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PubMed2006

Analysis of sphingolipids in potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) by reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS).

Ceramides and glucocerebrosides of potatoes (Solanum tuberosum L.) and sweet potatoes (Ipomoea batatas (L.) Lam.) were analyzed using RP-HPLC-ESI-MS/MS. Ceramides and glucocerebrosides containing the three different long-chain bases 4,8-sphingadienine (d18:2(delta4,delta8)), 4-hydroxy-8-sphingenine (t18:1(delta8)), and 8-sphingenine (d18:1(delta8)) acylated to saturated and unsaturated hydroxy- and nonhydroxy fatty acids with 16-26 carbon atoms were detected. For ceramides and glucocerebrosides 4,8-sphingadienine (d18:2(delta4,delta8)) was found as the major long-chain base, with lesser amounts of 4-hydroxy-8-sphingenine (t18:1(delta8)) and 8-sphingenine (d18:1(delta8)). 2-(Alpha-)hydroxypalmitic acid (C16:0h) was the major fatty acid, which was found to be acylated to the long-chain bases. For quantification of these compounds, an RP-HPLC-ESI-MS/MS method with an "echo-peak"-technique simulating internal standard injection was developed. The analyzed samples of potatoes and sweet potatoes showed amounts of approximately 0.1-8 microg/kg single ceramides and amounts up to 500 microg/kg glucocerebrosides, with C16:0h-glucosyl-4,8-sphingadienine as the major component.

Ceramides↗
PubMed1993

A feature of the coat protein of potato virus X affects both induced virus resistance in potato and viral fitness.

The coat protein of PVX determines whether isolates of PVX are affected by Rx-mediated resistance in potato. Isolates with the coat protein of PVXHB are not affected by the resistance; those with the coat protein of PVXUK3 elicit an extreme resistance in the Rx potato that prevents virus accumulation, even on the inoculated leaf. In this paper we describe the analysis of a series of hybrid and mutant isolates of PVXHB and PVXCP4 which were inoculated to plants and protoplasts of Rx and rx cultivars of potato. From the virulence phenotypes of these isolates we conclude that elicitation of the resistance is affected by amino acids 121 and 127 of the viral coat protein, with codon 121 being the major determinant. PVXHB and hybrid or mutant isolates with lysine and arginine at positions 121 and 127 were able to overcome the resistance of Rx, whereas those with threonine and arginine were resistance sensitive both on plants and in protoplasts. The viral isolates with single mutations at either codon 121 or 127 were less infectious than the wild-type or double mutant isolates although, in protoplasts of the susceptible cultivar of potato, they accumulated as well as the wild-type virus. Taken together these data suggest that amino acids 121 and 127 affect a feature of the viral coat protein which may interact with cellular components involved in the spread of PVX and with the product of the Rx resistance gene.

Amino Acid Sequence↗
PubMed1993

Activity of the promoter of the Lhca3.St.1 gene, encoding the potato apoprotein 2 of the light-harvesting complex of Photosystem I, in transgenic potato and tobacco plants.

We have isolated cDNA and genomic clones for the potato (Solanum tuberosum) apoprotein 2 of the light harvesting complex of Photosystem I, designated Lhca3.St.1. The protein shows all characteristics of the family of chlorophyll a/b-binding proteins. Potato Lhca3.1 gene expression occurs predominantly in leaves, and is transcriptionally regulated by light. One gene copy is present per haploid genome. The sequence of the 5' upstream region was determined. Most boxes identified in the promoter sequences of genes whose expression is light-regulated recur in the Lhca3.St.1 sequence. Functional analyses of the Lhca3.St.1 promoter and two deletion derivatives in transgenic potato transformed with a promoter-GUS fusion show high promoter activity in leaves and other green parts of the plant, which depends on light. Activity is absent in roots and potato tubers. The 500 bp promoter fragment is as active as the full 2.0 kb sequence, showing that all regulatory elements are present on the smallest deletion derivative. In transgenic tobacco (Nicotiana tabacum) plants carrying the largest promoter derivative a similar distribution of activity is found. Promoter activity is not restricted to the phloem, but also prominent in the xylem of the young stem, which contrasts with promoters of other photosynthesis-associated genes.

Amino Acid Sequence↗
PubMed1991

Expression of the potato leafroll luteovirus coat protein gene in transgenic potato plants inhibits viral infection.

Transgenic potato plants, cultivar Désirée, were produced that contained the coat protein gene of potato leafroll luteovirus (PLRV). The transformed potato plants expressed the PLRV coat protein (CP) RNA sequences but accumulation of coat protein in transgenic tissues could not be detected. Upon inoculation with PLRV, the PLRV CP RNA expressing potato plants showed a reduced rate of virus multiplication.

Base Sequence↗
PubMed1998

Multiple elements of the S2-RNase promoter from potato (Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco.

A functional analysis of the promoter of the S2-RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S2-RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S2 promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterized by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S1-RNase and S2-RNase promoters, termed motif and motif III, are located in a fragment of the S2 promoter extending from position of -200 to bp -100, and motif II, located between by -498 and -480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified with the motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer-either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S2-RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other issues.

Enhancer Elements, Genetic↗
PubMed1995

Digoxigenin-labelled cDNA probes for the detection of potato virus Y in dormant potato tubers.

An isolate (Y139) of the potato virus Y strain PVYO, common in the potato-growing regions of eastern Canada, was cloned and segments of about 1800 bases from the 3'-end of the PVY-genome were sequenced. These clones were used to prepare molecular detection probes by labelling with digoxigenin. Five methods reported to yield nucleic acid preparations from tuber tissue were compared. Two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. The other three methods yielded satisfactory nucleic acids which could be successfully detected with the PVYO probes. A method of nucleic acid extraction from tuber tissues was selected which is simple, reproducible and does not use phenol. Using this standardized procedure, PVY was detected in freshly harvested, greenhouse-grown tubers of 8 potato cultivars; the probe was specific to PVY; and the sensitivity of detection using purified viral RNA was down to 10 pg. The sensitivity of detection in tuber extracts was down to a dilution of 1:64, while that from potato leaves was to a dilution of 1:1024. Nucleic acids prepared by the standardized procedure and denatured prior to storage at 4 degrees C, -20 degrees C or at -70 degrees C remained suitable for hybridization for over 2 weeks.

DNA Probes↗
PubMed2006

Cytochrome c oxidase mRNA as an internal control for detection of Potato virus Y and Potato leafroll virus from single aphids by a co-amplification RT-PCR assay.

Using cytochrome c oxidase subunit 1 (COX1) mRNA as the internal control, a triplex reverse transcription-polymerase chain reaction (RT-PCR) for detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) with co-amplification of COX1 from single specimens of various aphid species has been developed. Partial length cDNA of COX1 from green peach aphid, Myzus persicae (Sulzer), potato aphid, Macrosiphum euphorbiae (Thomas), buckthorn aphid, Aphis nasturtii (Kaltenbach), and pea aphid, Acyrthosiphom pisum (Harris), was cloned and sequenced. These sequences, together with existing COX1 sequences from other aphid species capable or suspected to be capable of transmitting PVY and/or PLRV, were analyzed. The sequence identity between any two aphid species ranged from 97 to 100% at the putative protein level, and 89 to 94% at the nucleic acid level. Two highly conserved COX1 nucleotide sequence stretches were selected to design universal primers Aph F and Aph R. This primer pair, together with two existing universal primer pairs (C1-J-2183 and C1-N-2329; Favret F and Favret R), were evaluated at the optimal annealing temperature using RNA from M. persicase, M. euphorbiae, and A. nasturtii. The Aph primer pair performed well in the monoplex RT-PCR but poorly in the triplex RT-PCR in the presence of the PVY- and PLRV-specific primers. On the other hand, the Favret and C1 primer pairs performed well in both monoplex and triplex RT-PCR formats using single aphids of M. persicase, M. euphorbiae and A. nasturtii, demonstrating their suitability to indicate the successfulness of RT-PCR assays for PVY and PLRV. Using the Favret, PVY and PLRV primer sets, single aphids of M. persicase, M. euphorbiae and A. nasturtii that had been exposed to PLRV-infected and/or PVY-infected potato plants were assessed for their acquisition of the viruses by the triplex RT-PCR assay. Although majority (175/180) of the aphid samples produced the COX1 fragment, five aphid samples failed to produce either the COX1- or the virus-specific band, indicating failed RT-PCR in these samples. This method offers a sensitive tool for detection of viruliferous aphids combined to an effective quality control measure.

Animals↗
PubMed2006

A northward colonisation of the Andes by the potato cyst nematode during geological times suggests multiple host-shifts from wild to cultivated potatoes.

The cyst nematode Globodera pallida is a major pest of potato in South America where this specialist parasite is native. To investigate its phylogeography, we have genotyped individuals from 42 Peruvian populations using mitochondrial and nuclear molecular markers. A clear south-to-north phylogeographical pattern was revealed with five well-supported clades. The clade containing the southern populations is genetically more diverse and forms the most basal branch. The large divergence among cytochrome b haplotypes suggests that they diverged before human domestication of potato. As the nematodes studied have been sampled on cultivated potato, multiple host-shifts from wild to cultivated potatoes must have occurred independently in each clade. We hypothesise that this south-to-north pattern took place during the uplift of the Andes beginning 20 My ago and following the same direction. To our knowledge, this is the first study of a plant parasite sampled on cultivated plants revealing an ancient phylogeographical pattern.

Animals↗
PubMed1997

Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA.

NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 degrees C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The NASBA reaction products were visualized after electrophoresis by ethidium bromide or acridine orange staining. The specificity of the amplification products was validated by Northern blot analysis with a PLRV-specific 32P-labelled oligonucleotide probe. The procedure was coupled to immunocapture of PLRV virions from tuber extracts by immobilized antibodies in microtubes. It was possible to discriminate readily by this method between uninfected and primarily PLRV-infected potato tubers. NASBA is suitable for the direct detection of PLRV in potato tubers from primarily infected plants, offering the potential to considerably simplify the inspection of seed-potatoes for virus infection.

Animals↗
PubMed1993

Transgenic potato plants expressing mammalian 2'-5' oligoadenylate synthetase are protected from potato virus X infection under field conditions.

We cloned and sequenced a rat cDNA encoding the 2'-5' oligoadenylate synthetase, a component of the mammalian interferon-induced antiviral response, and used Agrobacterium-mediated transformation to generate transgenic potato clones expressing this mammalian enzyme. In transgenic plants infected with potato virus X and followed under field conditions, virus concentrations in leaves and in tubers were significantly lower than in nontransgenic controls. Additionally, virus concentration in the leaves of five transgenic clones and in tubers of one clone was also lower than in transgenic potatoes expressing potato virus X coat protein.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Potential adoption and management of insect-resistant potato in Peru, and implications for genetically engineered potato.

This paper analyzes some important issues surrounding possible deployment of genetically engineered (GE) insect-resistant potato in Peru, based on a large farmer survey held in Peru in 2003. We found that the formal seed system plays a limited role compared with the informal seed system, especially for smallholder farmers. Although 97% of smallholder farmers would buy seed of an insect-resistant variety, a majority would buy it only once every 2 to 4 years. Survey data show that farmers would be willing to pay a premium of 50% on seed cost for insect resistant varieties. Paying price premiums of 25% to 50%, farmers would still increase their net income, assuming insect resistance is high and pesticide use will be strongly reduced. Of all farmers, 55% indicated preference for insect-resistant potato in varieties other than their current varieties. The survey indicates that smallholder farmers are interested to experiment with new varieties and have a positive perception of improved varieties. Based on these findings, and considering the difficulties implementing existing biosafety regulatory systems such as those in place in the U.S. and E.U., we propose to develop a variety-based segregation system to separate GE from conventionally bred potatoes. In such a system, which would embrace the spread of GE potatoes through informal seed systems, only a limited number of sterile varieties would be introduced that are easily distinguishable from conventional varieties.

Agriculture↗
PubMed1980

Sequence homology between potato and rabbit muscle phosphroylases. Isolation of cysteinyl peptides by covalent chromatography from the potato enzyme and their amino acid sequences.

To elucidate the structural similarity between alpha-glucan phosphorylases from different sources, the amino acid sequences of the cysteinyl regions in potato phosphorylase were determined, and compared with the complete sequence of the rabbit muscle enzyme. Cysteinyl peptides were purified by covalent chromatography based on thiol-disulfide exchange. Potato phosphorylase was coupled to Thiopropyl-Sepharose 6B in the presence of urea, and, after tryptic digestion, 10 distinct cysteinyl peptides which accounted for all the cysteine present in the enzyme were finally isolated in high yields. From the sequence comparison, all of the peptides surrounding the cysteinyl residues in the potato enzyme are homologous with widely distributed specific regions in the rabbit muscle enzyme, although only 4 of 10 cysteinyl residues are conserved between the two enzymes. These observations, in addition to the previously established homology in the cofactor site, show that potato and rabbit muscle phosphorylases are similar in terms of the primary structure. The different properties of cysteinyl residues in the two enzymes are discussed based on the present sequence comparison and the recent X-ray crystallographic data for the rabbit muscle enzyme.

Amino Acid Sequence↗
PubMed1992

Resistance to phloem transport of potato leafroll virus in potato plants.

A 'double-graft sandwich' technique in which sections of potato stem from different potato cultivars were grafted between a susceptible healthy stock plant and a potato leafroll virus (PLRV)-infected scion was used to study the rate of phloem transport of PLRV in cultivars differing in resistance to PLRV infection (IR) and accumulation (AR). Resistance to phloem transport (i.e. delayed PLRV systemic movement) was found in Bismark cultivar (IR A(S)). This was independent of IR and AR as the rate of movement in Bismark cultivar was markedly slower than that in Omega and Spunta (IR AR), Delaware (I(S) AR), and Desiree and Renova (I(S) A(S)) cultivars. It operated in Bismark cultivar stems of two different ages, but did not operate against potato virus X (PVX) and was not influenced by previous infection with this virus. Aphid vector (Myzus persicae) feeding preferences and colonization rates differed between cultivars, but the cultivar characteristics responsible were unrelated to IR, AR or resistance to phloem transport. Delayed systemic movement of PLRV out of leaves inoculated with viruliferous aphids was independent of AR and resistance to phloem transport, and remained unaffected by previous infection with PVX. It was also independent of cultivar factors causing different aphid feeding preferences and colonization rates, but may be linked to IR.

Biological Transport↗
PubMed1995

Analysis of potato virus X coat protein genes in relation to resistance conferred by the genes Nx, Nb and Rx1 of potato.

The coat protein gene nucleotide sequences from eight previously uncharacterized strains of potato virus X (PVX) were determined. Comparison of the deduced amino acid sequences showed that two classes of PVX coat protein, designated types X and B, could be distinguished based on protein length and overall amino acid identities. In all there were 14 amino acid positions where all of the type X proteins differed from all of the type B proteins. The PVX coat protein is the principal viral determinant of the outcome of interactions between the virus and potatoes carrying either the Nx or Rx1 resistance genes. The different strains of PVX were tested for their ability to overcome resistance conferred by three potato resistance genes: Nx, Nb and Rx1. All of the strains that were avirulent on potato cultivars carrying the Nx resistance gene were found to have type X coat proteins whereas strains capable of overcoming the Nx resistance had type B coat proteins.

Amino Acid Sequence↗
PubMed1997

Short and long distance spread of potato leafroll luteovirus: effects of host genes and transgenes conferring resistance to virus accumulation in potato.

Potato leafroll luteovirus (PLRV) movement through phloem of PLRV-resistant potato clones was examined in experiments in which stem pieces were grafted either between infected rootstocks and virus-free susceptible scions or between infected scions and virus-free susceptible rootstocks. These test plants permitted either upwards or downwards virus movement into the susceptible tissue. Resistant potato clones had either host gene-mediated resistance (H-MR) or transgene-mediated resistance (T-NR, conferred by transformation with the PLRV coat protein gene) to PLRV accumulation. The rate of PLRV movement was similar whether stem tissue was taken from H-MR, T-MR or susceptible potato clones. Virus movement through two graft unions began around 7 days after grafting and was generally complete by about 14 to 16 days. Virus movement occurred soon after acquiring functional phloem continuity across grafts as demonstrated by tracing with 6(5)-carboxyfluorescein, a phloem-mobile dye. Most of the delay in virus detection after grafting probably resulted from the time necessary to develop new phloem strands across graft unions; subsequent movement of PLRV was rapid suggesting a passive process. PLRV infection was largely excluded from external phloem bundles in stem tissue of clones with either H-MR or T-MR. This trait was less pronounced as tissue aged. The mechanism limiting PLRV invasion of external phloem bundles of the T-MR clones appears to be similar to that operating in the H-MR clones. Results are discussed in the context of a proposed model of PLRV movement.

Antigens, Viral↗
PubMed2004

Potato virus X-induced gene silencing in leaves and tubers of potato.

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.

Base Sequence↗
PubMed1994

Posttranslational modification of an isoinhibitor from the potato proteinase inhibitor II gene family in transgenic tobacco yields a peptide with homology to potato chymotrypsin inhibitor I.

A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.

Amino Acid Sequence↗
PubMed1997

Pyrophosphorylases in potato. V. Allelic polymorphism of UDP-glucose pyrophosphorylase in potato cultivars and its association with tuber resistance to sweetening in the cold.

UDP-glucose pyrophosphorylase (UGPase) was cloned from six American and nine European potato (Solanum tuberosum L.) cultivars. Restriction mapping of the different UGPase-cDNAs with BamHI, HindIII, and EcoRI revealed that at least two mRNA populations were present in most cultivars. Staining for UGPase activity in nondenaturing gels of proteins extracted from developing potato tubers yielded two major isozymes that were highly active and appeared to be dimeric in nature. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all isozymes were disassociated into a single subunit with a molecular mass of 53 kD. Since UGPase has been demonstrated to be a single-copy gene in the haploid genome of potato (A.Y. Borovkov, P.E. McClean, J.R. Sowokinos, S.H. Ruud, G.A. Secor [1995] J Plant Physiol 147: 644-652), there must be allelic differences at the UGPase locus (chromosome 11). The two alleles, designated ugpA and ugpB, were identified by the absence and presence of a BamHI site, respectively. The relative band intensities of the two cDNA populations following polymerase chain reaction amplification and agarose gel electrophoresis were related to a potato cultivar's ability to resist sweetening when exposed to cold temperatures.

Alleles↗