Lettuce Mosaic Potyvirus Research Articles

Effects of Different Treatments on the Inactivation of Various Seedborne Viruses in Some Vegetables

In this study, we considered the efficacy of seed treatments for the inactivation of some seedborne viruses in tomato, pepper, melon, squash, bean and lettuce seeds, which are essential for human nutrition and seed production in our country. A total of 325 seed samples obtained from various farmers and foundations were tested by DAS-ELISA and RT-PCR procedures. Eight seed lots infected with Tomato mosaic tobamovirus (ToMV), Tobacco mosaic tobamovirus (TMV), Cucumber mosaic cucumovirus (CMV), Soybean mosaic potyvirus (SMV) and Lettuce mosaic potyvirus (LMV) were selected as research materials. Virus inactivation treatments were made by using acetic acid (CH3COOH), hydrogen peroxide (H2O2), hydrochloric acid (HCl), sodium hypochlorite (NaClO), Triton X 100, dry heat, heated water, ozone (O3), and UV (305 nm wavelength). The most effective treatments for reducing virus concentration were HCl, heated water (65 °C) and ozone (10 g m−3). These treatments reduced concentrations of seed-borne viruses in ranges of 51%, 42%, and 32%, respectively. Other treatments were less effective and reduced virus concentrations in the range of 27%–12%. HCl and ozone treatments were the most effective and applicable methods because they did not have negative effect on seed germination.

Detection of viruses in seeds of some vegetables by reverse transcriptase polymerase chain reaction (RT-PCR)

The present study was carried out to detect the presence of Alfalfa mosaic alfamovirus (AMV), cucumber mosaic cucumovirus (CMV), lettuce mosaic potyvirus (LMV), cucumber green mottle mosaic virus (CGMMV), tomato bushy stunt tombusvirus (TBSV), tobacco mosaic tobamovirus (TMV), tomato black ring nepovirus (TBRV) and tomato mosaic tobamovirus (ToMV) in seeds of pepper, tomato, cucumber and lettuce which are essential for human nutrition and seed production in Turkey. 50 seed samples for each vegetable obtained from various foundations and farmers were tested by reverse transcriptase polymerase chain reaction (RT-PCR) and double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) was conducted to compare the results. Besides, two total nucleic acid (TNA) extraction methods and a commercial RNA purification kit was compared to get high quality RNA for optimized RT-PCR. Purified total RNA extracts were used as template for cDNA synthesis, PCR products were analyzed by gel electrophoresis, and visualized by ethidium bromide staining. As a result of all, the optimum TNA extraction method was silica capture which is practical and easy to use for getting viral agents from tested vegetable seeds. RT-PCR was found rapid and sensitive in detecting viruses in seeds of vegetables when compared to DAS-ELISA. This study shows that RT-PCR can be successfully applied in certification programs and quarantine tests of vegetable seeds. Keywords : Seed, reverse transcriptase polymerase chain reaction (RT-PCR), total nucleic acid (TNA) extraction, optimization African Journal of Biotechnology Vol. 12(25), pp. 3891-3897

The RTM Resistance to Potyviruses in Arabidopsis thaliana: Natural Variation of the RTM Genes and Evidence for the Implication of Additional Genes

BackgroundThe non conventional RTM (Restricted Tobacco etch virus Movement) resistance which restricts long distance movement of some plant viruses in Arabidopsis thaliana is still poorly understood. Though at least three RTM genes have been identified, their precise role(s) in the process as well as whether other genes are involved needs to be elucidated.Methodology/Principal FindingsIn this study, the natural variation of the RTM genes was analysed at the amino acid level in relation with their functionality to restrict the long distance movement of Lettuce mosaic potyvirus (LMV). We identified non-functional RTM alleles in LMV-susceptible Arabidopsis accessions as well as some of the mutations leading to the non-functionality of the RTM proteins. Our data also indicate that more than 40% of the resistant accessions to LMV are controlled by the RTM genes. In addition, two new RTM loci were genetically identified.Conclusions/SignificanceOur results show that the RTM resistance seems to be a complex biological process which would involves at least five different proteins. The next challenges will be to understand how the different RTM protein domains are involved in the resistance mechanism and to characterise the new RTM genes for a better understanding of the blocking of the long distance transport of plant viruses.

The C terminus of lettuce mosaic potyvirus cylindrical inclusion helicase interacts with the viral VPg and with lettuce translation eukaryotic initiation factor 4E.

Recessive resistance to lettuce mosaic virus (LMV) is conferred in lettuce by the mo1 gene, encoding the eukaryotic translation initiation factor 4E (eIF4E). The C terminus of the viral cylindrical inclusion helicase (CI-Cter), together with the VPg, is involved directly in overcoming mo1 resistance. In this study, recombinant LMV VPg and CI-Cter proteins from wild-type or resistance-breaking isolates were expressed and purified from Escherichia coli. The allelic forms of eIF4E from susceptible or resistant lettuce cultivars were produced similarly and these proteins were used in ELISA-based assays to demonstrate the in vitro binding of the various forms of LMV CI-Cter to both lettuce eIF4E and LMV VPg proteins. All combinations tested displayed significant and specific interactions, and the interaction between the C-terminal part of the LMV CI and eIF4E was confirmed in vivo in bimolecular fluorescence complementation assays. Higher interaction signals for both CI-eIF4E and CI-VPg were observed for LMV-E, indicating that the eIF4E interaction network involving CI and VPg appears to be stronger in the case of this resistance-breaking isolate. This could suggest the need for a minimal interaction threshold for infection success in resistant lettuce, but more precise measurement of the interaction parameters linking eIF4E, VPg and CI is needed in order to reinforce such a hypothesis.

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein.

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.

Involvement of the cylindrical inclusion (CI) protein in the overcoming of an eIF4E-mediated resistance against Lettuce mosaic potyvirus.

The capacity of Lettuce mosaic virus to overcome the lettuce resistance conferred by the mo1(1) and mo1(2) alleles of the gene for eukaryotic translation initiation factor 4E (eIF4E) was analysed using reverse genetics. Mutations in the virus genome-linked protein (VPg) allowed mo1(1) only to be overcome, but mutations in the C-terminal portion of the cylindrical inclusion (CI) protein allowed both alleles to be overcome. Site-directed mutagenesis pinpointed a key role of the amino acid at position 621 in the virulence. This is the first example of the involvement of a potyviral CI protein in the breaking of an eIF4E-mediated resistance.

Central domain of a potyvirus VPg is involved in the interaction with the host translation initiation factor eIF4E and the viral protein HcPro.

Using recombinant proteins produced in bacteria or in infected plants, interactions between the VPg and HcPro of Lettuce mosaic potyvirus (LMV) and between LMV VPg and the lettuce translation initiation factor 4E, the cap-binding protein (eIF4E), were demonstrated in vitro. Interaction with eIF4E and HcPro both involved the same VPg central domain. The structure of this domain in the VPg context was predicted to include an amphiphilic alpha-helix, with the amino acids related to biological functions in various potyviruses exposed at the hydrophilic side.

The Canadian Phytopathological Society Annual Meeting, Waterton Lakes National Park, Alberta, 2002

The Canadian Phytopathological Society Annual Meeting, Waterton Lakes National Park, Alberta, 2002

Lettuce mosaic poty virus is the causal agent of a new disease inBupleurum spp.

Mosaic symptoms were seen on the ornamental plantBupleurum falcatum (Apiaceae) grown in a commercial nursery in Israel. Examination by electron microscopy (EM) of samples from diseased plants revealed elongated filamentous virus particles. Here we provide evidence thatB. falcatum is an alternate host for Lettuce mosaic potyvirus (LMV). Several lines of evidence indicated thatBupleurum was infected by LMV, including double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunosorbent electron microscopy, and sap and aphid transmission of LMV to lettuce and other host plants. Additionally, general primers derived from a conserved region of the 3’-terminus of the potyvirus genome were used in a reverse transcription-polymerase chain reaction (RT-PCR) assay and gave an expected amplification product of 672 bp. A similar approach was carried out to amplify the corresponding fragment from LMV-infected lettuce. The PCR product was cloned and sequenced; it encompassed an open reading frame encoding 153 amino acids and a 3’ untranslated region (UTR) of 273 nucleotides, very similar to the coat protein (CP) and the 3‵UTR nucleotide sequence, respectively, of other LMV isolates. Analysis of the amino acid sequences of the putative CP of theBupleurum isolate showed complete identity to the lettuce isolate of LMV. TheBupleurum isolate had no new nucleotide changes which have not been established in other published LMV strain sequences. It is concluded that LMV is the causal agent of the disease inB. falcatum in Israel.

Two diseases of dimorphotheca caused by lettuce mosaic potyvirus and tomato spotted wilt tospovirus

In 1997, virus-like symptoms were observed in dimorphotheca plants(Dimorphotheca sinuata) at two different locations in Greece. In a greenhouse near Heraklion (Crete), plants showed chlorotic patches mainly in the older leaves, and a mild stunting; near Katerini (Macedonia), garden plants showed chlorotic rings. Sap inoculation of healthy dimorphotheca plants with extracts from diseased plants from the two regions, reproduced each of the two diseases. Examination by electron microscopy (EM) of samples from diseased plants from the first region revealed filamentous particles, whereas samples from the second region showed quasi-spherical particles. The filamentous particles were decorated in EM with antibodies specific to lettuce mosaic potyvirus (LMV). Samples from the second location reacted with antibodies specific to tomato spotted wilt tospovirus (TSWV) in EL1SA tests. It is concluded that LMV and TSWV are the causal agents of the disease noticed in Heraklion and in Katerini, respectively.

Transformation of two potato cultivars ‘Spunta’ and ‘Claustar’ ( Solanum tuberosum) with lettuce mosaic virus coat protein gene and heterologous immunity to potato virus Y

The coat protein (CP) gene of the lettuce mosaic potyvirus (LMV) was introduced into two potato cultivars—Spunta and Claustar—via Agrobacterium tumefaciens-mediated transformation. Gene expression was verified by Southern. Northern and Western blot. The regenerated plants have been tested for resistance against potato virus Y (PVY-0) in greenhouse. We observed an immune phenotype in four transgenic lines expressing the CP-LMV protein at different detectable levels. However, in two other transgenic plant lines, symptoms were developed at a frequency of 25 and 60% with a delay in their appearance. Transgene transcripts of the CP-LMV gene were detected by Northern blot in these two lines, but the protein could not be detected by Western blot. The correlation between the presence of the CP protein at a detectable level and the expression of an immune phenotype is discussed.

Field resistance against potato virus Y infection using natural and genetically engineered resistance genes

Commercial tobacco cultivars BB16 (burley) and PBD6 (dark air cured) were transformed with the coat protein gene of lettuce mosaic potyvirus (LMV). Transgenic BB16 plants showed resistance to potato virus Y (PVY) infection, against the necrotic strain PVY-N Versailles, as well as the resistance breaking necrotic strain PVY-N 107. Transgenic PBD6, which carries the recessive ‘va’ gene conferring resistance to PVY, was also protected against PVY-N 107. In the progenies of most BB16 and PBD6 transformants, 45% to 100% of the inoculated plants were immune to PVY infection. The rest of the plants were tolerant, with atypical attenuated non necrotic symptoms and reduced virus accumulation. No recovery was observed in the tolerant plants, which stably expressed detectable level of LMV coat protein. This suggested a protein-mediated mechanism of heterologous protection.

Cytopathology of Developing Anthers and Pollen Mother Cells from Lettuce Plants Infected by Lettuce Mosaic Potyvirus

AbstractUltrastructural studies of developing anthers from young flowers of lettuce (Lactuca saliva L.) infected with lettuce mosaic potyvirus (LMV) revealed infection in the pollen mother cells of one of three plat. Within the infected pollen mother cells, structures reminiscent of bundles and partly formed pinwheel inclusions were observed, Immunogold labelling showed that particles of LMV were associated with these structures. Neither large aggregates of LMV particles nor complete pinwheel inclusions were observed in pollen mother cells. Infection of pollen mother cells was consistently correlated with infection of the adjacent tapetal cell layer. Pollen mother cells of two other LMV‐infected plants were not infected. This study supports the hypothesis that early invasion of the floral meristem is necessary for virus infection of mature pollen grains.

Capsid Protein and Helper Component-Proteinase Function as Potyvirus Cell-to-Cell Movement Proteins

The role of bean common mosaic necrosis potyvirus (BCMNV) and lettuce mosaic potyvirus (LMV) proteins was investigated in terms of their capacity to function as viral movement proteins (MPs). UsingEscherichia coli-expressed proteins and microinjection techniques, direct evidence was obtained that both the potyviral capsid protein (CP) and helper component- proteinase (HC-Pro) function in this capacity, in that both proteins (a) trafficked from cell to cell, (b) induced an increase in plasmodesmal size exclusion limit, and (c) facilitated cell-to-cell movement of viral RNA. CP and HC-Pro mutants were also produced and used in microinjection experiments. Mutations in the core region of the CP either impaired (single and double amino acid substitution mutants) or abolished (triple amino acid substitution mutant) cell-to-cell movement, as did C-terminal deletion mutants in HC-Pro. The BCMNV P1, CI, NIa, and NIb proteins did not exhibit viral MP properties, but NIa and NIb proteins were found to accumulate within the nuclei of injected cells. These results further establish the multifunctional nature of the potyvirus CP and HC-Pro.

Gazania spp.: A New Host of Lettuce Mosaic Potyvirus, and a Potential Inoculum Source for Recent Lettuce Mosaic Outbreaks in the Salinas Valley of California.

Lettuce mosaic potyvirus (LMV) outbreaks in the Salinas Valley of California have increased in recent years despite a comprehensive lettuce mosaic management program. In a previous study, it was established that new serologically and/or genetically distinct LMV strains were not responsible for these outbreaks. Therefore, the role of alternate hosts was investigated. Here we provide evidence that the ornamental plant, Gazania spp., is an alternate host of LMV. A number of lines of evidence were used to establish that gazanias were infected by LMV, including indirect enzyme-linked immunosorbent assay, dot blot hybridization with an LMV coat protein (CP) probe, and sap- and aphid-transmission of LMV from gazania to lettuce and Nicotiana benthamiana. Additionally, an LMV CP fragment was amplified from an infected gazania plant by reverse transcription-polymerase chain reaction, and the nucleotide sequence of this fragment was 99% identical to the CP sequence of an LMV isolate from the Salinas Valley. We further show that LMV-infected gazanias are widely distributed in the Salinas Valley, and are present near areas where LMV outbreaks have occurred. Field experiments demonstrated that LMV could be transmitted from infected gazania to lettuce. LMV-infected gazanias also were found in local nurseries, suggesting that infection of gazania with LMV had occurred prior to planting in the Salinas Valley (i.e., lettuce was not serving as the source of inoculum for infection of gazania in the Salinas Valley). These results establish that gazania is an alternate host of LMV, and suggest that gazanias may be a primary inoculum source for recent lettuce mosaic outbreaks in the Salinas Valley.

Survey of chickpea for viruses in Morocco

Chickpea was surveyed for viruses in the main growing areas of the crop in Morocco. A total of 36 fields was examined and 167 samples with symptoms suggestive of virus infection were collected. Serological testing using polyclonal antisera and monoclonal antibodies revealed the presence of the following viruses: alfalfa mosaic alfamovirus (AMV), broad bean mottle bromovirus (BBMV), broad bean stain comovirus (BBSV), bean yellow mosaic potyvirus, cucumber mosaic cucumovirus (CMV), faba bean necrotic yellows nanavirus (FBNYV), lettuce mosaic potyvirus, pea seed‐borne mosaic potyvirus (PSbMV), and luteoviruses. Field symptoms of these viruses in chickpea comprised mosaics, yellowing, stunting, narrowing of leaflets and bushy growth. AMV, luteoviruses and potyviruses were found spread over the chickpea‐growing regions, while BBSV, BBMV, and FBNYV were confined to some areas. Virus incidence per field varied from 1 to 40% for luteoviruses and from 1 to 20% for mosaic‐inducing viruses. AMV, luteoviruses, potyviruses and CMV were detected in 89, 83, 75 and 42%, respectively, of the surveyed fields. BBMV, BBSV and FBNYV were less widespread at the national level. This is the first report of the occurrence of viruses other than CMV and BBMV in chickpea in Morocco, and the first record of the natural occurrence of BBSV and PSbMV in chickpea.

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.

Coat protein gene-mediated protection in Lactuca sativa against lettuce mosaic potyvirus strains

Lettuce mosaic potyvirus (LMV) can be very destructive on lettuce crops worldwide. The LMV strain 0 (LMV-0) coat protein (CP) gene was engineered for expression in plants. It was introduced into three susceptible cultivars of Lactuca sativa using an improved procedure for transformation and regeneration of lettuce, by co-cultivation of leaf explants with Agrobacterium tumefaciens. Several transformants accumulated detectable levels of LMV CP. The R1 progeny of twelve R0 transformants (four plants per cultivar) with T-DNA integration at one single locus, was studied for protection against LMV. The progeny from five R0 transformants showed resistance to LMV-0, with the effectiveness of resistance depending on the development stage of the plants at the time of inoculation. The R1 and R2 progeny from one of these R0 transformants, Cocarde-9a, were more extensively analysed. The homozygous but not the hemizygous R1 plants displayed protection to LMV-0. The R2 progeny from one homozygous R1 plant were shown to be resistant to infection by LMV-0 and other LMV strains. As previously observed in other cases of potyvirus sequence-mediated protection, a phenomenon of recovery was observed in some plants, as well as complete resistance. However, this recovery phenotype was not always maintained, as opposed to the previous described cases, leading to a late progression of viral infection.

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 mo11 and mo12 recessive resistance genes, whereas LMV-E is able to induce disease in the same plants. The genomes of these two isolates are 10 080 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.

A Recent Outbreak of Lettuce Mosaic Potyvirus in Commercial Lettuce Production in Florida.

A Recent Outbreak of Lettuce Mosaic Potyvirus in Commercial Lettuce Production in Florida.