Sweet Potato Feathery Mottle Potyvirus Research Articles

The P1N-PISPO trans-Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor.

The positive-sense RNA genome of Sweet potato feathery mottle virus (SPFMV) (genus Potyvirus, family Potyviridae) contains a large open reading frame (ORF) of 3,494 codons translatable as a polyprotein and two embedded shorter ORFs in the -1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located in the P1 and P3 regions, respectively. PISPO is specific to some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6 motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D. B. Baulcombe, J. J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965-6967, 2015, doi:10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infected Ipomoea batatas plants. Interestingly, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene in Nicotiana benthamiana revealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within the Potyviridae family. Gene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb, and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In sweet potato feathery mottle virus (SPFMV), another out-of-frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectrometry analysis that P1N-PISPO is indeed produced in SPFMV-infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of the Potyviridae family.

CHARACTERISTICS AND DIVERSITY IN SWEETPOTATO-INFECTING VIRUSES IN AFRICA

In Africa, seven viruses occurring singly or in combination have been identified in sweetpotato. The viruses include Sweet potato feathery mottle potyvirus (SPFMV), Sweet potato chlorotic stunt crinivirus (SPCSV), Sweet potato mild mottle ipoitiovirus (SPMMV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato virus II (SPVII), Sweet potato caulimo-like virus (SPCaLV) and Sweet potato virus G (SPVG). Out of these, the presence of SPFMV was detected in most countries surveyed, while SPCSV occurred in Nigeria, Gabon, Kenya, Uganda, Tanzania, Zambia, Madagascar and Egypt. SPCFV and SPMMV had limited distribution, mainly in East Africa. SPCaLV was only found in Uganda, SPVII in South Africa and SPVG in Ethiopia and Egypt. Molecular characterisation showed that most of the virus isolates have wide genetic variations, although some such as SPCSV are highly conserved. SPCSV is unique among these viruses because it has the ability to synergise others, therefore, is considered to be the most important virus responsible for the development of severe symptoms characteristic of sweetpotato virus disease, hence, it should be a major target for control.

Resistance to Sweetpotato Chlorotic Stunt Virus and Sweetpotato Feathery Mottle Virus Is Mediated by Two Separate Recessive Genes in Sweetpotato

When sweetpotato chlorotic stunt crinivirus (SPCSV) and sweetpotato feathery mottle potyvirus (SPFMV) infect sweetpotato [Ipomoea batatas (L.) Lam.], they interact synergistically and cause sweetpotato virus disease (SPVD), a major constraint to food productivity in east Africa. The genetic basis of resistance to these diseases was investigated in 15 sweetpotato diallel families (1352 genotypes) in Uganda, and in two families of the same diallel at the International Potato Center (CIP), Lima, Peru. Graft inoculation with SPCSV and SPFMV resulted in severe SPVD symptoms in all the families in Uganda. The distribution of SPVD scores was skewed toward highly susceptible categories (SPVD scores 4 and 5), eliminating almost all the resistant genotypes (scores 1 and 2). Likewise, when two promising diallel families (`Tanzania' × `Bikilamaliya' and `Tanzania' × `Wagabolige') were graft inoculated with SPCSV and SPFMV at CIP, severe SPVD was observed in most of the progenies. Individual inoculation of these two families with SPCSV or SPFMV, and Mendelian segregation analysis for resistant vs. susceptible categories led us to hypothesize that resistance to SPCSV and SPFMV was conditioned by two separate recessive genes inherited in a hexasomic or tetradisomic manner. Subsequent molecular marker studies yielded two genetic markers associated with resistance to SPCSV and SPFMV. The AFLP and RAPD markers linked to SPCSV and SPFMV resistance explained 70% and 72% of the variation in resistance, respectively. We propose naming these genes as spcsv1 and spfmv1. Our results also suggest that, in the presence of both of these viruses, additional genes mediate oligogenic or multigenic horizontal (quantitative) effects in the progenies studied for resistance to SPVD.

NATURE OF RESISTANCE OF SWEETPOTATO TO SWEETPOTATO VIRUS DISEASE

Sweetpotato virus disease (SPVD) is a disease syndrome due to the dual infection and synergistic interaction of sweetpotato chlorotic stunt crinivirus (SPCSV) and sweetpotato feathery mottle potyvirus (SPFMV). SPVD causes up to 98% yield loss in East Africa. Uganda’s sweetpotato breeding program released six sweetpotato cultivars with moderate to high levels of field SPVD resistance in 1995. A multidisciplinary partnership involving Namulonge Agricultural and Animal Production Research Institute in Uganda, North Carolina State University, the US Vegetable laboratory (USV, USDA-ARS) and Clemson University, Charleston, South Carolina, and the International Potato Center (CIP), Lima, Peru, has investigated the genetic basis of SPVD resistance. This paper highlights research findings of the response of sweetpotato to sweetpotato virus disease. General combining ability (GCA) and specific combining ability of (SCA) of 45 sweetpotato diallel families, and the GCA to SCA variance component ratios were high, indicating that additive gene effects are predominant in the inheritance of SPVD and recovery. The distribution of SPVD scores in the promising families was skewed toward highly susceptible categories in Uganda and Peru. In the proposed model for inheritance, two genes are unlinked and they are inherited in a hexosamic or tetradisomic manner. Based on amplified fragment length polymorphism (AFLP) and quantitative trait loci (QTL) analyses, two unlinked AFLP markers were associated with the loci conferring resistance to SPCSV and SPFMV.

Diallel analysis of sweetpotatoes for resistance to sweetpotato virus disease

Sweetpotato virus disease (SPVD) is due to the dual infection and synergistic interaction of Sweetpotato feathery mottle potyvirus (SPFMV) and Sweetpotato chlorotic stunt crinivirus(SPCSV), and causes up to 98% yield loss in sweetpotato in East Africa. This study was conducted to determine the inheritance of resistance to SPVD in sweetpotato and to estimate the nature of genetic variance. Ten parental clones varying in reaction to SPVD were crossed in a half diallel mating design to generate 45 full-sib families. The families were graft-inoculated with SPCSV and SPFMV to induce SPVD and evaluated for resistance in a randomized complete block design at two sites in Namulonge, Uganda during 1998–2000. In serological assays for SPFMV and SPCSV,resistance to symptom development and recovery from initial systemic SPVD symptoms, characterised resistant genotypes. Genetic component analysis showed significant effects for both general combining ability (GCA) and specific combining ability (SCA) for resistance to SPVD. GCA to SCA variance component ratios were large (0.51–0.87), hence GCA effects were more important than SCA effects. Resistant parents exhibited high GCA indicating that additive gene effects were predominant in the inheritance of resistance to SPVD and recovery. Narrow-sense heritability (31–41%) and broad-sense heritability (73–98%) were moderate to high, indicating that rapid genetic gains for SPVD resistance could be accomplished by mass selection breeding techniques. Two genotypes, New Kawogo and Sowola, had high negative GCA effects and had several families in specific crosses,which exhibited rapid recovery from SPVD,and are promising parents for enhancement of SPVD resistance and recovery.

Delayed activation of post-transcriptional gene silencing and de novo transgene methylation in plants with the coat protein gene of sweet potato feathery mottle potyvirus

The relationship between post-transcriptional gene silencing (PTGS) and DNA methylation was examined using Nicotiana benthamiana transformed with the coat protein gene including the 3′ non-translated region of sweet potato feathery mottle potyvirus. Line 4.28 showed a delayed activation of the transgene silencing in comparison with the other silenced lines, and showed complete resistance against the recombinant potato virus X engineered to contain the sequence homologous to the transgene when the silencing was activated. The transgene methylation in line 4.28 was less extensive in comparison with those of the other silenced lines before the silencing was activated. However, the extent of methylation increased in the course of plant development and became comparable with those in the other silenced lines. The activated silencing and the increased transgene methylation were reset after meiosis. However, the characters of delayed activation of the silencing and developmentally increased transgene methylation were meiotically transmitted to the next generation. These results suggest that transgene(s) itself has a potential to trigger and reset DNA methylation, which could determine a state of PTGS.

Erratum

Volume 21, Number 1, January 2000, pages 1–8, Sonoda and NishiguchiIncorrect text was added to the Summary of the above paper. The correct Summary is printed below.The publisher apologises for any confusion caused.SummaryUsing the grafting procedure, we examined the transmission of post‐transcriptional gene silencing (PTGS) in Nicotiana benthamiana which had been transformed with the coat protein gene, including the 3′ non‐translated region of the sweet potato feathery mottle potyvirus. Transmission of PTGS from silenced lines to non‐silenced ones was bidirectional, but occurred efficiently from root stocks to scions. The level of transgene methylation in non‐silenced scions grafted onto silenced root stocks was not increased. When grafted scions which had become silenced were removed from silenced root stocks and regrafted onto non‐silenced or vector‐transformed root stocks, PTGS was maintained. However, their progeny did not show PTGS. Previously we reported that our transgenic lines had different target specificities of PTGS for RNA degradation: one line recognized only the 3′ part of the transgene mRNA while others involved the whole transgene mRNA (Sonoda et al. 1999, Phytopathology, 89, 385–391). Using these lines, we showed that target specificity of PTGS induced in non‐silenced scions after grafting was determined by that in silenced root stocks. However, unexpectedly, target specificity of PTGS in silenced scions was not changed by grafting onto silenced root stocks showing different target specificity, indicating that the second PTGS from silenced root stocks was not superimposed to silenced scions.

Graft transmission of post-transcriptional gene silencing: target specificity for RNA degradation is transmissible between silenced and non-silenced plants, but not between silenced plants.

Using the grafting procedure, we examined the transmission of post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana which had been transformed with the coat protein gene, including the 3' non-translated region of the sweet potato feathery mottle potyvirus. Transmission of PTGS from silenced lines to non-silenced ones was bidirectional, but occurred efficiently from root stocks to scions. The level of transgene methylation in non-silenced scions grafted onto silenced root stocks was not increased. When grafted scions which had become silenced were removed from silenced root stocks and regrafted onto non-silenced or vector-transformed root stocks, PTGS was maintained. However, their progeny did not show PTGS. Previously we reported that our transgenic lines had different target specificities of PTGS for RNA degradation: one line recognized only the 3' part of the transgene mRNA while others involved the whole transgene mRNA (Sonoda et al. 1999, Phytopathology, 89, 385-391). Using these lines, we showed that target specificity of PTGS induced in non-silenced scions after grafting was determined by that in silenced root stocks. However, unexpectedly, target specificity of PTGS induced in silenced scions was not changed [corrected] by grafting onto silenced root stocks showing different target specificity, indicating that the second PTGS from silenced root stocks was not superimposed to silenced scions.

Homology-Dependent Virus Resistance in Transgenic Plants with the Coat Protein Gene of Sweet Potato Feathery Mottle Potyvirus: Target Specificity and Transgene Methylation

ABSTRACT Nicotiana benthamiana plants were transformed with the coat protein (CP) coding sequence and the 3' nontranslated region (NTR) of the severe strain of sweet potato feathery mottle potyvirus (SPFMV-S). Regenerated lines were screened for virus resistance using recombinant potato virus X (PVX) engineered to contain the sequence homologous to the transgene. Out of 19 transgenic lines, 7 showed virus resistance after inoculation by the recombinant PVX. In most of the resistant lines, relatively low steady-state accumulation of the CP gene mRNA and little or no protein products were observed, suggesting that the resistance was manifested by a post-transcriptional gene-silencing mechanism. The resistant lines could be divided into two groups according to the target specificity of the silencing mechanism; one group recognizing the 3' part of the transgene mRNA and the other not only the 3' part, but also the 5' and the central part of the transgene mRNA. Particular regions of the transgene corresponding to the RNA target in the resistant lines were differentially methylated compared with the transgene sequence in a susceptible line.

Virus Resistance in Gene-silenced Transgenic Plants with the Coat Protein Gene of Sweet Potato Feathery Mottle Potyvirus.

Virus Resistance in Gene-silenced Transgenic Plants with the Coat Protein Gene of Sweet Potato Feathery Mottle Potyvirus.

Identification of the East African Strain of Sweet Potato Chlorotic Stunt Virus as a Major Component of Sweet Potato Virus Disease in Southern Africa.

Sweet potato virus disease (SPVD) is the most damaging disease of sweet potato Ipomoea batatas (L.) Lam. in Africa. It is caused by sweet potato feathery mottle potyvirus (SPFMV) plus either the West African strain of sweet potato chlorotic stunt crinivirus (Closteroviridae) (SPCSV-WA) (2) or the serologically distinct and apparently more severe East African strain (SPCSV-EA) (1). Typical symptoms of SPVD include severe plant stunting, leaf distortion, chlorosis, mosaic, or vein clearing (1). During a survey done in February 1998 of 48 farmers' fields in Lusaka Province and North Western Province of Zambia, sweet potato plants with typical SPVD symptoms were observed. Incidence was generally 1 to 5% but occasionally >20%. To determine which viruses (SPFMV, SPCSV-EA, SPCSV-WA) were present in symptomatic plants, enzyme-linked immunosorbent assays (ELISAs) were done on leaf sap extracts. Twenty-two SPVD-affected plants from Lusaka Province and 15 from North Western Province were tested and SPFMV and SPCSV-EA (but not SPCSV-WA) were detected in all samples. SPCSV-EA by itself may cause purpling or yellowing of lower or middle leaves (1). Eight plants showing these symptoms were collected from North Western Province, and SPCSV-EA only was detected in six of the samples. SPVD was also observed in a 1997 survey of crops near Antsirable, Madagascar; incidence was generally <1% but occasionally >20%; SPFMV and SPCSV-EA, but not SPCSV-WA, were detected in two SPVD samples tested. Our results are the first report of SPCSV in southern Africa. SPVD in the regions surveyed appears to be due to SPFMV and SPCSV-EA; SPCSV-WA was not detected.

Resistance to sweet potato virus disease (SPVD) in wild East African Ipomoea

Summary.Sweet potato virus disease (SPVD), the most harmful disease of sweet potatoes in East Africa, is caused by mixed infection with sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Wild Ipomoea spp. native to East Africa (J cairica, I. hildebrandtii, I. involucra and J wightii) were graft‐inoculated with SPVD‐affected sweet potato scions. Inoculated plants were monitored for symptom development and tested for SPFMV and SPCSV by grafting to the indicator plant J setosa, and by enzyme‐linked immunosorbent assay (ELISA). Virus‐free scions of sweet potato cv. Jersey were grafted onto these wild Ipomoea spp. in the field, and scions collected 3 wk later were rooted in the greenhouse and tested for viruses using serological tests and bioassays. In all virus tests, J cairica and J involucra were not infected with either SPFMV or SPCSV. J wightii was infected with SPFMV, but not SPCSV, in the field and following experimental inoculation; J hildebrandtii was infected with SPCSV, but not SPFMV, following experimental inoculation. These data provide the first evidence of East African wild Ipomoea germplasm resistant to the viruses causing SPVD.

The incidence of sweet potato virus disease and virus resistance of sweet potato grown in Uganda

SummarySweet potato virus disease (SPVD) was common (25–30% average incidences), and farmers recognised it as an important disease, in sweet potato crops in southern Mpigi, Masaka and Rakai Districts in Uganda, but SPVD was rare in Soroti and Tororo Districts. Whiteflies, which are the vector of sweet potato chlorotic stunt crinivirus (SPCSV) a component cause of SPVD, were correspondingly common on sweet potato crops in Mpigi and rare on crops in Tororo. However, aphids, which are the vectors of sweet potato feathery mottle potyvirus (SPFMV), the other component cause of SPVD, were not found colonising sweet potato crops, and itinerant alate aphids may be the means of transmission. Different sweet potato cultivars were predominant in the different districts surveyed and four local cultivars obtained from Kanoni in S. Mpigi, where whiteflies and SPVD were common, were more resistant to SPVD than four cultivars from Busia in Tororo District, where whiteflies and SPVD were rare. However, nationally released cultivars were even more resistant than the local cultivars from Kanoni. Yield results and interviews with farmers indicated that farmers in S. Mpigi were making compromises in their choice of cultivars to grow, some key factors being SPVD susceptibility, and the yield, taste, and marketability, duration of harvest and in‐ground storability of the storage roots. These compromises need to be included in an assessment of yield losses attributable to SPVD.

Nucleotide sequence analysis of the coat protein genes of two Korean isolates of sweet potato feathery mottle potyvirus.

The coat protein (CP) genes of the genomic RNA of two Korean isolates of sweet potato feathery mottle potyvirus (SPFMV), SPFMV-K1 and SPFMV-K2, were cloned and their complete nucleotide sequences were determined. Sequence comparisons of the two Korean isolates showed 97.8% amino acid identity in the CP cistron, and 79.9% to 99.0% identity with those of 6 other known SPFMV strains. Of 74 amino acid changes totally among the SPFMV strains, 39 changes were located at the N-terminal region. Pairwise amino acid sequence comparison revealed sequence similarities of 48.6 to 70.2% between SPFMV and 20 other potyviruses, indicating SPFMV to be a quite distinct species. Multiple alignment of the CP cistrons from other potyviruses showed that most of the conserved amino acid residues of the genus Potyvirus are well preserved in the corresponding locations.

Symptoms, aetiology and serological analysis of sweet potato virus disease in Uganda

Sweet potato virus disease (SPVD) is the name used to describe a range of severe symptoms in different cultivars of sweet potato, comprising overall plant stunting combined with leaf narrowing and distortion, and chlorosis, mosaic or vein‐clearing. Affected plants of various cultivars were collected from several regions of Uganda. All samples contained the aphid‐borne sweet potato feathery mottle potyvirus (SPFMV) and almost all contained the whitefly‐borne sweet potato chlorotic stunt closterovirus (SPCSV). SPCSV was detected by a mix of monoclonal antibodies (MAb) previously shown to react only to a Kenyan isolate of SPCSV, but not by a mixture of MAb that detected SPCSV isolates from Nigeria and other countries. Sweet potato chlorotic fleck virus (SPCFV) and sweet potato mild mottle ipomovirus (SPMMV) were seldom detected in SPVD‐affected plants, while sweet potato latent virus (SPLV) was never detected. Isolates of SPFMV and SPCSV obtained by insect transmissions together induced typical symptoms of SPVD when graft‐inoculated to virus‐free sweet potato. SPCSV alone caused stunting and either purpling or yellowing of middle and lower leaves when graft‐inoculated to virus‐free plants of two cultivars. Similarly diseased naturally inoculated field plants were shown consistently to contain SPCSV. Both this disease and SPVD spread rapidly in a sweet potato crop.

Resistance against a Heterologous Potyvirus in Transgenic Tobacco Plants Expressing Antisense RNA of HC-Pro Gene of Sweet Potato Feathery Mottle Potyvirus.

Resistance against a Heterologous Potyvirus in Transgenic Tobacco Plants Expressing Antisense RNA of HC-Pro Gene of Sweet Potato Feathery Mottle Potyvirus.

Sequencing and characterization of the coat protein and 3' non-coding region of a new sweet potato potyvirus.

A cDNA library was constructed from viral genomic RNA purified from sweet potato plants affected by "Sweet Potato Chlorotic Dwarf disease" in an attempt to clarify the etiology of this viral complex in Argentina. By sequence analysis, some of the obtained clones were found to belong to sweet potato feathery mottle potyvirus (SPFMV), to a closterovirus and to a new potyvirus. A cDNA clone of 1,103 bp representing the coat protein cistron and 3' non-coding region of the newly identified potyvirus was further characterized. The sequence contained an ORF of 855 nucleotides with a coding capacity of 285 amino acids, followed by a 3' untranslated tail of 248 nucleotides. The core and C-terminal regions have sequences well conserved among potyviruses. Furthermore, amino acid sequence comparisons of the capsid protein with those of other described potyviruses showed 63% homology with SPFMV, 68 to 70% with two different isolates of sweet potato latent potyvirus (SPLV), 57% with sweet potato G potyvirus (SPGV) and 73% with potato virus Y (PVY). These data allowed us to propose the inclusion of this virus as a new member of the family Potyviridae, genus Potyvirus with the designation sweet potato mild speckling potyvirus (SPMSV).

Detection of Sweet Potato Virus Disease-Associated Closterovirus in a Sweet Potato Accession in the United States

A closterovirus (sweet potato virus disease-associated closterovirus [SPVD-aC]) that, in association with sweet potato feathery mottle potyvirus (SPFMV), causes the Nigerian sweet potato virus disease (SPVD), was detected in sweet potato (Ipomoea setosa) following grafting of infected scions of sweet potato cv. White Bunch, an accession in the USDA sweet potato repository. Confirmation of infection with SPVD-aC was by a riboprobe hybridization assay specific for the viral RNA coupled with chemiluminescent detection of the bound RNA, and SPFMV was detected by enzyme-linked immunosorbent assay (ELISA) using a specific antiserum. This is the first report of SPVD-aC and of SPVD in sweet potato in North America. Use of the riboprobe hybridization assay should facilitate a more rapid detection of SPVD-aC infected sweet potato and subsequent selection of virus-free germ plasm for international exchange.

A Preliminary Assessment of Sweet Potato Cultivars for Sweet Potato Feathery Mottle Virus (Spfmv).

Four cultivars of sweet potato (Ipomoea batatas) at the Gatton Research Station (Queensland) were screened for sweet potato feathery mottle virus (SPFMV). Both mother stock and field grown material were indexed by grafting to the indicator plant Ipomoea setosa. Within the limitations of our testing procedures, virus was not detected in mother stock of cw. Rojo Blanco (27 grafts), Resisto (20 grafts), LO-323 (22 grafts) and Red Abundance (23 grafts). Four of 35 grafts made with field-grown material were positive suggesting that virus infection of commercial planting material is more likely to occur by infection during field multiplication than from infected mother stock. Virus infection was detected by symptoms on and the presence of flexuous, filamentous particles in sap extracts of I. setosa. The virus was identified as sweet potato feathery mottle potyvirus by serology.

Sweet potato feathery mottle potyvirus (C1 isolate) virion and RNA purification

A procedure for the purification of a Peruvian isolate (C1) of sweet potato feathery mottle potyvirus (SPFMV) and infective RNA has been developed. The use of Hepes [ N-(2-hydroxyethyl)piperazine- N′-2-ethanesulfonic acid] buffer containing urea and sodium EDTA as a base for tissue extraction and virus suspension enabled good yields of virus (35–50 mg/100 g) to be obtained from Nicotiana benthamiana L. Domin. A short RNA isolation procedure yielded infectious RNA, from which ds cDNA of nearly genome size could be obtained. Sweet potato feathery mottle potyvirus, Purification, RNA isolation, cDNA synthesis.