the nucleoprotein of tomato spotted wilt tospovirus expressed as a recombinant fusion protein in E. coli. In indirect plate trapping ELISA, the antiserum gave similar titres against purified TSWV nucleocapsids in native form, the fusion protein and the carrier protein. The crude antiserum was also tested by Western blotting, indirect plate trapping ELISA and immunogold electron microscopy of thin sections: purified immunoglobulins were tested by DAS-ELISA. In all cases, with both glasshouse and field material, the antibodies had good detectability and specificity. By ELISA and Western blots against other tospoviruses, impatiens necrotic spot and groundnut bud necrosis viruses did not react but there was a reaction with groundnut ringspot virus, reflecting the nucleoprotein amino acid sequence similarity. These antibodies combine specificity to the target protein and versatility with regard to all the more important serological techniques. There were no undesired reactions resulting from immunization using a complex virus purified from infected host material.

Animals↗

PubMed1994

High similarity among the tomato yellow leaf curl virus isolates from the west Mediterranean basin: the nucleotide sequence of an infectious clone from Spain.

An isolate of tomato yellow leaf curl geminivirus, from the first epidemic outbreaks that occurred in Murcia, Spain (TYLCV-M) in 1992, was cloned and its nucleotide sequence was determined. The circular single stranded DNA consisted of 2777 nucleotides. The genome organization resembled that of other TYLCV sequenced so far; regulatory signal sequences for bidirectional transcription and for polyadenylation of the transcripts were localized in the sequence. Infectivity of the cloned DNA was demonstrated by subcloning a 1.8 mer of TYLCV-M in pBin19 and agroinoculating it into tomato and Nicotiana benthamiana plants. Symptoms and viral DNA forms in agroinfected plants did not differ from those of field infected ones. Sequence comparisons with other TYLCV isolates show a high homogeneity between isolates from the West Mediterranean Basin, suggesting the presence of a geographical cluster.

Amino Acid Sequence↗

PubMed1994

Advances in diagnosing tomato yellow leaf curl geminivirus infection.

As a result of the spread of TYLCV on tomato crops, reliable and rapid diagnostic tools to identify and isolate new sources of infection are necessary. We tested several methods, based both on antibodies and on nonradioactive DNA probes. Indirect plate-trapping ELISA was only effective in detecting the virus in purified preparations, but not in crude extracts. Dot-ELISA with chemiluminescence detection gave satisfactory results when young stems were directly squashed on membranes. A digoxigenin-labeled probe, detected with chemiluminescence, was used in leaf squashes and dot blots. Best results were obtained with dot blots of total nucleic acids prepared with a fast and safe procedure. TYLCV DNA was readily and reliably detected in spots corresponding to 15 micrograms fresh weight. When weak signals were observed, total extracts were analyzed by Southern blotting, to confirm the presence of viral DNA forms.

Antibodies, Viral↗

PubMed1993

Digitaria streak geminivirus replicative forms are abundant in S-phase nuclei of infected cells.

We have analyzed the replication of Digitaria streak geminivirus (DSV) using flow cytometry. Nuclei from infected plants were sorted on the basis of their DNA content in the different phases of the cell cycle and viral DNA forms in them were analyzed. DSV replicative forms were much more abundant (up to 20 times in one experiment) in S-phase nuclei than in G0-G1 and G2 nuclei, while single-stranded viral DNA did not show such dramatic increase during any phase. DSV replicative forms constitute almost 90% of total viral DNA forms in S-phase nuclei. DSV replication seems to be synchronized with host DNA replication and might be primarily under control of host factors becoming available only at the G1/S boundary.

Blotting, Southern↗

PubMed1991

Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus.

The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced. The single circular DNA molecule comprises 2770 nucleotides. Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome. A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants. Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation. The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule. Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.

Agrobacterium tumefaciens↗

PubMed1990

Processing of complementary sense RNAs of Digitaria streak virus in its host and in transgenic tobacco.

We have used a polymerase chain reaction (PCR) procedure to analyse low abundance complementary sense RNAs of Digitaria streak virus (DSV) from infected leaves of Digitaria setigera. This study has confirmed that both spliced and unspliced RNAs are synthesised by the same transcription unit. The position of the intron has been proven from sequencing cDNAs corresponding to the spliced RNA. Although the majority of cDNAs have 3' ends at coordinate 1063, downstream from a consensus polyadenylation sequence, a minor population of RNAs with heterogeneous 3' ends has also been identified. Two major RNA species with alternative splice sites or 3' ends, previously identified by nuclease S1 protection assays, could not be detected, but a cDNA species was observed with an apparent 90bp insertion at the 5' end of the intron. In transgenic tobacco containing integrated dimers of DSV DNA, the major unspliced RNA could readily be detected, but no spliced RNA was present. This may be a reason why DSV DNA did not replicate in tobacco. In addition, neither the minor population of heterogeneous RNAs nor the cDNA species with the insertion could be detected. The failure of the intron to be spliced in tobacco and its low activity in Digitaria is discussed in relation to recent studies on RNA splicing in plants and has led us to the conclusion that the geminivirus introns may be intrinsically inefficient.

Base Sequence↗