Pea Seed-borne Mosaic Potyvirus Research Articles

DETECTION, BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF SOME VIRUSES TRANSMITTED BY EDIBLE LEGUMINOUS SEEDS

Bu çalışma, 2017 yılında Türkiye’nin farklı illerindeki üreticilerden ve firmalardan temin edilen bakla, bezelye, mercimek ve nohut tohum örneklerinde Alfalfa mosaic alfamovirus (AMV), Bean yellow mosaic potyvirus (BpYMV), Cucumber mosaic cucumovirus (CMV) ve Pea seed-borne mosaic potyvirus (PSbMV) virüslerinin bulunma durumunun belirlenmesi ve tespit edilen virüslerin biyolojik ve moleküler karakterizasyonunun yapılması amacıyla gerçekleştirilmiştir.
 Tohum örneklerinde virüslerin saptanmasında, DAS-ELISA ve RT-PCR yöntemleri kullanılmıştır. Tespit edilen virüsler mekanik inokulasyonla test bitkilerine taşınarak biyolojik karakterizasyonu yapılmıştır. Daha sonra RT-PCR yöntemiyle çoğaltılan virüslerin CP gen bölgelerinin dizi analizleri yapılarak, MEGA programı vasıtasıyla dünya izolatlarıyla karşılaştırılmalı olarak filogenetik analizi gerçekleştirilmiştir.
 Bakla, mercimek ve nohut tohum örneklerinde virüslere rastlanırken, bezelye tohumlarında herhangi bir virüse rastlanmamıştır. Baklada PSbMV, Nohutta BYMV, CMV ve PSbMV virüsleri ülkemizde ilk kez belirlenmiştir. Biyolojik karakterizasyon çalışmalarında, AMV, BYMV, CMV ve PSbMV izolatları dünya izolatlarıyla farklılıklar ve benzerlikler göstermiştir. Ayrıca, dünya izolatlarıyla yüksek oranda nükleotit ve aminoasit benzerliği tespit edilmiştir.

Molecular characterisation of Pea seed borne mosaic poytvirus associated with apical rosette in field pea (Pisum sativum L.) in India

Symptoms of apical rosette were seen in field pea grown as winter season crop in India. Symptomatic leaves were processed for the identification of associated virus. The primers pair (PSbMV-F/PSbMV-R) targeting part of NIb gene of Pea seed borne mosaic potyvirus (PSbMV) was used in RT-PCR. The targeted fragment was successfully amplified cloned and sequenced. Sequencing of amplicons revealed it to be 795 bp long and identical to each other and hence only one was deposited to the NCBI database. Blast results of sequence showed it to be a portion of NIb region of the PSbMV and had 100–88.5% nt similarity with known isolates of the PSbMV. Phylogenetic analysis revealed that all the PSbMV isolates used group together. However, the PSbMV-IN:Knp isolate sub-clustered with the known PSbMV isolates of pathotype 1. This is the first report of the characterisation of an Indian isolate of PSbMV based on genomic sequence.

A comparison between virus replication and abiotic stress (heat) as modifiers of host gene expression in pea

Abstract Pea embryonic tissues respond to active replication of pea seed-borne mosaic potyvirus (PSbMV) by the down-regulation of a range of genes and the induction of others. Both of these responses can be seen when tissues are subjected to abiotic stress, particularly heat. We have compared the effects of the two inducers to assess whether the host alterations following virus replication represent generic responses to stress, or more specific effects. Five classes of response were identified: (i) genes induced by both stresses (e.g. heat shock protein 70, hsp70); (ii) genes induced by virus replication but unaffected by heat (e.g. glutathione reductase 2, gor2); (iii) genes induced by heat but unaffected by virus replication (e.g. heat shock factor, hsf); (iv) genes down-regulated by virus replication and unaffected by heat (e.g. vicilin, vic); and (v) genes unaffected by both inducers (e.g. actin, act and beta-tubulin, tub). A change in the appearance and organization of the endoplasmic reticulum (ER) was also seen in cells actively replicating PSbMV RNA. Heat treatment of pea embryonic tissues also produced altered ER, although the changes were different from those seen following virus infection. Collectively, these data show that, while there are some common features of the responses to virus infection and heat, there are also substantial differences. Hence, it appears that the host response to virus replication is not a general stress response.

Pea Embryonic Tissues Show Common Responses to the Replication of a Wide Range of Viruses

The response of pea embryonic tissues to the replication of a range of different viruses was investigated using in situ hybridization to analyze changes in the expression of two host genes, heat shock protein 70 (hsp70) and lipoxygenase (lox1). Excised pea embryos were infected using microprojectile bombardment with a nonseed transmissible strain of Pea seed-borne mosaic potyvirus, or with Pea early browning tobravirus (PEBV), White Clover mosaic potexvirus, or Beet curly top geminivirus. Collectively, these examples represent families of viruses with differing genomic features, differing numbers of genomic components and differing replication strategies. In all cases, there was an induction of hsp70 associated with virus replication and, in most cases, a downregulation of lox1. Hence, either each virus has a direct inducer of these common responses or the induction is indirectly the result of a generic feature of virus infection. By exploiting the bipartite nature of the PEBV genome, the coat protein gene and genes involved in vector transmission were excluded as potential inducers.

A heat shock transcription factor in pea is differentially controlled by heat and virus replication.

Since some heat-inducible genes [heat shock (hs) genes] can be induced by virus infection in pea [e.g. Hsp70; Aranda et al. 1996, Proc. Natl Acad. Sci. USA 93, 15289-15293], we have investigated the effect that heat and virus replication may have on the expression of a heat-shock transcription factor gene (Hsf). We have characterized what appears to be the only member of the Hsf family in pea, PsHsfA. Similar to Hsp70, PsHsfA is heat-inducible in vegetative and embryonic tissues, which is concordant with the presence of heat shock elements (HSEs) and stress responsive elements (STREs) on its promoter sequence. The expression of PsHsfA during virus replication was studied in pea cotyledons and leaves, and compared to that of Hsp70. In situ hybridization experiments showed that whereas Hsp70 is induced, there is no detectable increased accumulation of PsHsfA RNA associated with the replication of pea seed-borne mosaic potyvirus (PSbMV). These experiments indicate that there is a selective control of virus-induced hs gene expression, and suggest that different regulatory pathways control hs gene expression during heat shock and virus replication.

Two Cases of Non‐host Resistance against Potyviruses Operating at the Single Cell Level

AbstractNonhost resistance of plants against viruses is a very common but poorly understood phenomenon. In this study Nicotiana tabacum L. and pea seed‐borne mosaic potyvirus (PSbMV) were chosen as a model system for a closer examination of nonhost resistance. Nicotiana tahacum was confirmed to be a nonhost for PSbMV by mechanical inoculations followed by analysis by enzyme‐linked immunosorbent assay and by immunofluorescence staining of mesophyll and epidermal protoplasts isolated from inoculated leaves. When tobacco protoplasts were inoculated with PSbMV virions or genomic RNA, only very few eells became infected. On the other hand, both PSbMV virions and genomic RNA could infeet protoplasts from the host plant Pisum sativum L. In parallel experiments, virions of another potyvirus, potato virus Y (PVY), infected tobacco protoplasts but not protoplasts from pea, a nonhost for PVY. The results suggest, that the inabihty of PSbMV to infect tobacco, and of PVY to infect pea, is at least partly caused by a failure of virus replication in the inoculated cells.

Specificity of resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (Nlb) gene.

Transgenic pea lines carrying the replicase (NIb) gene of pea seed-borne mosaic potyvirus (PSbMV) were generated and used in experiments to determine the effectiveness of induced resistance upon heterologous isolates. Three pea lines showed inducible resistance in which an initial infection by the homologous isolate (PSbMV-DPD1) was followed by a highly resistant state. Resistance was observed in plants in either the homozygous or hemizygous condition and resulted in no overall yield loss despite the initial infection. Resistance was associated with a loss of both viral and transgene RNA, which is indicative of a mechanism based upon post-transcriptional gene silencing. There was no correlation between the steady-state levels of transgene RNA and ability of the plants to show resistance. To test the specificity of the resistance, plants were also inoculated with the most distantly related sequenced PSbMV isolate, NY. PSbMV-NY varied between experiments in its ability to induce resistance, suggesting that the sequence identity in the NIb gene is borderline for the specificity required for triggering gene silencing. Upon challenge inoculation of virus-free recovered leaves, the specificity of the induced resistance varied between the two isolates and indicated that the virus and transgene additively determined the resistant state. These results suggest that the sequence requirements for triggering gene silencing may differ from those involved in the degradation process.

Potyvirus genome-linked protein (VPg) determines pea seed-borne mosaic virus pathotype-specific virulence in Pisum sativum.

The mechanism of Pisum sativum pathotype-specific resistance to pea seed-borne mosaic potyvirus (PSbMV) was investigated and the coding region determinant of PSbMV virulence was defined. Homozygous recessive sbm-1 peas are unable to support replication of PSbMV pathotype 1 (P-1), whereas biochemically and serologically related pathotype 4 (P-4) is fully infectious in the sbm-1/sbm-1 genotype. We were unable to detect viral coat protein or RNA with double antibody sandwich-enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction in sbm-1/sbm-1 P-1-inoculated protoplasts and plants. Lack of viral coat protein or RNA in P-1 transfected sbm-1/sbm-1 protoplasts suggests that sbm-1 resistance is occurring at the cellular level and that inhibition of cell-to-cell virus movement is not the operating form of resistance. In addition, because virus products were not detected at any time post-inoculation, resistance must either be constitutive or expressed very early in the virus infection process. P-1-resistant peas challenged with full-length, infectious P-1/P-4 recombinant clones demonstrated that a specific P-4 coding region, the 21-kDa, genome-linked protein (VPg), was capable of overcoming sbm-1 resistance, whereas clones containing the P-1 VPg coding region were noninfectious to sbm-1/sbm-1 peas. VPg is believed to be involved in potyvirus replication and its identification as the PSbMV determinant of infectivity in sbm-1/sbm-1 peas is consistent with disruption of an early P-1 replication event.

A Single Conserved Amino Acid in the Coat Protein Gene of Pea Seed-Borne Mosaic Potyvirus Modulates the Ability of the Virus to Move Systemically inChenopodium quinoa

Two isolates of pea seed-borne mosaic potyvirus, DPD1 and NY, which both infect pea (Pisum sativum) systemically, differ in their ability to move long distance inChenopodium quinoa.DPD1 spreads to uninoculated leaves, whereas NY is restricted to the inoculated leaves. The NY isolate was found to move from cell to cell infecting all parts of the inoculated leaves, including the petiole. The coat protein (CP) coding region was identified as the determinant of long-distance movement. Virus chimeras containing the CP coding sequence of NY were restricted to inoculated leaves, whereas chimeras containing the CP coding sequence of DPD1 infectedC. quinoasystemically. Mutational analysis of the CP demonstrated that changing the serine at position 47 of the NY CP to proline was sufficient to permit systemic spread of the NY(S47P) mutant. The reverse mutant, DPD1(P47S), in which the proline at position 47 of the CP was changed to serine, was restricted to inoculated leaves. The movement characteristics and CP sequences of 10 additional PSbMV isolates were determined. All isolates caused systemic infection in pea. InC. quinoa6 of the isolates that were restricted to inoculated leaves had a serine at position 47. Two isolates that infectedC. quinoasystemically had a proline at position 47. Two isolates, S6 and NEP-1, infectedC. quinoasystemically, but had a serine at position 47 of the CP. This shows that although a proline/serine difference at position 47 of the CP determined systemic spread of the isolates DPD1 and NY, this amino acid alone does not govern the spread of PSbMV inC. quinoa.

Sensitive detection of pea seed borne mosaic potyvirus by dot blot and tissue print hybridisation assays

Dot blot and tissue print hybridisation assays were developed for the rapid and sensitive detection of pea seed borne mosaic virus (PSbMV). Radioactive (32P-labelled) and non-radioactive (DIG-labelled) random primed cDNA probes representing the entire genome of PSbMV were used in hybridisation assays. A comparison of detection sensitivity was made between these probes and they were found equally sensitive. PSbMV was readily detected in dot blots by their homologous cDNA probes to 50 fg in purified preparations and to a 1 : 3125 dilution in purfied total nucleic acid or crude sap from infected plants. The sensitivity of heterologous probes was significantly less than that of homologous probes in purified preparation but nearly the same in purified total nucleic acid or crude sap from infected plants. The virus was also specifically detected in tissue squashes by tissue print hybridisation using cDNA probes. Non-radioactive probes were found suitable for PSbMV diagnosis without significant loss of detection sensitivity. There was no non-specific hybridisation against the healthy control in hybridisation assays using either radioactive or non-radioactive probes.

Contrasting patterns in the spread of two seed‐borne viruses in pea embryos

The temporal and spatial patterns of pea seed‐borne mosaic potyvirus (PSbMV) and pea early browning tobra‐virus (PEBV) accumulation in pea embryos were analyzed using in‐situ hybridization and immunohistochemistry. For PSbMV, which infects embryos after fertilization, the distribution changed as the embryo developed and some tissues remained free of virus infection. In contrast, embryos were uniformly infected with PEBV from the earliest stages of embryo development, and PEBV was detected in the egg cell and pollen grains, indicative of gametic transmission into the embryo. These observations suggest that gametically transmitted viruses may be appropriate as potential vectors for the ectopic and uniform expression of novel genes in embryonic tissues. Functional complementarity in the two processes of embryo invasion was tested following co‐inoculation with PSbMV and PEBV. Instead of complementation, interference in PSbMV seed transmission by PEBV was observed; PEBV seed transmission remained unaffected by PSbMV.

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.

Early Season Survey of Pea Viruses in Pakistan and the Detection of Two New Pathotypes of Pea Seedborne Mosaic Potyvirus.

Sixty-two commercial pea fields or experimental plots located in eight districts of the major pea-growing areas of North West Frontier Province (NWFP) of Pakistan were surveyed for pea viruses in the early winter growing season of 1995. Samples were analyzed by dot-immunobinding assay (DIBA) using antisera to 14 different viruses. Of the 713 plants sampled, 82 were positive for either bean yellow mosaic potyvirus, cucumber mosaic cucumovirus, or pea seedborne mosaic potyvirus (PSbMV), with an average incidence of 9.4, 0.57, and 1.5%, respectively. PSbMV was also detected in 1 to 5% of dry seed from five of the 12 pea varieties tested and in 8 to 20% of seedlings raised from seed of three of these varieties. The infectivities of 12 PSbMV isolates found in the survey of pea varieties from Pakistan were compared using a standard range of pea differential genotypes, and the isolates were classified into four distinct pathotypes. Four isolates were classified as pathotype P-1 and two as P-4. The remainder did not fit into the existing PSbMV pathotype classification and were tentatively placed into two other groups named U-1 and U-2.

Multiple viral determinants affect seed transmission of pea seedborne mosaic virus in Pisum sativum.

Two pea seedborne mosaic potyvirus (PSbMV) isolates, P-1 DPD1 (P-1), which is highly seed-transmitted, and P-4 NY (P-4), which is rarely seed-transmitted, and chimeras between P-1 and P-4 were analysed to map the viral genetic determinants of seed transmission. Infectivity of chimeric viruses was evaluated by inoculating Pisum sativum with RNA transcribed in vitro from recombinant full-length cDNA clones. The chimeric viruses that were used demonstrated that a genomic segment encoding the 49 kDa protease and putative RNA polymerase was responsible for symptom induction. Attempts to determine transmission of the chimeric viruses in P. sativum cultivars known to transmit P1 at high frequencies showed that seed transmission is a quantitative character influenced by multiple viral determinants. Seed transmission frequency did not correlate with accumulation of virus in vegetative tissue. The 5' 2.5 kb of the 10 kb PSbMV genome had a major influence on the seed transmission frequency and was analysed further. This showed that, while the helper-component protease was a major determinant of seed transmission, the potyviral P1 -protease exerted no measurable influence.

Biological and molecular properties of a pathotype P-1 and a pathotype P-4 isolate of pea seed-borne mosaic virus.

Two isolates of pea seed-borne mosaic potyvirus, DPD1 and NY, were identified as pathotypes P-1 and P-4, respectively, by their infectivity on Pisum sativum L. lines homozygous for the recessive resistance genes sbm-1 and sbm-4. The two isolates differed in several biological characteristics. DPD1 induced transient vein clearing, downward rolling of leaflets and internode shortening on P. sativum, whereas NY only caused a slight growth reduction. DPD1 moved systemically in Chenopodium quinoa whereas NY was restricted to inoculated leaves. DPD1 was frequently transmitted by seeds whereas NY was rarely seed-transmitted: 24% and 0.3%, respectively, in P. sativum '549'. Both DPD1 and NY were transmitted by aphids (Myzus persicae), though a DAG triplet was not present in the N terminus of the coat protein. The nucleotide sequence and deduced amino acid sequence of NY were determined and compared to the corresponding sequences of DPD1.

Some studies on cowpea aphid‐borne mosaic and pea seed‐borne mosaic potyviruses in Nepal

Abstract Various virus‐like symptoms were recorded on field‐grown cowpea (Vigna unguiculata L. Walp) and pea (Pisum sativum L.) during 1989–1990 at the experimental farm of the Institute of Agriculture and Animal Sciences (IAAS), Rampur, Chitwan valley, in the central terai plains of Nepal. The causal agent(s) causing narrow and/or curled leaves, and stunting of pea, and prominent mosaic and vein banding of cowpea were transmitted by both aphids and sap inoculations. Electron microscopic examination of leaf extracts from affected cowpea and pea plants revealed the presence of flexuous filamentous virus particles c. 700 nm long, similar to those of potyviruses. The virions in the leaf extracts from cowpea samples reacted strongly to antibodies against cowpea aphid‐borne mosaic potyvirus (CABMV) in immunosorbent electron microscopy, while the extract from the pea samples reacted strongly to antibodies against pea seed‐borne mosaic potyvirus (PSbMV) in enzyme‐linked immunosorbent assay (ELISA). Time of plant...

International programme on the serological detection of bacterial, fungal and viral pathogens of protein pea seeds 1

Arable (protein) peas are widely grown in northern Europe, specifically for use in animal feed. The main production areas (1 million ha) are France, UK and Denmark; The Netherlands is a major seed producer. There are a number of important seed‐borne pathogens, including bacterial blight (Pseudomonas syringae pv. pisi), leaf and pod spot (Mycosphaerella pinodes) and pea seed‐borne mosaic potyvirus (PSbMV), that cause crop losses. There is a need for improved rapid methods for the detection of these pathogens both to monitor the production of high‐quality seed and for quarantine purposes. Using knowledge of the specificity of antibodies raised to these organisms, techniques were developed for simultaneous detection of the pathogens from the same seed sample. Methods were developed that permitted the detection of 101–102 bacterial cfu ml‐1 in seed‐soak fluid, soluble antigen from seed with and without obvious fungal infection and the presence of PSbMV within the embryo of homogenized seed.

Characterization and Molecular Detection of the P4 Pathotype of Pea Seedborne Mosaic Potyvirus

The New York isolate of pea seedborne mosaic potyvirus (PSbMV) pathotype P4 was differentiated from the more prevalent PSbMV-P1 pathotype on selected pea cultivars, serologically and by sequence-specific reverse transcription-polymerase chain reaction (RT-PCR). PSbMV-P4 was seed-transmissible at much lower rates than P1 (0 to 0.7% versus 0 to 32%) in selected pea cultivars and was not detectable by enzyme-linked immunosorbent assay (ELISA) in reproductive tissues (pollen or embryo axes) of pea, whereas P1 was readily detectable in these tissues. P4 also was aphid-transmitted at lower rates (4 to 16%) than those of Pl (11 to 40%) and had distinct virus acquisition-time optima. RT-PCR detected approximately 10 pg of P4 RNA per 1.0 μg of total nucleic acids from P4-infected tissues and provided positive pathotype identification in single or mixed infections. RT-PCR and pathotype-selective ELISA were used to determine seed-transmission rates of the two pathotypes in seedling progeny of pea (Pisum sativum) cultivars inoculated with P1, P4, or P1 + P4. There were slight variations in the frequency of P1 seed transmission in the presence of P4; P4 seed-transmission rates did not increase in the presence of P1.

A surprising effect of extraction conditions on the mobility of some plant virus coat proteins in SDS-PAGE

The coat protein from purified particles of pea seedborne mosaic potyvirus (PSbMV) moves in SDS-PAGE with an apparent molecular weight ( M r) of 36 kDa. However, extracts of PSbMV infected plants prepared with SDS or urea contain PSbMV immunoreactive proteins with apparent ( M r) 39 kDa as well as 36 kDa. The low mobility form may be generated from the apparent ( M r) 36 kDa form by incubating purified PSbMV particles with healthy plant sap in the presence of denaturing agents. A similar effect is observed with bean yellow mosaic potyvirus, but not with three viruses outside the potyvirus group. Experiments suggest that a soluble plant enzyme is responsible for the conversion, which apparently takes place only in vitro under denaturing conditions. This phenomenon may lead to erroneous conclusions about the ( M r) of some viral coat proteins. However, the conversion can be prevented by heat treatment of the plant tissue prior to extraction.

Detection of a 45 kD protein derived from the N terminus of the pea seedborne mosaic potyvirus polyprotein in vivo and in vitro.

A 45 kD protein (Pro1) derived from the N terminus of the pea seedborne mosaic potyvirus (PSbMV) polyprotein has been detected in extracts of infected pea plants and among in vitro translation products of PSbMV genomic RNA. The genomic region coding for the first 231 amino acids of the PSbMV polyprotein was cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase. A rabbit antiserum raised against the fusion protein recognized an approximately 45 kD protein in immunoblots of extracts of PSbMV-infected pea leaves that was not present in extracts of healthy leaves. The highest concentration of the 45 kD protein was found in extracts of young leaves, suggesting the protein may be rapidly degraded in vivo. After in vitro translation of PSbMV genomic RNA in a wheat germ extract, the antiserum immunoprecipitated a 45 kD polypeptide as well as some lower molecular weight translation products. On the other hand, an approximately 90 kD polypeptide was immunoprecipitated from in vitro translation products of genomic RNA in a rabbit reticulocyte lysate, corresponding to the combined molecular weights of Pro1 and the helper component predicted from genomic sequence data.