Transfer of resistance to potato leafroll virus, potato virus Y and potato virus X from Solarium brevidens to S. tuberosum through symmetric and designed asymmetric somatic hybridisation

Resistance to potato leafroll virus (PLRV), potato virus Y (PVY^o) and potato virus X (PVX) was studied in symmetric and asymmetric somatic hybrids produced by electrofusion between Solanum brevidens (2n=2×=24) and dihaploid S. tuberosum (2n=2×=24), and also in regenerants (B-hybrids) derived through protoplast culture from a single somatic hybrid (chromosome number 48). All of the somatic hybrids between 5. brevidens and the two dihaploid lines of potato cv. Pito were extremely resistant to PLRV and PVY^oand moderately resistant to PVX, irrespective of their chromosome number and ploidy level (tetraploid or hexaploid). Most (56%) of the asymmetric hybrids of irradiated S. brevidens and the dihaploid line of potato cv. Pentland Crown (PDH40) had high titres of PVY^osimilar to those of PDH40, whereas the rest of the hybrids had PVY^otitres less than a tenth of those in PDH40. Three B-hybrids had a highly reduced chromosome number (27, 30 and 34), but were however as resistant to PLRV, PVY^oand PVX as 5. brevidens. Two asymmetric hybrids and one B-hybrid were extremely resistant to PLRV but susceptible to both PVY and PVX. The results suggested that resistance to PLRV in 5. brevidens is controlled by a gene or genes different from those controlling resistance to PVY and PVX, and the gene(s) for resistance to PVY and PVX are linked in S. brevidens.     

Accumulation of potato virus Y is enhanced in Solatium brevidens also infected with tobacco mosaic virus or potato spindle tuber viroid

The accumulation of potato virus Y?(PVY?) and potato leaf roll virus (PLRV) was studied in plants of Solanum brevidens co-infected with each of six viruses or a viroid. Virus could not be detected by ELISA in plants of S. brevidens infected solely with PVY. However, accumulation of PVY was increased c. 1000-fold in plants doubly infected with tobacco mosaic virus or potato spindle tuber viroid (PSTVd). PVY titres in doubly infected plants of S. brevidens were between 1% and 0.1% of those found in the PVY-susceptible interspecific Solanum hybrid DTO-33. Double infections of 5. brevidens by PVY and alfalfa mosaic virus or potato viruses M, S, T or X did not significantly enhance PVY accumulation. Accumulation of PLRV was not enhanced in plants co-infected with any of the six viruses or PSTVd.     

马铃薯Y病毒蚜传辅助成分介导PVX/PVY协生作用

构建了马铃薯Y病毒中国株系（PVY－C）蚜传辅助成分（HC－Pro）基因的正义、反义和缺失三种植物表达载体,通过农杆菌介导法转化烟草品种NC89。Southern blot分析表明,HC－Pro基因及其突变体已经整合到烟草染色体中,Western blot分析证明,正义HC－Pro基因及其缺失突变体在转基因烟草中有表达产物,攻毒试验结果表明,转正义,HC－Pro基因及其缺失突变体不仅能够提高T1转基因烟草中PVY－C的病毒积累和致病,而且对异源病毒PVX具有同样的作用,而转反义HC－Pro基因烟草对PVY－C和PVX的致病性无影响,因此,PVY－C HC－Pro基因介导PVX／PVY的协作作用。     

Resistance to cucumber mosaic virus in potato

Reactions to two subgroup I isolates (Fny-CMV and Pf-CMV) and two subgroup II isolates (A9-CMV and LS-CMV) of cucumber mosaic virus (CMV) were studied in three non tuber-bearing wild potato species (Solanum spp.) of the series Etuberosa, and in two tuber-bearing interspecific potato hybrids and four potato cultivars using graft-inoculation. Three classes of phenotypic reactions (susceptible, hypersensitive, extreme resistance) were observed in the tuber-bearing genotypes. Susceptible genotypes developed mosaic or severe mosaic with leaf malformation and had high CMV titres. Hypersensitive genotypes developed either top necrosis or vein necrosis and/or necrotic spots on apical leaves, and had low CMV titres. Extremely resistant genotypes had no symptoms and no CMV was detected. The hybrid 87HW13.7 (S. tuberosum×S. multidissectum) developed top necrosis specific to infection with Fny-CMV. The hybrid ‘A6’ (S. demissum×S. tuberosum cv. Aquila) was hypersensitive to all CMV isolates tested. Extreme resistance was not functional against all CMV isolates. Neither hypersensitivity nor extreme resistance were related to the CMV subgroup.     

Engineering virus resistance in agricultural crops

Plant viral genomes are relatively small and in the past decade many have been characterized at the molecular level. This has prompted research into the development of virus resistance based on interference with the viral multiplication cycle by the introduction of viral sequences into the plant genome. Several strategies have been tested. The most successful one so far involves the constitutive expression of the coat protein gene of the virus against which resistance is desired. In this review we describe progress made in engineering virus resistance into potato, an important agricultural crop. To this end the molecular structure of the potato viruses X and Y and leafroll is discussed as well as the introduction of resistance against potato virus X into potato. In addition, we address the question of preservation of cultivar-specific characteristics, an important prerequisite for commercial application. Finally, recent investigations for alternative forms of virus resistance are described against the background of the results of coat protein-mediated protection.     

Economic importance of potato virus diseases distribution in the republic of Belarus

The phytopathological situation in potato plantings in Belarus is analysed. A wide distribution of mosaic potato viruses and their strains is indicated. A complex of measures directed to virus disease spread limitation under modern ecological and economic conditions is detennined. It is stressed that the best solution of the problem is the foundation and growing of resistant to virus diseases potato varieties.     

Resistance to potato leaf roll virus multiplication in potato is under major gene control

The concentration of potato leaf roll virus (PLRV), as measured by a quantitative enzyme-linked immunosorbent assay, in the foliage of potato plants (Solanum tuberosum) of cv Maris Piper with secondary infection was 2900 ng/g leaf, whereas in clones G7445(1) and G7032(5) it was 180 ng/g leaf and 120 ng/g leaf, respectively. To examine the genetic control of resistance to PLRV multiplication, reciprocal crosses were made between the susceptible cultivar Maris Piper and the two resistant clones, and the three parents were selfed. Seedling progenies of these families were grown to generate tubers of individual genotypes (clones). Clonally propagated plants were graft-inoculated, and their daughter tubers were collected and used to grow plants with secondary infection in which PLRV concentration was estimated. The expression of resistance to PLRV multiplication had a bimodal distribution in progenies from crosses between Maris Piper and either resistant clone, and also in progeny from selfing the resistant parents, with genotypes segregating into high and low virus titre groups. Only the progeny obtained from selfing Maris Piper did not segregate, all genotypes being susceptible to PLRV multiplication. The pattern of segregation obtained from these progenies fits more closely with the genetical hypothesis that resistance to PLRV multiplication is controlled by two unlinked dominant complementary genes, both of which are required for resistance, than with the simpler hypothesis that resistance is conferred by a single dominant gene, as published previously.     

应用Dot—ELISA检测PVX,PVY和PVS

以ＮＣＭ为固相载体、应用间接ＥＬＩＳＡ法测定了纯化的ＰＶＸ、ＰＶＹ和ＰＶＳ;对接种的烟草,马铃薯块茎的芽、休眠块茎顶端的稀释度ＰＶＸ分别为：１／２０４８０－１／８１９２０、１／５１２０;ＰＶＹ分别为１／８１９２０、１／２０４８０和１／５１２０;ＰＶＳ分别为１／８１９２０－１／３２７６８０、１／２０４８０－１／８１９２０和１／５１２０－１／２０４８０,和Ｃｏｃｋｔａｉｌ－ＥＬＩＳＡ相关,检测ＰＶＸ和     

Spread of potato leafroll virus is decreased from plants of potato clones in which virus accumulation is restricted

Tubers of eight potato clones infected with potato leafroll luteovirus (PLRV) were planted as ‘infectors’ in a field crop grown, at Invergowrie, of virus-free potato cv. Maris Piper in 1989. The mean PLRV contents of the infector clones, determined by enzyme-linked immunosorbent assay (ELISA) of leaf tissue, ranged from c. 65 to 2400 ng/g leaf. Myzus persicae colonised the crop shortly after shoot emergence in late May and established large populations on all plants, exceeding 2000/plant by 27 June. Aphid infestations were controlled on 30 June by insecticide sprays. Aphid-borne spread of PLRV from plants of the infector clones was assessed in August by ELISA of foliage samples from the neighbouring Maris Piper ‘receptors’. Up to 89% infection occurred in receptor plots containing infector clones with high concentrations of PLRV. Spread was least (as little as 6%) in plots containing infectors in which PLRV concentrations were low. Primary PLRV infection in guard areas of the crop away from infectors was 4%. Some receptor plants became infected where no leaf contact was established with the infectors, suggesting that some virus spread may have been initiated by aphids walking across the soil.     

Novel resistances to four potyviruses in tuber-bearing potato species, and temperature-sensitive expression of hypersensitive resistance to potato virus Y

Novel potyvirus resistance specificities were found in eight tested wild potato species (clones): hypersensitive resistance (HR) to potato Y potyvirus (PVY) strain groups PVY^O in Solanum megistacrolobum and S. polyadenium and PVY^N in S. stoloniferum; HR to potato V potyvirus (PW) in S. maglia, S. polyadenium, S. stoloniferum, S. sparsipilum and S. sucrense, HR to potato A potyvirus (PVA) strain group 1 in S. sucrense, and extreme resistance (ER) to PVA in S. polyadenium. S. commersonii and S. stoloniferum expressed HR to tobacco etch potyvirus (TEV) which has not been reported previously in potato species. The studied clone of S. stoloniferum expressed HR to all potyviruses and potyvirus strains tested. The clone of S. stoloniferum (2n = 48; nuclear DNA content (2C) = 3.6 pg) and S. chacoense (2n = 24; 2C=1.9 pg) were crossed and one hybrid (2n = 36; 2C = 2.9 pg) was obtained. The hybrid expressed HR to all tested potyviruses except PVA, which indicated that HR to PVA was controlled by a gene which is different from the genes (or gene) controlling HR to PVY^O, PVY^N, PVV and TEV in S. stoloniferum. On the other hand, S. chacoense and the hybrid expressed ER to cucumber mosaic cucumovirus (CMV), whereas S. stoloniferum was susceptible to CMV. All tested wild species and the six tested potato cultivars (S. tuberosum subsp. tuberosum) expressed HR to PVV. Expression of HR following infection with PVY^N induced systemic acquired resistance (SAR) in S. chacoense. HR to PVY^N in S. sparsipilum and S. sucrense and to PVY^O in potato cv. Pito was efficiently expressed at lower temperatures (16/18°C) indicated by the development of distinct necrotic lesions and/or vein necrosis in inoculated leaves, whereas the HR was rendered less effective at higher temperatures (19/24°C) which was indicated by the development of systemic infection with leaf-drop and mosaic symptoms.     

Distribution and accumulation of PVM and PVY,PVX in infected potato plants

The ability of PVM, PVY and PVX viruses and their progeny to distribute themselves and accumulate in primary infected potato plants by mono‐ and mixed infections is analysed. It is shown, that the transport and accumulation of virus in inoculated potato plants depends on variety resistance, combination of viruses and sequence of their application.     

Aspects of resistance to sweet potato virus disease in sweet potato

In field trials during the first and the second rainy season of 1996 in Uganda, whiteflies were similarly abundant and aphids were absent on three clones of sweet potato (NIS-93–63, cv. Tanzania and cv. New Kawogo) although the three clones differed considerably in their resistance to sweet potato virus disease (SPVD), a complex disease resulting from infection by both the aphid-borne sweet potato feathery mottle virus (SPFMV) and the whitefly-borne sweet potato chlorotic stunt virus (SPCSV). This suggests that vector resistance does not determine the relative SPVD resistance of these genotypes. SPFMV alone had only a low virus titre in sweet potato cvs Tanzania and New Kawogo, became increasingly difficult to detect in plants of these cultivars and was seldom acquired by aphids. However, this resistance to SPFMV was not apparent in plants which were also infected with SPCSV. Plants then had a high SPFMV titre, appeared unable to eliminate SPFMV and provided good sources for aphids to acquire it.     

Interactions of the Solanum spp. of the Etuberosa group and nine potato-infecting viruses and a viroid

All 26 accessions of Solanum brevidens, one accession of S. etuberosum and one accession of S. fernandezianum tested were all extremely resistant to potato leafroll virus (PLRV) and potato viruses Y (PVY) and A (PVA). S. brevidens and S. etuberosum were also resistant to Andean potato mottle virus (APMV) and moderately resistant to potato virus X (PVX), whereas S. fernandezianum was susceptible to these viruses. Additionally, S. brevidens was resistant to sap-inoculated potato viruses M (PVM) and S (PVS). All the Etuberosa accessions were susceptible by graft-inoculation to PVM, PVS, potato virus T (PVT) and Andean potato latent virus (APLV). Infections by the above mentioned viruses were symptomless in all of the Etuberosa spp. S. etuberosum and S. fernandezianum were infected by mechanical inoculation with potato spindle tuber viroid, S. etuberosum developing severe stunting and leaf-curl symptoms, but S. brevidens was infected only by graft-inoculation. The genes conferring resistance to PVY and PVX in S. brevidens and S. etuberosum appeared to be different from those currently utilised by plant breeders.     

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

Sweet 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.     

Facile assessment of cDNA constructs for expression of functional antibodies in plants using the potato virus X vector

Antibodies have been expressed in plants to confer novel traits such as virus resistance or altered phenotype. However, not every antibody is suitable for plant expression, and successful intracellular expression of antibody fragments depends primarily on their amino acid sequence in a way that is as yet unpredictable. Therefore it is desirable to assess different constructs before embarking on the production of transgenic plants. We have used a transient expression system based on potato virus X to compare different cDNA constructs for expression and stability of antibody variable gene fragments in plants. Constructs contained an anti-plant enzyme (granule-bound starch synthase I) scFv sequence derived from a naive phage display library together with different combinations of sequences encoding the human IgG constant domain, a murine IgG secretory signal sequence, or the endoplasmic reticulum retention signal peptide KDEL. The results obtained with the potato virus X vector correlated with those from Agrobacterium-mediated stable transformation of potato. The best expression levels were obtained by incorporating sequences that target scFv to the lumen of the endoplasmic reticulum and the secretory pathway. The anti-enzyme scFv retained activity during storage of potato tubers for more than five months. The results demonstrate the utility of the potato virus X vector for the analysis and comparison of many scFv with different epitope specificities or sequence modifications. Evaluation of scFv by transient expression from the PVX vector should aid progress in selection of functional scFv for applications in plant biotechnology.     

利用RNA干涉技术及微束激光转化法培育抗病毒马铃薯

针对马铃薯生产中危害严重、分布广泛并且具有强烈协生作用的马铃薯X病毒和马铃薯Y病毒,开展了抗病毒病的育种研究。采用RT-PCR技术分别克隆了267 bp的编码马铃薯X病毒外壳蛋白(PVX-cp)基因的相对保守区段X和294 bp的编码马铃薯Y病毒外壳蛋白(PVY-cp)基因的相对保守区段Y两个片段,将其按顺序连接,形成融合基因XY,然后分别将融合基因XY正向和反向插入到载体pHANN IBAL中,得到了载体pH IV,再用NoⅠt对pH IV进行酶切,将产生的大片段插入到载体pART27中,得到同时以PVX-cp基因和PVY-cp基因为靶标的XY的RNA i载体pAR IV。采用微束激光穿刺转化技术转化马铃薯品种Favorita的愈伤组织,得到了14株表型正常的转基因植株,转基因植株的southern b lot分析表明,导入的外源融合基因拷贝数介于2~4之间。攻毒实验表明,其中11株转基因植株对PVX、PVYo以及PVX和PVYo混合侵染均表现免疫,而其它3株的病毒浓度也低于对照。这一结果将为利用RNA i干涉技术开展植物抗多种病毒病的育种提供重要依据,验证了所构建的RNA干涉(RNA interference RNA i)载体具有良好的功能,同时又一次证明微束激光穿刺法是一种有效的遗传转化手段。     

Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing

In this study we applied RNA silencing to engineer potato plants that are resistant to potato virus Y (PVY). We expressed double-stranded (ds) RNA derived from the 3 terminal part of the coat protein gene of PVY, which is highly conserved in sequence amongst different PVY isolates, in transgenic potatoes of the commercial variety Spunta. Transgenic plants were analyzed for generation of transgene-derived short interfering RNAs (siRNAs) prior to virus inoculation. Twelve of fifteen transgenic lines produced siRNAs and were highly resistant to three strains of PVY, each belonging to three different subtypes of the virus (PVY^N, PVY^O and PVY^NTN). Infection of transgenic plants with Potato virus X (PVX) simultaneously or prior to the challenge with PVY did not interfere with PVY-resistance.Anastasia Missiou: M.A. and K.K. have contributed equally to this workKriton Kalantidis: M.A. and K.K. have contributed equally to this work     

Incidence of potato virus Y infection in seed and ware tubers of the potato cv. Record

The incidence of potato virus Y (PVY) infection was assessed in samples of potato tubers, cv. Record, taken from Scottish seed stocks and English ware crops grown from some of these seed stocks. PVY was readily detected by ELISA of tuber sprouts. PVY-infected tubers were found in 10 seed stocks of 84 tested. The mean level of virus infection was 0.23%, 0.76% and 0.56% in Super Elite, Elite and AA stocks respectively. In 46 commercial ware crops grown from some of these seed stocks, a substantial proportion of the harvested tubers in all but one of the crops were infected with PVY, the mean percentage of infected tubers was 58.5%. Ware crops grown from seven seed stocks in which PVY had been detected (mean 6.2% infection in seed) contained a mean of 70% infected tubers, compared with 56% infection in crops grown from 39 stocks in which PVY was not detected in the seed tubers. The predominant PVY strain detected in the ware crops was the veinal necrosis strain (PVY^vn).     

Behavior of potato gametoclonal plants against the necrotic strain of potato Y potyvirus

A total of 59 Solanum tuberosum androgenetic plants have been obtained through anther culture, 47 of which derived from a tetraploid clone, seven from a diploid hybrid, and five from an anther-derived clone. About two thirds of the anther-derived plants were dihaploids, a few were monohaploids (5.08%) or aneuploids (6.78%), whereas the tetraploid genotype generated about a third of tetraploids. Seven hundred twenty seven R₁ plants arisen from tubers of the androgenetic potatoes were mechanically inoculated with the necrotic strain of the potato Y potyvirus (PVY^N) and grown in a glasshouse. Fifty days after inoculation, the presence of PVY^N in R₁ plants was detected by DAS-ELISA (Double Autibody Sandwich). Only three plants (0.4%) of genotype H2-258 exhibited local necrotic symptoms (hypersensitivity reaction) suggesting the presence of the N _y gene, and this extreme resistance is epistatic to hypersensitive resistance. The immunity (R _y-gene) to PVY^N was retained through anther culturing and present at all levels of ploidy. The pattern of segregation for immunity was differentiated according to the ploidy level of the anther-derived plants. This changed segregation pattern may be due to a loss of resistance during the culturing, when an endoreduplication has taken place or to the possible regeneration from Second-division restituted unreduced microspores. Anyway, this segregation pattern must be taken into account when gametoclones are used in genetic studies. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 572–578. The text was submitted by the authors in English.